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Sample records for overexpression increases dopamine

  1. Increased expression of the dopamine transporter leads to loss of dopamine neurons, oxidative stress and l-DOPA reversible motor deficits.

    PubMed

    Masoud, S T; Vecchio, L M; Bergeron, Y; Hossain, M M; Nguyen, L T; Bermejo, M K; Kile, B; Sotnikova, T D; Siesser, W B; Gainetdinov, R R; Wightman, R M; Caron, M G; Richardson, J R; Miller, G W; Ramsey, A J; Cyr, M; Salahpour, A

    2015-02-01

    The dopamine transporter is a key protein responsible for regulating dopamine homeostasis. Its function is to transport dopamine from the extracellular space into the presynaptic neuron. Studies have suggested that accumulation of dopamine in the cytosol can trigger oxidative stress and neurotoxicity. Previously, ectopic expression of the dopamine transporter was shown to cause damage in non-dopaminergic neurons due to their inability to handle cytosolic dopamine. However, it is unknown whether increasing dopamine transporter activity will be detrimental to dopamine neurons that are inherently capable of storing and degrading dopamine. To address this issue, we characterized transgenic mice that over-express the dopamine transporter selectively in dopamine neurons. We report that dopamine transporter over-expressing (DAT-tg) mice display spontaneous loss of midbrain dopamine neurons that is accompanied by increases in oxidative stress markers, 5-S-cysteinyl-dopamine and 5-S-cysteinyl-DOPAC. In addition, metabolite-to-dopamine ratios are increased and VMAT2 protein expression is decreased in the striatum of these animals. Furthermore, DAT-tg mice also show fine motor deficits on challenging beam traversal that are reversed with l-DOPA treatment. Collectively, our findings demonstrate that even in neurons that routinely handle dopamine, increased uptake of this neurotransmitter through the dopamine transporter results in oxidative damage, neuronal loss and l-DOPA reversible motor deficits. In addition, DAT over-expressing animals are highly sensitive to MPTP-induced neurotoxicity. The effects of increased dopamine uptake in these transgenic mice could shed light on the unique vulnerability of dopamine neurons in Parkinson's disease.

  2. Increased brain dopamine and dopamine receptors in schizophrenia

    SciTech Connect

    Mackay, A.V.; Iversen, L.L.; Rossor, M.; Spokes, E.; Bird, E.; Arregui, A.; Creese, I.; Synder, S.H.

    1982-09-01

    In postmortem samples of caudate nucleus and nucleus accumbens from 48 schizophrenic patients, there were significant increases in both the maximum number of binding sites (Bmax) and the apparent dissociation constant (KD) for tritiated spiperone. The increase in apparent KD probably reflects the presence of residual neuroleptic drugs, but changes in Bmax for tritiated spiperone reflect genuine changes in receptor numbers. The increases in receptors were seen only in patients in whom neuroleptic medication had been maintained until the time of death, indicating that they may be entirely iatrogenic. Dopamine measurements for a larger series of schizophrenic and control cases (n greater than 60) show significantly increased concentrations in both the nucleus accumbens and caudate nucleus. The changes in dopamine were not obviously related to neuroleptic medication and, unlike the receptor changes, were most severe in younger patients.

  3. How does angiotensin II increase cardiac dopamine-beta-hydroxylation?

    PubMed

    Chevillard, C; Duchene, N; Alexandre, J M

    1975-03-01

    The potent accelerating effect of angiotensin II (Ang II) on caridac dopamine beta-hydroxylation was studied on slices of rat heart. Ang II did not affect the kinetics of beta-hydroxylation but it increased the axonal uptake of dopamine, and, concomitant with the acceleration of biosynthesis, it enhanced the accumulation of dopamine into tissue. Puromycin, in contrast to actinomycin D, antagonized the stimulation of dopamine beta-hydroxylation by Ang II, but did not suppress the rise in cardiac dopamine. Therefore, to promote the acceleration of dopamine beta-hydroxylation, (i) the rise in tissue dopamine available for conversion appeared to be insufficient, (ii) the formation of new proteins by activation of traduction seemed to constitute the basic mechanism of Ang II action.

  4. Contribution of vesicular and cytosolic dopamine to the increased striatal dopamine efflux elicited by intrastriatal injection of SKF38393.

    PubMed

    Saigusa, Tadashi; Aono, Yuri; Sekino, Reiko; Uchida, Takuya; Takada, Koji; Oi, Yoshiyuki; Koshikawa, Noriaki; Cools, Alexander R

    2009-12-10

    Like dexamphetamine, SKF38393 induces an increase in striatal dopamine efflux which is insensitive for tetrodotoxin, Ca(2+) independent and prevented by a dopamine transporter inhibitor. The dexamphetamine-induced striatal dopamine efflux originates from both the reserpine-sensitive vesicular dopamine pool and the alpha-methyl-para-tyrosine-sensitive cytosolic dopamine pool. Given the similarities between dexamphetamine and SKF38393, we hypothesized that both types of pool also contribute to the striatally applied SKF38393-induced dopamine efflux. Using in vivo microdialysis technique, we analysed the contribution of these pools to the SKF38393-induced striatal dopamine efflux in freely moving rats. The increase of dopamine efflux induced by 1.5 microg SKF38393 was largely prevented by either reserpine (5mg/kg i.p., given 24h earlier) or alpha-methyl-para-tyrosine (250 mg/kg i.p., given 2h earlier), showing that both the vesicular dopamine pool and the cytosolic dopamine pool contribute to the SKF38393-induced increase in striatal dopamine efflux. The sum of the amounts of dopamine that was sensitive to either reserpine or alpha-methyl-para-tyrosine, was greater than 100%, namely 137.6% of the basal dopamine level and 143.9% of the SKF38393-induced dopamine level, suggesting that striatally applied SKF38393 promotes the redistribution of dopamine from vesicles to the cytosol, and vice versa. The finding that the combined treatment of reserpine and alpha-methyl-para-tyrosine only inhibited the SKF38393-induced striatal dopamine efflux till 86.0% of the control, is ascribed to the notion that SKF38393 can also inhibit the re-uptake of dopamine. The latter conclusion has far-reaching consequences for studies in which the effects of SKF38393 are simply ascribed to its dopamine D1 receptor stimulation capacity.

  5. Dopamine Increases a Value-Independent Gambling Propensity.

    PubMed

    Rigoli, Francesco; Rutledge, Robb B; Chew, Benjamin; Ousdal, Olga T; Dayan, Peter; Dolan, Raymond J

    2016-10-01

    Although the impact of dopamine on reward learning is well documented, its influence on other aspects of behavior remains the subject of much ongoing work. Dopaminergic drugs are known to increase risk-taking behavior, but the underlying mechanisms for this effect are not clear. We probed dopamine's role by examining the effect of its precursor L-DOPA on the choices of healthy human participants in an experimental paradigm that allowed particular components of risk to be distinguished. We show that choice behavior depended on a baseline (ie, value-independent) gambling propensity, a gambling preference scaling with the amount/variance, and a value normalization factor. Boosting dopamine levels specifically increased just the value-independent baseline gambling propensity, leaving the other components unaffected. Our results indicate that the influence of dopamine on choice behavior involves a specific modulation of the attractiveness of risky options-a finding with implications for understanding a range of reward-related psychopathologies including addiction.

  6. LRRK2 overexpression alters glutamatergic presynaptic plasticity, striatal dopamine tone, postsynaptic signal transduction, motor activity and memory.

    PubMed

    Beccano-Kelly, Dayne A; Volta, Mattia; Munsie, Lise N; Paschall, Sarah A; Tatarnikov, Igor; Co, Kimberley; Chou, Patrick; Cao, Li-Ping; Bergeron, Sabrina; Mitchell, Emma; Han, Heather; Melrose, Heather L; Tapia, Lucia; Raymond, Lynn A; Farrer, Matthew J; Milnerwood, Austen J

    2015-03-01

    Mutations in leucine-rich repeat kinase 2 (Lrrk2) are the most common genetic cause of Parkinson's disease (PD), a neurodegenerative disorder affecting 1-2% of those >65 years old. The neurophysiology of LRRK2 remains largely elusive, although protein loss suggests a role in glutamatergic synapse transmission and overexpression studies show altered dopamine release in aged mice. We show that glutamate transmission is unaltered onto striatal projection neurons (SPNs) of adult LRRK2 knockout mice and that adult animals exhibit no detectable cognitive or motor deficits. Basal synaptic transmission is also unaltered in SPNs of LRRK2 overexpressing mice, but they do exhibit clear alterations to D2-receptor-mediated short-term synaptic plasticity, behavioral hypoactivity and impaired recognition memory. These phenomena are associated with decreased striatal dopamine tone and abnormal dopamine- and cAMP-regulated phosphoprotein 32 kDa signal integration. The data suggest that LRRK2 acts at the nexus of dopamine and glutamate signaling in the adult striatum, where it regulates dopamine levels, presynaptic glutamate release via D2-dependent synaptic plasticity and dopamine-receptor signal transduction. PMID:25343991

  7. LRRK2 overexpression alters glutamatergic presynaptic plasticity, striatal dopamine tone, postsynaptic signal transduction, motor activity and memory.

    PubMed

    Beccano-Kelly, Dayne A; Volta, Mattia; Munsie, Lise N; Paschall, Sarah A; Tatarnikov, Igor; Co, Kimberley; Chou, Patrick; Cao, Li-Ping; Bergeron, Sabrina; Mitchell, Emma; Han, Heather; Melrose, Heather L; Tapia, Lucia; Raymond, Lynn A; Farrer, Matthew J; Milnerwood, Austen J

    2015-03-01

    Mutations in leucine-rich repeat kinase 2 (Lrrk2) are the most common genetic cause of Parkinson's disease (PD), a neurodegenerative disorder affecting 1-2% of those >65 years old. The neurophysiology of LRRK2 remains largely elusive, although protein loss suggests a role in glutamatergic synapse transmission and overexpression studies show altered dopamine release in aged mice. We show that glutamate transmission is unaltered onto striatal projection neurons (SPNs) of adult LRRK2 knockout mice and that adult animals exhibit no detectable cognitive or motor deficits. Basal synaptic transmission is also unaltered in SPNs of LRRK2 overexpressing mice, but they do exhibit clear alterations to D2-receptor-mediated short-term synaptic plasticity, behavioral hypoactivity and impaired recognition memory. These phenomena are associated with decreased striatal dopamine tone and abnormal dopamine- and cAMP-regulated phosphoprotein 32 kDa signal integration. The data suggest that LRRK2 acts at the nexus of dopamine and glutamate signaling in the adult striatum, where it regulates dopamine levels, presynaptic glutamate release via D2-dependent synaptic plasticity and dopamine-receptor signal transduction.

  8. Increased dopamine tone during meditation-induced change of consciousness.

    PubMed

    Kjaer, Troels W; Bertelsen, Camilla; Piccini, Paola; Brooks, David; Alving, Jørgen; Lou, Hans C

    2002-04-01

    This is the first in vivo demonstration of an association between endogenous neurotransmitter release and conscious experience. Using 11C-raclopride PET we demonstrated increased endogenous dopamine release in the ventral striatum during Yoga Nidra meditation. Yoga Nidra is characterized by a depressed level of desire for action, associated with decreased blood flow in prefrontal, cerebellar and subcortical regions, structures thought to be organized in open loops subserving executive control. In the striatum, dopamine modulates excitatory glutamatergic synapses of the projections from the frontal cortex to striatal neurons, which in turn project back to the frontal cortex via the pallidum and ventral thalamus. The present study was designed to investigate whether endogenous dopamine release increases during loss of executive control in meditation. Participants underwent two 11C-raclopride PET scans: one while attending to speech with eyes closed, and one during active meditation. The tracer competes with endogenous dopamine for access to dopamine D2 receptors predominantly found in the basal ganglia. During meditation, 11C-raclopride binding in ventral striatum decreased by 7.9%. This corresponds to a 65% increase in endogenous dopamine release. The reduced raclopride binding correlated significantly with a concomitant increase in EEG theta activity, a characteristic feature of meditation. All participants reported a decreased desire for action during meditation, along with heightened sensory imagery. The level of gratification and the depth of relaxation did not differ between the attention and meditation conditions. Here we show increased striatal dopamine release during meditation associated with the experience of reduced readiness for action. It is suggested that being in the conscious state of meditation causes a suppression of cortico-striatal glutamatergic transmission. To our knowledge this is the first time in vivo evidence has been provided for

  9. Overexpression of GRK6 rescues L-DOPA-induced signaling abnormalities in the dopamine-depleted striatum of hemiparkinsonian rats

    PubMed Central

    Ahmed, M. Rafiuddin; Bychkov, Evgeny; Kook, Seunghyi; Zurkovsky, Lilia; Dalby, Kevin N.; Gurevich, Eugenia V.

    2015-01-01

    L-DOPA therapy in Parkinson’s disease often results in side effects such as L-DOPA-induced dyskinesia (LID). Our previous studies demonstrated that defective desensitization of dopamine receptors caused by decreased expression of G protein-coupled receptor kinases (GRKs) plays a role. Overexpression of GRK6, the isoform regulating dopamine receptors, in parkinsonian rats and monkeys alleviated LID and reduced LID-associated changes in gene expression. Here we show that 2-fold lentivirus-mediated overexpression of GRK6 in the dopamine-depleted striatum in rats unilaterally lesioned with 6-hydroxydopamine ameliorated supersensitive ERK response to L-DOPA challenge caused by loss of dopamine. A somewhat stronger effect of GRK6 was observed in drug-naïve than in chronically L-DOPA-treated animals. GRK6 reduced the responsiveness of p38 MAP kinase to L-DOPA challenge rendered supersensitive by dopamine depletion. The JNK MAP kinase was unaffected by loss of dopamine, chronic or acute L-DOPA, or GRK6. Overexpressed GRK6 suppressed enhanced activity of Akt in the lesioned striatum by reducing elevated phosphorylation at its major activating residue Thr308. Finally, GRK6 reduced accumulation of ΔFosB in the lesioned striatum, the effect that paralleled a decrease in locomotor sensitization to L-DOPA in GRK6-expressing rats. The results suggest that elevated GRK6 facilitate desensitization of DA receptors, thereby normalizing of the activity of multiple signaling pathways implicated in LID. Thus, improving the regulation of dopamine receptor function via the desensitization mechanism could be an effective way of managing LID. PMID:25687550

  10. Increased dopamine level enhances male-male courtship in Drosophila.

    PubMed

    Liu, Tong; Dartevelle, Laurence; Yuan, Chunyan; Wei, Hongping; Wang, Ying; Ferveur, Jean-François; Guo, Aike

    2008-05-21

    Sexual behavior between males is observed in many species, but the biological factors involved are poorly known. In mammals, manipulation of dopamine has revealed the role of this neuromodulator on male sexual behavior. We used genetic and pharmacological approaches to manipulate the dopamine level in dopaminergic cells in Drosophila and investigated the consequence of this manipulation on male-male courtship behavior. Males with increased dopamine level showed enhanced propensity to court other males but did not change their courtship toward virgin females, general olfactory response, general gustatory response, or locomotor activity. Our results indicate that the high intensity of male-male interaction shown by these manipulated males was related to their altered sensory perception of other males.

  11. Increased dopamine level enhances male-male courtship in Drosophila.

    PubMed

    Liu, Tong; Dartevelle, Laurence; Yuan, Chunyan; Wei, Hongping; Wang, Ying; Ferveur, Jean-François; Guo, Aike

    2008-05-21

    Sexual behavior between males is observed in many species, but the biological factors involved are poorly known. In mammals, manipulation of dopamine has revealed the role of this neuromodulator on male sexual behavior. We used genetic and pharmacological approaches to manipulate the dopamine level in dopaminergic cells in Drosophila and investigated the consequence of this manipulation on male-male courtship behavior. Males with increased dopamine level showed enhanced propensity to court other males but did not change their courtship toward virgin females, general olfactory response, general gustatory response, or locomotor activity. Our results indicate that the high intensity of male-male interaction shown by these manipulated males was related to their altered sensory perception of other males. PMID:18495888

  12. Increasing dopamine D2 receptor expression in the adult nucleus accumbens enhances motivation.

    PubMed

    Trifilieff, P; Feng, B; Urizar, E; Winiger, V; Ward, R D; Taylor, K M; Martinez, D; Moore, H; Balsam, P D; Simpson, E H; Javitch, J A

    2013-09-01

    A decrease in dopamine D2 receptor (D2R) binding in the striatum is one of the most common findings in disorders that involve a dysregulation of motivation, including obesity, addiction and attention deficit hyperactivity disorder. As disruption of D2R signaling in the ventral striatum--including the nucleus accumbens (NAc)--impairs motivation, we sought to determine whether potentiating postsynaptic D2R-dependent signaling in the NAc would improve motivation. In this study, we used a viral vector strategy to overexpress postsynaptic D2Rs in either the NAc or the dorsal striatum. We investigated the effects of D2R overexpression on instrumental learning, willingness to work, use of reward value representations and modulation of motivation by reward associated cues. Overexpression of postsynaptic D2R in the NAc selectively increased motivation without altering consummatory behavior, the representation of the value of the reinforcer, or the capacity to use reward associated cues in flexible ways. In contrast, D2R overexpression in the dorsal striatum did not alter performance on any of the tasks. Thus, consistent with numerous studies showing that reduced D2R signaling impairs motivated behavior, our data show that postsynaptic D2R overexpression in the NAc specifically increases an animal's willingness to expend effort to obtain a goal. Taken together, these results provide insight into the potential impact of future therapeutic strategies that enhance D2R signaling in the NAc. PMID:23711983

  13. The neurotropic parasite Toxoplasma gondii increases dopamine metabolism

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The common parasite Toxoplasma gondii induces behavioral alterations in its hosts including phenotypes increasing the likelihood of its transmission in rodents and reports of psychobehavioral alterations in humans. We have found that elevated levels of dopamine are associated with the encysted stage...

  14. Dopamine Increases a Value-Independent Gambling Propensity.

    PubMed

    Rigoli, Francesco; Rutledge, Robb B; Chew, Benjamin; Ousdal, Olga T; Dayan, Peter; Dolan, Raymond J

    2016-10-01

    Although the impact of dopamine on reward learning is well documented, its influence on other aspects of behavior remains the subject of much ongoing work. Dopaminergic drugs are known to increase risk-taking behavior, but the underlying mechanisms for this effect are not clear. We probed dopamine's role by examining the effect of its precursor L-DOPA on the choices of healthy human participants in an experimental paradigm that allowed particular components of risk to be distinguished. We show that choice behavior depended on a baseline (ie, value-independent) gambling propensity, a gambling preference scaling with the amount/variance, and a value normalization factor. Boosting dopamine levels specifically increased just the value-independent baseline gambling propensity, leaving the other components unaffected. Our results indicate that the influence of dopamine on choice behavior involves a specific modulation of the attractiveness of risky options-a finding with implications for understanding a range of reward-related psychopathologies including addiction. PMID:27149935

  15. Dopamine Increases a Value-Independent Gambling Propensity

    PubMed Central

    Rigoli, Francesco; Rutledge, Robb B; Chew, Benjamin; Ousdal, Olga T; Dayan, Peter; Dolan, Raymond J

    2016-01-01

    Although the impact of dopamine on reward learning is well documented, its influence on other aspects of behavior remains the subject of much ongoing work. Dopaminergic drugs are known to increase risk-taking behavior, but the underlying mechanisms for this effect are not clear. We probed dopamine's role by examining the effect of its precursor L-DOPA on the choices of healthy human participants in an experimental paradigm that allowed particular components of risk to be distinguished. We show that choice behavior depended on a baseline (ie, value-independent) gambling propensity, a gambling preference scaling with the amount/variance, and a value normalization factor. Boosting dopamine levels specifically increased just the value-independent baseline gambling propensity, leaving the other components unaffected. Our results indicate that the influence of dopamine on choice behavior involves a specific modulation of the attractiveness of risky options—a finding with implications for understanding a range of reward-related psychopathologies including addiction. PMID:27149935

  16. Hypothyroidism leads to increased dopamine receptor sensitivity and concentration

    SciTech Connect

    Crocker, A.D.; Overstreet, D.H.; Crocker, J.M.

    1986-06-01

    Rats treated with iodine-131 were confirmed to be hypothyroid by their reduced baseline core body temperatures, reduced serum thyroxine concentrations and elevated serum thyroid stimulating hormone concentrations. When hypothyroid rats were compared to euthyroid controls they were more sensitive to the effects of apomorphine (1.0 mumol/kg) on stereotypy, operant responding and body temperature and showed a smaller reduction in locomotor activity after injection of haloperidol (0.25 mumol/kg). Receptor binding studies on striatal homogenates indicated that hypothyroid rats had increased concentrations of D2 dopamine receptors but there was no change in the affinity. It is concluded that hypothyroidism increases dopamine receptor sensitivity by increasing receptor concentration.

  17. Elevated dopamine concentration in light-adapted zebrafish retinas is correlated with increased dopamine synthesis and metabolism.

    PubMed

    Connaughton, Victoria P; Wetzell, Bradley; Arneson, Lynne S; DeLucia, Vittoria; Riley, Anthony L

    2015-10-01

    Probing zebrafish (Danio rerio) retinal cryostat sections, collected either 8 h into the light or dark cycle, with an antibody against tyrosine hydroxylase (TH) identified a single population of immunopositive cells in the inner retina. However, the observed labeling patterns were not identical in both sets of tissues - label intensity was brighter in light-adapted tissue. This difference was quantified by probing western blots of retinal homogenates with the same TH antibody, which showed that TH expression increased by 42% in light-adapted tissue. High-performance liquid chromatography with electrochemical detection revealed that the concentrations of both dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) are also elevated in light-adapted zebrafish retinal tissue. Dopamine levels increased by 14% and DOPAC levels increased by 25% when measured in retinal homogenates harvested during the light cycle. These results indicate that dopamine levels in zebrafish retina are significantly increased in light-adapted tissue. The increase in dopamine content is correlated with an increase in both TH and DOPAC, suggesting that changes in dopamine concentration are due to light-adaptive changes in the synthesis, release and metabolism of dopamine. Dopamine concentration is elevated in lighted-adapted zebrafish retinas. This increase is correlated with an increase in both tyrosine hydroxylase (TH) and DOPAC (3,4-dihydroxyphenylacetic acid), suggesting that changes in dopamine concentration are due to light-adaptive changes in the synthesis, release and metabolism of dopamine. This is applicable to studies examining retinal mutants, the role of dopamine in disease or visual system development.

  18. Acrylamide increases dopamine levels by affecting dopamine transport and metabolism related genes in the striatal dopaminergic system.

    PubMed

    Pan, Xiaoqi; Guo, Xiongxiong; Xiong, Fei; Cheng, Guihong; Lu, Qing; Yan, Hong

    2015-07-01

    Dopaminergic system dysfunction is proved to be a possible mechanism in acrylamide (ACR) -induced neurotoxicity. The neurotransmitter dopamine (DA) has an increasingly important role in the dopaminergic system. Thus, the goal of this study is to evaluate effects of ACR on dopamine and its metabolite levels, dopamine transport and metabolic gene expression in dopaminergic neurons. Male Sprague-Dawley (SD) rats were dosed orally with ACR at 0 (saline), 20, 30, and 40 mg/kg/day for 20 days. Splayed hind limbs, reduced tail flick time and abnormal gait which preceded other neurologic parameters were observed in the above rats. ACR significantly increased dopamine levels, decreased 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanilic acid (HVA) contents in an area dependent manner in rat striatum. Immunohistochemical staining of the striatum revealed that the number of tyrosine hydroxylase (TH) positive cells significantly increased, while monoamine oxidase (MAO) positive cells were drastically reduced, which was consistent with changes in their mRNA and protein expressions. In addition, dopamine transporter (DAT) and vesicular monoamine transporter 2 (VMAT2) expression levels were both down-regulated in the striatum. These results suggest that dopamine levels increase significantly in response to ACR, presumably due to changes in the dopamine transport and metabolism related genes expression in the striatal dopaminergic neurons.

  19. Acute fasting increases somatodendritic dopamine release in the ventral tegmental area.

    PubMed

    Roseberry, Aaron G

    2015-08-01

    Fasting and food restriction alter the activity of the mesolimbic dopamine system to affect multiple reward-related behaviors. Food restriction decreases baseline dopamine levels in efferent target sites and enhances dopamine release in response to rewards such as food and drugs. In addition to releasing dopamine from axon terminals, dopamine neurons in the ventral tegmental area (VTA) also release dopamine from their soma and dendrites, and this somatodendritic dopamine release acts as an autoinhibitory signal to inhibit neighboring VTA dopamine neurons. It is unknown whether acute fasting also affects dopamine release, including the local inhibitory somatodendritic dopamine release in the VTA. In these studies, I have tested whether fasting affects the inhibitory somatodendritic dopamine release within the VTA by examining whether an acute 24-h fast affects the inhibitory postsynaptic current mediated by evoked somatodendritic dopamine release (D2R IPSC). Fasting increased the contribution of the first action potential to the overall D2R IPSC and increased the ratio of repeated D2R IPSCs evoked at short intervals. Fasting also reduced the effect of forskolin on the D2R IPSC and led to a significantly bigger decrease in the D2R IPSC in low extracellular calcium. Finally, fasting resulted in an increase in the D2R IPSCs when a more physiologically relevant train of D2R IPSCs was used. Taken together, these results indicate that fasting caused a change in the properties of somatodendritic dopamine release, possibly by increasing dopamine release, and that this increased release can be sustained under conditions where dopamine neurons are highly active.

  20. Increased baseline occupancy of D2 receptors by dopamine in schizophrenia

    PubMed Central

    Abi-Dargham, Anissa; Rodenhiser, Janine; Printz, David; Zea-Ponce, Yolanda; Gil, Roberto; Kegeles, Lawrence S.; Weiss, Richard; Cooper, Thomas B.; Mann, J. John; Van Heertum, Ronald L.; Gorman, Jack M.; Laruelle, Marc

    2000-01-01

    The classical dopamine hypothesis of schizophrenia postulates a hyperactivity of dopaminergic transmission at the D2 receptor. We measured in vivo occupancy of striatal D2 receptors by dopamine in 18 untreated patients with schizophrenia and 18 matched controls, by comparing D2 receptor availability before and during pharmacologically induced acute dopamine depletion. Acute depletion of intrasynaptic dopamine resulted in a larger increase in D2 receptor availability in patients with schizophrenia (19% ± 11%) compared with control subjects (9% ± 7%, P = 0.003). The increased occupancy of D2 receptors by dopamine occurred both in first-episode neuroleptic-naive patients and in previously treated chronic patients experiencing an episode of illness exacerbation. In addition, elevated synaptic dopamine was predictive of good treatment response of positive symptoms to antipsychotic drugs. This finding provides direct evidence of increased stimulation of D2 receptors by dopamine in schizophrenia, consistent with increased phasic activity of dopaminergic neurons. PMID:10884434

  1. Nicotine decreases ethanol-induced dopamine signaling and increases self-administration via stress hormones.

    PubMed

    Doyon, William M; Dong, Yu; Ostroumov, Alexey; Thomas, Alyse M; Zhang, Tao A; Dani, John A

    2013-08-01

    Tobacco smoking is a well-known risk factor for subsequent alcohol abuse, but the neural events underlying this risk remain largely unknown. Alcohol and nicotine reinforcement involve common neural circuitry, including the mesolimbic dopamine system. We demonstrate in rodents that pre-exposure to nicotine increases alcohol self-administration and decreases alcohol-induced dopamine responses. The blunted dopamine response was due to increased inhibitory synaptic transmission onto dopamine neurons. Blocking stress hormone receptors prior to nicotine exposure prevented all interactions with alcohol that we measured, including the increased inhibition onto dopamine neurons, the decreased dopamine responses, and the increased alcohol self-administration. These results indicate that nicotine recruits neuroendocrine systems to influence neurotransmission and behavior associated with alcohol reinforcement.

  2. Propentophylline increases striatal dopamine release but dampens methamphetamine-induced dopamine dynamics: A microdialysis study.

    PubMed

    Gough, B; Pereira, F C; Fontes Ribeiro, C A; Ali, S F; Binienda, Z K

    2014-10-01

    While there are currently no medications approved for methamphetamine (METH) addiction, it has been shown that propentofylline (PPF), an atypical methylxanthine, can suppress the rewarding effects of methamphetamine (METH) in mice. This experiment studied the interactions of PPF with METH in striatal dopaminergic transmission. Herein, the impact of PPF (10-40mM, intrastriatally perfused (80min) on the effect of METH (5mg/kg, i.p.) on striatal dopamine (DA) release was evaluated using brain microdialysis in Sprague-Dawley adult rats. METH was injected at the 60min time point of the 80min PPF perfusion. The extracellular levels of DA and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were determined using high performance liquid chromatography with electrochemical detection (HPLC-ED). PPF induced a concentration-dependent increase in DA release beginning 30min after the onset of PPF perfusion. DA peak levels evoked by 40mM PPF were similar to those induced by 5mg/kg METH i.p. Only the highest concentration of PPF decreased the METH-induced DA peak (circa 70%). The significant decreases in extracellular levels of DOPAC and HVA evoked by METH were partially blocked by 10 and 20mM PPF. Although 40mM of PPF also partially blocked the METH-induced DOPAC decrease, it completely blocked HVA depletion after a transient increase in HVA levels in METH-treated rats. Data indicates for the first time that while PPF increases presynaptic striatal DA dynamics it attenuates METH-induced striatal DA release and metabolism.

  3. Characterization of the phencyclidine-induced increase in prefrontal cortical dopamine metabolism in the rat

    PubMed Central

    Umino, Asami; Takahashi, Kiyohisa; Nishikawa, Toru

    1998-01-01

    We have investigated the effects of a schizophrenomimetic drug phencyclidine (PCP) and N-methyl-D-aspartate (NMDA)-related agents alone or in combination on dopamine metabolism in the medial prefrontal cortex and striatum of the rats by measuring the tissue concentrations of dopamine and its metabolite, 3,4-dihyroxyphenylacetic acid (DOPAC), and the rate of dopamine disappearance (dopamine utilization) after its synthesis inhibition.Systemic injection of PCP and selective, non-competitive, NMDA antagonists caused an increase of both tissue concentrations of DOPAC and dopamine utilization in the prefrontal cortex but not in the striatum. The PCP-induced augmentation of cortical dopamine metabolism was not influenced by selective lesion of ascending noradrenergic neurones.Intra-prefrontal cortical infusion of PCP or selective competitive or non-competitive antagonists of the NMDA receptor mimicked the ability of systemic PCP injection to enhance DOPAC levels and dopamine utilization in the prefrontal cortex. However, an NMDA antagonist injected into the cell body area of the mesocortical dopaminergic neurones failed to affect dopamine metabolism in the prefrontal cortex.The increasing effects of PCP and selective NMDA antagonists on cortical dopamine utilization were not additive, although a dopamine receptor antagonist, haloperidol, still accelerated the disappearance of dopamine, even in the presence of PCP.Intra-cortical or intra-ventricular infusion of NMDA or D-alanine but not L-alanine, attenuated the ability of systemic PCP administration to facilitate prefrontal dopamine utilization.These data suggest that PCP might activate prefrontal cortical dopaminergic neurones, at least in part, by blocking the NMDA receptor in the prefrontal cortex which participates in a tonic inhibitory control of the mesoprefrontal dopaminergic projections. PMID:9641556

  4. Stimulant-induced dopamine increases are markedly blunted in active cocaine abusers.

    PubMed

    Volkow, N D; Tomasi, D; Wang, G-J; Logan, J; Alexoff, D L; Jayne, M; Fowler, J S; Wong, C; Yin, P; Du, C

    2014-09-01

    Dopamine signaling in nucleus accumbens is essential for cocaine reward. Interestingly, imaging studies have reported blunted dopamine increases in striatum (assessed as reduced binding of [(11)C]raclopride to D2/D3 receptors) in detoxified cocaine abusers. Here, we evaluate whether the blunted dopamine response reflected the effects of detoxification and the lack of cocaine-cues during stimulant exposure. For this purpose we studied 62 participants (43 non-detoxified cocaine abusers and 19 controls) using positron emission tomography and [(11)C]raclopride (radioligand sensitive to endogenous dopamine) to measure dopamine increases induced by intravenous methylphenidate and in 24 of the cocaine abusers, we also compared dopamine increases when methylphenidate was administered concomitantly with a cocaine cue-video versus a neutral-video. In controls, methylphenidate increased dopamine in dorsal (effect size 1.4; P<0.001) and ventral striatum (location of accumbens) (effect size 0.89; P<0.001), but in cocaine abusers methylphenidate's effects did not differ from placebo and were similar whether cocaine-cues were present or not. In cocaine abusers despite the markedly attenuated dopaminergic effects, the methylphenidate-induced changes in ventral striatum were associated with intense drug craving. Our findings are consistent with markedly reduced signaling through D2 receptors during intoxication in active cocaine abusers regardless of cues exposure, which might contribute to compulsive drug use. PMID:24912491

  5. Stimulant-induced dopamine increases are markedly blunted in active cocaine abusers

    PubMed Central

    Volkow, ND; Tomasi, D; Wang, G-J; Logan, J; Alexoff, DL; Jayne, M; Fowler, JS; Wong, C; Yin, P; Du, C

    2016-01-01

    Dopamine signaling in nucleus accumbens is essential for cocaine reward. Interestingly, imaging studies have reported blunted dopamine increases in striatum (assessed as reduced binding of [11C]raclopride to D2/D3 receptors) in detoxified cocaine abusers. Here, we evaluate whether the blunted dopamine response reflected the effects of detoxification and the lack of cocaine-cues during stimulant exposure. For this purpose we studied 62 participants (43 non-detoxified cocaine abusers and 19 controls) using positron emission tomography and [11C]raclopride (radioligand sensitive to endogenous dopamine) to measure dopamine increases induced by intravenous methylphenidate and in 24 of the cocaine abusers, we also compared dopamine increases when methylphenidate was administered concomitantly with a cocaine cue-video versus a neutral-video. In controls, methylphenidate increased dopamine in dorsal (effect size 1.4; P < 0.001) and ventral striatum (location of accumbens) (effect size 0.89; P < 0.001), but in cocaine abusers methylphenidate’s effects did not differ from placebo and were similar whether cocaine-cues were present or not. In cocaine abusers despite the markedly attenuated dopaminergic effects, the methylphenidate-induced changes in ventral striatum were associated with intense drug craving. Our findings are consistent with markedly reduced signaling through D2 receptors during intoxication in active cocaine abusers regardless of cues exposure, which might contribute to compulsive drug use. PMID:24912491

  6. CCK2 receptor-deficient mice have increased sensitivity of dopamine D2 receptors.

    PubMed

    Kõks, S; Abramov, U; Veraksits, A; Bourin, M; Matsui, T; Vasar, E

    2003-02-01

    The present study supports a role of CCK(2) receptors in the regulation of dopamine neurones. In pharmacological studies conducted on male CCK(2) receptor-deficient mice the changes in the activity of dopamine system were established. A low dose of dopamine agonist apomorphine (0.1 mg/kg), stimulating the pre-synaptic dopamine receptors, induced significantly stronger suppression of locomotor activity in mutant mice (-/-) compared to their wild-type littermates (+/+). The administration of amphetamine (3-6 mg/kg), a drug increasing dopamine release, caused a dose-dependent stimulation of locomotor activity in wild-type mice. In mice lacking CCK(2) receptors, a lower dose of amphetamine (3 mg/kg) tended to suppress the motor activity, whereas the higher dose (6 mg/kg) induced the significantly stronger motor stimulation in mutant mice. Moreover, in the CCK(2) receptor-deficient mice the affinity of dopamine D(2) receptors, but not 5-HT(2) receptors, was increased. Altogether, the targeted genetic suppression of CCK(2) receptors increased the sensitivity of pre- and post-synaptic dopamine D(2) receptors.

  7. A53T human α-synuclein overexpression in transgenic mice induces pervasive mitochondria macroautophagy defects preceding dopamine neuron degeneration.

    PubMed

    Chen, Linan; Xie, Zhiguo; Turkson, Susie; Zhuang, Xiaoxi

    2015-01-21

    In vitro evidence suggests that the inefficient removal of damaged mitochondria by macroautophagy contributes to Parkinson's disease (PD). Using a tissue-specific gene amplification strategy, we generated a transgenic mouse line with human α-synuclein A53T overexpression specifically in dopamine (DA) neurons. Transgenic mice showed profound early-onset mitochondria abnormalities, characterized by macroautophagy marker-positive cytoplasmic inclusions containing mainly mitochondrial remnants, which preceded the degeneration of DA neurons. Genetic deletion of either parkin or PINK1 in these transgenic mice significantly worsened mitochondrial pathologies, including drastically enlarged inclusions and loss of total mitochondria contents. These data suggest that mitochondria are the main targets of α-synuclein and their defective autophagic clearance plays a significant role during pathogenesis. Moreover, endogenous PINK1 or parkin is indispensable for the proper autophagic removal of damaged mitochondria. Our data for the first time establish an essential link between mitochondria macroautophagy impairments and DA neuron degeneration in an in vivo model based on known PD genetics. The model, its well-defined pathologies, and the demonstration of a main pathogenesis pathway in the present study have set the stage and direction of emphasis for future studies.

  8. Long-term controlled GDNF over-expression reduces dopamine transporter activity without affecting tyrosine hydroxylase expression in the rat mesostriatal system.

    PubMed

    Barroso-Chinea, Pedro; Cruz-Muros, Ignacio; Afonso-Oramas, Domingo; Castro-Hernández, Javier; Salas-Hernández, Josmar; Chtarto, Abdelwahed; Luis-Ravelo, Diego; Humbert-Claude, Marie; Tenenbaum, Liliane; González-Hernández, Tomás

    2016-04-01

    The dopamine (DA) transporter (DAT) is a plasma membrane glycoprotein expressed in dopaminergic (DA-) cells that takes back DA into presynaptic neurons after its release. DAT dysfunction has been involved in different neuro-psychiatric disorders including Parkinson's disease (PD). On the other hand, numerous studies support that the glial cell line-derived neurotrophic factor (GDNF) has a protective effect on DA-cells. However, studies in rodents show that prolonged GDNF over-expression may cause a tyrosine hydroxylase (TH, the limiting enzyme in DA synthesis) decline. The evidence of TH down-regulation suggests that another player in DA handling, DAT, may also be regulated by prolonged GDNF over-expression, and the possibility that this effect is induced at GDNF expression levels lower than those inducing TH down-regulation. This issue was investigated here using intrastriatal injections of a tetracycline-inducible adeno-associated viral vector expressing human GDNF cDNA (AAV-tetON-GDNF) in rats, and doxycycline (DOX; 0.01, 0.03, 0.5 and 3mg/ml) in the drinking water during 5weeks. We found that 3mg/ml DOX promotes an increase in striatal GDNF expression of 12× basal GDNF levels and both DA uptake decrease and TH down-regulation in its native and Ser40 phosphorylated forms. However, 0.5mg/ml DOX promotes a GDNF expression increase of 3× basal GDNF levels with DA uptake decrease but not TH down-regulation. The use of western-blot under non-reducing conditions, co-immunoprecipitation and in situ proximity ligation assay revealed that the DA uptake decrease is associated with the formation of DAT dimers and an increase in DAT-α-synuclein interactions, without changes in total DAT levels or its compartmental distribution. In conclusion, at appropriate GDNF transduction levels, DA uptake is regulated through DAT protein-protein interactions without interfering with DA synthesis. PMID:26777664

  9. Dopamine disruption increases negotiation for cooperative interactions in a fish.

    PubMed

    Messias, João P M; Paula, José R; Grutter, Alexandra S; Bshary, Redouan; Soares, Marta C

    2016-01-01

    Humans and other animals use previous experiences to make behavioural decisions, balancing the probabilities of receiving rewards or punishments with alternative actions. The dopaminergic system plays a key role in this assessment: for instance, a decrease in dopamine transmission, which is signalled by the failure of an expected reward, may elicit a distinct behavioural response. Here, we tested the effect of exogenously administered dopaminergic compounds on a cooperative vertebrate's decision-making process, in a natural setting. We show, in the Indo-Pacific bluestreak cleaner wrasse Labroides dimidiatus, that blocking dopamine receptors in the wild induces cleaners to initiate more interactions with and to provide greater amounts of physical contact to their client fish partners. This costly form of tactile stimulation using their fins is typically used to prolong interactions and to reconcile with clients after cheating. Interestingly, client jolt rate, a correlate of cheating by cleaners, remained unaffected. Thus, in low effective dopaminergic transmission conditions cleaners may renegotiate the occurrence and duration of the interaction with a costly offer. Our results provide first evidence for a prominent role of the dopaminergic system in decision-making in the context of cooperation in fish. PMID:26853241

  10. Dopamine disruption increases negotiation for cooperative interactions in a fish

    PubMed Central

    Messias, João P. M.; Paula, José R.; Grutter, Alexandra S.; Bshary, Redouan; Soares, Marta C.

    2016-01-01

    Humans and other animals use previous experiences to make behavioural decisions, balancing the probabilities of receiving rewards or punishments with alternative actions. The dopaminergic system plays a key role in this assessment: for instance, a decrease in dopamine transmission, which is signalled by the failure of an expected reward, may elicit a distinct behavioural response. Here, we tested the effect of exogenously administered dopaminergic compounds on a cooperative vertebrate’s decision-making process, in a natural setting. We show, in the Indo-Pacific bluestreak cleaner wrasse Labroides dimidiatus, that blocking dopamine receptors in the wild induces cleaners to initiate more interactions with and to provide greater amounts of physical contact to their client fish partners. This costly form of tactile stimulation using their fins is typically used to prolong interactions and to reconcile with clients after cheating. Interestingly, client jolt rate, a correlate of cheating by cleaners, remained unaffected. Thus, in low effective dopaminergic transmission conditions cleaners may renegotiate the occurrence and duration of the interaction with a costly offer. Our results provide first evidence for a prominent role of the dopaminergic system in decision-making in the context of cooperation in fish. PMID:26853241

  11. Dopamine Transporter Blockade Increases LTP in the CA1 Region of the Rat Hippocampus via Activation of the D3 Dopamine Receptor

    ERIC Educational Resources Information Center

    Swant, Jarod; Wagner, John J.

    2006-01-01

    Dopamine has been demonstrated to be involved in the modulation of long-term potentiation (LTP) in the CA1 region of the hippocampus. As monoamine transporter blockade will increase the actions of endogenous monoamine neurotransmitters, the effect of a dopamine transporter (DAT) antagonist on LTP was assessed using field excitatory postsynaptic…

  12. Food Restriction Increases Glutamate Receptor-Mediated Burst Firing of Dopamine Neurons

    PubMed Central

    Branch, Sarah Y.; Goertz, R. Brandon; Sharpe, Amanda L.; Pierce, Janie; Roy, Sudip; Ko, Daijin; Paladini, Carlos A.

    2013-01-01

    Restriction of food intake increases the acquisition of drug abuse behavior and enhances the reinforcing efficacy of those drugs. However, the neurophysiological mechanisms responsible for the interactions between feeding state and drug use are largely unknown. Here we show that chronic mild food restriction increases the burst firing of dopamine neurons in the substantia nigra. Dopamine neurons from food-restricted mice exhibited increased burst firing in vivo, an effect that was enhanced by an injection of the psychomotor stimulant cocaine (10 mg/kg, i.p.). Food restriction also enhanced aspartic acid-induced burst firing of dopamine neurons in an ex vivo brain slice preparation, consistent with an adaptation occurring in the somatodendritic compartment and independent of a circuit mechanism. Enhanced burst firing persisted after 10 d of free feeding following chronic food restriction but was not observed following a single overnight fast. Whole-cell patch-clamp recordings indicated that food restriction also increased electrically evoked AMPAR/NMDAR ratios and increased D2 autoreceptor-mediated desensitization in dopamine neurons. These results identify dopamine neurons in the substantia nigra as a convergence point for the interactions between feeding state and drugs of abuse. Furthermore, increased glutamate transmission combined with decreased autoreceptor inhibition could work in concert to enhance drug efficacy in response to food restriction. PMID:23966705

  13. An increase in renal dopamine does not stimulate natriuresis after fava bean ingestion123

    PubMed Central

    Garland, Emily M; Cesar, Tericka S; Lonce, Suzanna; Ferguson, Marcus C; Robertson, David

    2013-01-01

    Background: Fava beans (Vicia faba) contain dihydroxyphenylalanine (dopa), and their ingestion may increase dopamine stores. Renal dopamine regulates blood pressure and blood volume via a natriuretic effect. Objective: The objective was to determine the relation between dietary fava beans, plasma and urinary catechols, and urinary sodium excretion in 13 healthy volunteers. Design: Catechol and sodium data were compared by using a longitudinal design in which all participants consumed a fixed-sodium study diet on day 1 and the fixed-sodium diet plus fava beans on day 2. Blood was sampled at 1, 2, 4, and 6 h after a meal, and 3 consecutive 4-h urine samples were collected. Results: Mean (±SD) plasma dopa was significantly greater 1 h after fava bean consumption (11,670 ± 5440 compared with 1705 ± 530 pg/mL; P = 0.001) and remained elevated at 6 h. Plasma dopamine increased nearly 15-fold during this period. Fava bean consumption also increased urinary dopamine excretion to 306 ± 116, 360 ± 235, and 159 ± 111 μg/4-h urine sample compared with 45 ± 21, 54 ± 29, and 44 ± 17 μg in the 3 consecutive 4-h samples after the control diet (P ≤ 0.005). These substantial increases in plasma and urinary dopa and dopamine were unexpectedly associated with decreased urinary sodium. Conclusion: The failure of fava bean consumption to provoke natriuresis may indicate that dopa concentrations in commercially available beans do not raise renal dopamine sufficiently to stimulate sodium excretion, at least when beans are added to a moderate-sodium diet in healthy volunteers. This trial was registered at clinicaltrials.gov as NCT01064739. PMID:23553159

  14. Increased isobutanol production in Saccharomyces cerevisiae by overexpression of genes in valine metabolism

    PubMed Central

    2011-01-01

    Background Isobutanol can be a better biofuel than ethanol due to its higher energy density and lower hygroscopicity. Furthermore, the branched-chain structure of isobutanol gives a higher octane number than the isomeric n-butanol. Saccharomyces cerevisiae was chosen as the production host because of its relative tolerance to alcohols, robustness in industrial fermentations, and the possibility for future combination of isobutanol production with fermentation of lignocellulosic materials. Results The yield of isobutanol was improved from 0.16 to 0.97 mg per g glucose by simultaneous overexpression of biosynthetic genes ILV2, ILV3, and ILV5 in valine metabolism in anaerobic fermentation of glucose in mineral medium in S. cerevisiae. Isobutanol yield was further improved by twofold by the additional overexpression of BAT2, encoding the cytoplasmic branched-chain amino-acid aminotransferase. Overexpression of ILV6, encoding the regulatory subunit of Ilv2, in the ILV2 ILV3 ILV5 overexpression strain decreased isobutanol production yield by threefold. In aerobic cultivations in shake flasks in mineral medium, the isobutanol yield of the ILV2 ILV3 ILV5 overexpression strain and the reference strain were 3.86 and 0.28 mg per g glucose, respectively. They increased to 4.12 and 2.4 mg per g glucose in yeast extract/peptone/dextrose (YPD) complex medium under aerobic conditions, respectively. Conclusions Overexpression of genes ILV2, ILV3, ILV5, and BAT2 in valine metabolism led to an increase in isobutanol production in S. cerevisiae. Additional overexpression of ILV6 in the ILV2 ILV3 ILV5 overexpression strain had a negative effect, presumably by increasing the sensitivity of Ilv2 to valine inhibition, thus weakening the positive impact of overexpression of ILV2, ILV3, and ILV5 on isobutanol production. Aerobic cultivations of the ILV2 ILV3 ILV5 overexpression strain and the reference strain showed that supplying amino acids in cultivation media gave a substantial

  15. Intrahippocampal Infusions of Anisomycin Produce Amnesia: Contribution of Increased Release of Norepinephrine, Dopamine, and Acetylcholine

    ERIC Educational Resources Information Center

    Qi, Zhenghan; Gold, Paul E.

    2009-01-01

    Intra-amygdala injections of anisomycin produce large increases in the release of norepinephrine (NE), dopamine (DA), and serotonin in the amygdala. Pretreatment with intra-amygdala injections of the beta-adrenergic receptor antagonist propranolol attenuates anisomycin-induced amnesia without reversing the inhibition of protein synthesis, and…

  16. Her-2 overexpression increases the metastatic outgrowth of breast cancer cells in the brain.

    PubMed

    Palmieri, Diane; Bronder, Julie L; Herring, Jeanne M; Yoneda, Toshiyuki; Weil, Robert J; Stark, Andreas M; Kurek, Raffael; Vega-Valle, Eleazar; Feigenbaum, Lionel; Halverson, Douglas; Vortmeyer, Alexander O; Steinberg, Seth M; Aldape, Kenneth; Steeg, Patricia S

    2007-05-01

    Retrospective studies of breast cancer patients suggest that primary tumor Her-2 overexpression or trastuzumab therapy is associated with a devastating complication: the development of central nervous system (brain) metastases. Herein, we present Her-2 expression trends from resected human brain metastases and data from an experimental brain metastasis assay, both indicative of a functional contribution of Her-2 to brain metastatic colonization. Of 124 archival resected brain metastases from breast cancer patients, 36.2% overexpressed Her-2, indicating an enrichment in the frequency of tumor Her-2 overexpression at this metastatic site. Using quantitative real-time PCR of laser capture microdissected epithelial cells, Her-2 and epidermal growth factor receptor (EGFR) mRNA levels in a cohort of 12 frozen brain metastases were increased up to 5- and 9-fold, respectively, over those of Her-2-amplified primary tumors. Co-overexpression of Her-2 and EGFR was also observed in a subset of brain metastases. We then tested the hypothesis that overexpression of Her-2 increases the colonization of breast cancer cells in the brain in vivo. A subclone of MDA-MB-231 human breast carcinoma cells that selectively metastasizes to brain (231-BR) overexpressed EGFR; 231-BR cells were transfected with low (4- to 8-fold) or high (22- to 28-fold) levels of Her-2. In vivo, in a model of brain metastasis, low or high Her-2-overexpressing 231-BR clones produced comparable numbers of micrometastases in the brain as control transfectants; however, the Her-2 transfectants yielded 3-fold greater large metastases (>50 microm(2); P < 0.001). Our data indicate that Her-2 overexpression increases the outgrowth of metastatic tumor cells in the brain in this model system. PMID:17483330

  17. Enduring increases in anxiety-like behavior and rapid nucleus accumbens dopamine signaling in socially isolated rats

    PubMed Central

    Yorgason, Jordan T.; España, Rodrigo A.; Konstantopoulos, Joanne K.; Weiner, Jeffrey L.; Jones, Sara R.

    2013-01-01

    Social isolation (SI) rearing, a model of early life stress, results in profound behavioral alterations, including increased anxiety-like behavior, impaired sensorimotor gating and increased self-administration of addictive substances. These changes are accompanied by alterations in mesolimbic dopamine function, such as increased dopamine and metabolite tissue content, increased dopamine responses to cues and psychostimulants, and increased dopamine neuron burst firing. Using voltammetric techniques, we examined the effects of SI rearing on dopamine transporter activity, vesicular release and dopamine D2-type autoreceptor activity in the nucleus accumbens core. Long–Evans rats were housed in group (GH; 4/cage) or SI (1/cage) conditions from weaning into early adulthood [postnatal day (PD) 28–77]. After this initial housing period, rats were assessed on the elevated plus-maze for an anxiety-like phenotype, and then slice voltammetry experiments were performed. To study the enduring effects of SI rearing on anxiety-like behavior and dopamine terminal function, another cohort of similarly reared rats was isolated for an additional 4 months (until PD 174) and then tested. Our findings demonstrate that SI rearing results in lasting increases in anxiety-like behavior, dopamine release and dopamine transporter activity, but not D2 activity. Interestingly, GH-reared rats that were isolated as adults did not develop the anxiety-like behavior or dopamine changes seen in SI-reared rats. Together, our data suggest that early life stress results in an anxiety-like phenotype, with lasting increases in dopamine terminal function. PMID:23294165

  18. Lamin B1 overexpression increases nuclear rigidity in autosomal dominant leukodystrophy fibroblasts

    PubMed Central

    Ferrera, Denise; Canale, Claudio; Marotta, Roberto; Mazzaro, Nadia; Gritti, Marta; Mazzanti, Michele; Capellari, Sabina; Cortelli, Pietro; Gasparini, Laura

    2014-01-01

    The architecture and structural mechanics of the cell nucleus are defined by the nuclear lamina, which is formed by A- and B-type lamins. Recently, gene duplication and protein overexpression of lamin B1 (LB1) have been reported in pedigrees with autosomal dominant leukodystrophy (ADLD). However, how the overexpression of LB1 affects nuclear mechanics and function and how it may result in pathology remain unexplored. Here, we report that in primary human skin fibroblasts derived from ADLD patients, LB1, but not other lamins, is overexpressed at the nuclear lamina and specifically enhances nuclear stiffness. Transient transfection of LB1 in HEK293 and neuronal N2a cells mimics the mechanical phenotype of ADLD nuclei. Notably, in ADLD fibroblasts, reducing LB1 protein levels by shRNA knockdown restores elasticity values to those indistinguishable from control fibroblasts. Moreover, isolated nuclei from ADLD fibroblasts display a reduced nuclear ion channel open probability on voltage-step application, suggesting that biophysical changes induced by LB1 overexpression may alter nuclear signaling cascades in somatic cells. Overall, the overexpression of LB1 in ADLD cells alters nuclear mechanics and is linked to changes in nuclear signaling, which could help explain the pathogenesis of this disease.—Ferrera, D., Canale, C., Marotta, R., Mazzaro, N., Gritti, M., Mazzanti, M., Capellari, S., Cortelli, P., Gasparini, L. Lamin B1 overexpression increases nuclear rigidity in autosomal dominant leukodystrophy fibroblasts. PMID:24858279

  19. Increased dopaminergic innervation in the brain of conditional mutant mice overexpressing Otx2: effects on locomotor behavior and seizure susceptibility.

    PubMed

    Tripathi, P P; Di Giovannantonio, L G; Sanguinetti, E; Acampora, D; Allegra, M; Caleo, M; Wurst, W; Simeone, A; Bozzi, Y

    2014-03-01

    The homeobox-containing transcription factor Otx2 controls the identity, fate and proliferation of mesencephalic dopaminergic (mesDA) neurons. Transgenic mice, in which Otx2 was conditionally overexpressed by a Cre recombinase expressed under the transcriptional control of the Engrailed1 gene (En1(Cre/+); tOtx2(ov/+)), show an increased number of mesDA neurons during development. In adult mice, Otx2 is expressed in a subset of neurons in the ventral tegmental area (VTA) and its overexpression renders mesDA more resistant to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-HCl (MPTP) neurotoxin. Here we further investigated the neurological consequences of the increased number of mesDA neurons in En1(Cre/+); tOtx2(ov/+) adult mice. Immunohistochemistry for the active, glycosylated form of the dopamine transporter (glyco-Dat) showed that En1(Cre/+); tOtx2(ov/+) adult mice display an increased density of mesocortical DAergic fibers, as compared to control animals. Increased glyco-Dat staining was accompanied by a marked hypolocomotion in En1(Cre/+); tOtx2(ov/+) mice, as detected in the open field test. Since conditional knockout mice lacking Otx2 in mesDA precursors (En1(Cre/+); Otx2(floxv/flox) mice) show a marked resistance to kainic acid (KA)-induced seizures, we investigated the behavioral response to KA in En1(Cre/+); tOtx2(ov/+) and control mice. No difference was observed between mutant and control mice, but En1(Cre/+); tOtx2(ov/+) mice showed a markedly different c-fos mRNA induction profile in the cerebral cortex and hippocampus after KA seizures, as compared to controls. Accordingly, an increased density of parvalbumin (PV)-positive inhibitory interneurons was detected in the deep layers of the frontal cortex of naïve En1(Cre/+); tOtx2(ov/+) mice, as compared to controls. These data indicate that Otx2 overexpression results in increased DAergic innervation and PV cell density in the fronto-parietal cortex, with important consequences on spontaneous locomotor

  20. Overexpression of acetyl-CoA synthetase in Saccharomyces cerevisiae increases acetic acid tolerance

    PubMed Central

    Ding, Jun; Holzwarth, Garrett; Penner, Michael H.; Patton-Vogt, Jana; Bakalinsky, Alan T.

    2015-01-01

    Acetic acid-mediated inhibition of the fermentation of lignocellulose-derived sugars impedes development of plant biomass as a source of renewable ethanol. In order to overcome this inhibition, the capacity of Saccharomyces cerevisiae to synthesize acetyl-CoA from acetic acid was increased by overexpressing ACS2 encoding acetyl-coenzyme A synthetase. Overexpression of ACS2 resulted in higher resistance to acetic acid as measured by an increased growth rate and shorter lag phase relative to a wild-type control strain, suggesting that Acs2-mediated consumption of acetic acid during fermentation contributes to acetic acid detoxification. PMID:25673654

  1. Overexpression of acetyl-CoA synthetase in Saccharomyces cerevisiae increases acetic acid tolerance.

    PubMed

    Ding, Jun; Holzwarth, Garrett; Penner, Michael H; Patton-Vogt, Jana; Bakalinsky, Alan T

    2015-01-01

    Acetic acid-mediated inhibition of the fermentation of lignocellulose-derived sugars impedes development of plant biomass as a source of renewable ethanol. In order to overcome this inhibition, the capacity of Saccharomyces cerevisiae to synthesize acetyl-CoA from acetic acid was increased by overexpressing ACS2 encoding acetyl-coenzyme A synthetase. Overexpression of ACS2 resulted in higher resistance to acetic acid as measured by an increased growth rate and shorter lag phase relative to a wild-type control strain, suggesting that Acs2-mediated consumption of acetic acid during fermentation contributes to acetic acid detoxification.

  2. Overexpression of acetyl-CoA synthetase in Saccharomyces cerevisiae increases acetic acid tolerance.

    PubMed

    Ding, Jun; Holzwarth, Garrett; Penner, Michael H; Patton-Vogt, Jana; Bakalinsky, Alan T

    2015-01-01

    Acetic acid-mediated inhibition of the fermentation of lignocellulose-derived sugars impedes development of plant biomass as a source of renewable ethanol. In order to overcome this inhibition, the capacity of Saccharomyces cerevisiae to synthesize acetyl-CoA from acetic acid was increased by overexpressing ACS2 encoding acetyl-coenzyme A synthetase. Overexpression of ACS2 resulted in higher resistance to acetic acid as measured by an increased growth rate and shorter lag phase relative to a wild-type control strain, suggesting that Acs2-mediated consumption of acetic acid during fermentation contributes to acetic acid detoxification. PMID:25673654

  3. Overexpression of MMP-7 increases collagen 1A2 in the aging kidney

    PubMed Central

    Ślusarz, Anna; Nichols, LaNita A; Grunz-Borgmann, Elizabeth A; Chen, Gang; Akintola, Adebayo D; Catania, Jeffery M; Burghardt, Robert C; Trzeciakowski, Jerome P; Parrish, Alan R

    2013-01-01

    The percentage of the U.S. population over 65 is rapidly increasing, as is the incidence of chronic kidney disease (CKD). The kidney is susceptible to age-dependent alterations in structure, specifically tubulointerstitial fibrosis that leads to CKD. Matrix metalloproteinases (MMPs) were initially characterized as extracellular matrix (ECM) proteinases; however, it is clear that their biological role is much larger. We have observed increased gene expression of several MMPs in the aging kidney, including MMP-7. MMP-7 overexpression was observed starting at 16 months, with over a 500-fold upregulation in 2-year-old animals. Overexpression of MMP-7 is not observed in age-matched, calorically restricted controls that do not develop fibrosis and renal dysfunction, suggesting a role in the pathogenesis. In order to delineate the contributions of MMP-7 to renal dysfunction, we overexpressed MMP-7 in NRK-52E cells. High-throughput sequencing of the cells revealed that two collagen genes, Col1a2 and Col3a1, were elevated in the MMP-7 overexpressing cells. These two collagen genes were also elevated in aging rat kidneys and temporally correlated with increased MMP-7 expression. Addition of exogenous MMP-7, or conditioned media from MMP-7 overexpressing cells also increased Col1A2 expression. Inhibition of protein kinase A (PKA), src, and MAPK signaling at p38 and ERK was able to attenuate the MMP-7 upregulation of Col1a2. Consistent with this finding, increased phosphorylation of PKA, src, and ERK was seen in MMP-7 overexpressing cells and upon exogenous MMP-7 treatment of NRK-52E cells. These data suggest a novel mechanism by which MMP-7 contributes to the development of fibrosis leading to CKD. PMID:24273653

  4. Overexpression of MMP-7 Increases Collagen 1A2 in the Aging Kidney.

    PubMed

    Oelusarz, Anna; Nichols, Lanita A; Grunz-Borgmann, Elizabeth A; Chen, Gang; Akintola, Adebayo D; Catania, Jeffery M; Burghardt, Robert C; Trzeciakowski, Jerome P; Parrish, Alan R

    2013-10-01

    The percentage of the U.S. population over 65 is rapidly increasing, as is the incidence of chronic kidney disease (CKD). The kidney is susceptible to age-dependent alterations in structure, specifically tubulointerstitial fibrosis, that lead to CKD. Matrix metalloproteinases (MMPs) were initially characterized as extracellular matrix (ECM) proteinases; however it is clear that their biological role is much larger. We have observed increased gene expression of several MMPs in the aging kidney, including MMP-7. MMP-7 overexpression was observed starting at 16 months, and over a 500 fold up-regulation in 2 year-old animals. Overexpression of MMP-7 is not observed in age-matched, calorically restricted controls that do not develop fibrosis and renal dysfunction, suggesting a role in the pathogenesis. In order to delineate the contributions of MMP-7 to renal dysfunction, we overexpressed MMP-7 in NRK-52E cells. High-throughput sequencing of the cells revealed that two collagen genes, Col1a2 and Col3a1, were elevated in the MMP-7 overexpressing cells. These two collagen genes were also elevated in aging rat kidneys and temporally correlated with increased MMP-7 expression. Addition of exogenous MMP-7, or conditioned media from MMP-7 overexpressing cells also increased Col1A2 expression. Inhibition of PKA, src, and MAPK signaling at p38 and ERK was able to attenuate the MMP-7 up-regulation of Col1a2. Consistent with this finding, increased phosphorylation of PKA, src and ERK was seen in MMP-7 overexpressing cells and upon exogenous MMP-7 treatment of NRK-52E cells. These data suggest a novel mechanism by which MMP-7 contributes to the development of fibrosis leading to CKD. PMID:24273653

  5. Increased Impulsivity Retards the Transition to Dorsolateral Striatal Dopamine Control of Cocaine Seeking

    PubMed Central

    Murray, Jennifer E.; Dilleen, Ruth; Pelloux, Yann; Economidou, Daina; Dalley, Jeffrey W.; Belin, David; Everitt, Barry J.

    2014-01-01

    Background Development of maladaptive drug-seeking habits occurs in conjunction with a ventral-to-dorsal striatal shift in dopaminergic control over behavior. Although these habits readily develop as drug use continues, high impulsivity predicts loss of control over drug seeking and taking. However, whether impulsivity facilitates the transition to dorsolateral striatum (DLS) dopamine-dependent cocaine-seeking habits or whether impulsivity and cocaine-induced intrastriatal shifts are additive processes is unknown. Methods High- and low-impulsive rats identified in the five-choice serial reaction-time task were trained to self-administer cocaine (.25 mg/infusion) with infusions occurring in the presence of a cue-light conditioned stimulus. Dopamine transmission was blocked in the DLS after three stages of training: early, transition, and late-stage, by bilateral intracranial infusions of α-flupenthixol (0, 5, 10, or 15 μg/side) during 15-min cocaine-seeking test sessions in which each response was reinforced by a cocaine-associated conditioned stimulus presentation. Results In early-stage tests, neither group was affected by DLS dopamine receptor blockade. In transition-stage tests, low-impulsive rats showed a significant dose-dependent reduction in cocaine seeking, whereas high-impulsive rats were still unaffected by α-flupenthixol infusions. In the final, late-stage seeking test, both groups showed dose-dependent sensitivity to dopamine receptor blockade. Conclusions The results demonstrate that high impulsivity is associated with a delayed transition to DLS-dopamine-dependent control over cocaine seeking. This suggests that, if impulsivity confers an increased propensity to addiction, it is not simply through a more rapid development of habits but instead through interacting corticostriatal and striato-striatal processes that result ultimately in maladaptive drug-seeking habits. PMID:24157338

  6. Evidence that amphetamine and Na+ gradient reversal increase striatal synaptosomal dopamine synthesis through carrier-mediated efflux of dopamine.

    PubMed

    Connor, C E; Kuczenski, R

    1986-09-15

    Amphetamine (AMPH) releases dopamine (DA) from striatal synaptosomes and concomitantly increases DA synthesis. Since AMPH may release DA through carrier-mediated diffusion via reversal of the DA uptake system, the increase in DA synthesis might depend on a functioning uptake carrier. Consistent with such a mechanism, the uptake inhibitors nomifensine (NMF) and benztropine (BZT) completely prevented the AMPH-induced increase in DA synthesis at concentrations known to inhibit DA uptake. Changes in the Na+ gradient across the synaptosomal membrane also promote DA release, since DA and Na+ are cotransported by the neuronal uptake carrier. Incubation of synaptosomes in medium containing decreasing Na+ increased DA synthesis inversely proportional to Na+ over the range 128 to 20 mM. Similarly, incubations in the presence of 10(-4) M ouabain to inhibit Na+, K+-ATPase and allow intracellular accumulation of Na+ also increased DA synthesis. These changes in DA synthesis could also be prevented by BZT and were non-additive with the AMPH-induced increase in DA synthesis. However, a concentration of ouabain (10(-6) M) which by itself did not increase DA synthesis, and does not promote DA release, potentiated the AMPH-induced increase in DA synthesis. Further, the increased DA synthesis promoted by all three manipulations was only marginally dependent on the presence of Ca2+ in the incubation medium. However, at 5 and 10 mM Na+, a second component of increased DA synthesis was observed which was insensitive to BZT, but was prevented by Ca2+ removal. These results suggest that the increase in DA synthesis, and presumably DA release promoted by AMPH, lowered Na+, and ouabain, depend on the availability of the DA carrier at the internal face of the neuronal membrane and the intracellular content of Na+. The second component of increased DA synthesis which is evident at 5 and 10 mM Na+ is discussed in terms of a possible Ca2+-mediated change in DA synthesis which is independent of

  7. Selective Deletion of the Leptin Receptor in Dopamine Neurons Produces Anxiogenic-like Behavior and Increases Dopaminergic Activity in Amygdala

    PubMed Central

    Liu, Jing; Perez, Stephanie M.; Zhang, Wei; Lodge, Daniel J.; Lu, Xin-Yun

    2012-01-01

    Leptin receptors (Lepr) are expressed on midbrain dopamine neurons. However, the specific role of Lepr signaling in dopamine neurons remains to be clarified. In the present study, we generated a line of conditional knockout mice lacking functional leptin receptors selectively on dopamine neurons (LeprDAT-Cre). These mice exhibit normal body weight and feeding. Behaviorally, LeprDAT-Cre mice display an anxiogenic-like phenotype in the elevated plus-maze, light-dark box, social interaction and novelty-suppressed feeding tests. Depression-related behaviors in the chronic stress-induced anhedonia, forced swim and tail-suspension tests were not affected by deletion of Lepr in dopamine neurons. In vivo electrophysiological recordings of dopamine neurons in the ventral tegmental area (VTA) revealed an increase in burst firing in LeprDAT-Cre mice. Moreover, blockade of D1-dependent dopamine transmission in the central amygdala by local microinjection of the D1 antagonist SCH23390 attenuated the anxiogenic phenotype of LeprDAT-Cre mice. These findings suggest that leptin receptor signaling in midbrain dopamine neurons has a crucial role for the expression of anxiety and for the dopamine modulation of amygdala function. PMID:21483433

  8. Increased α-tocotrienol content in seeds of transgenic rice overexpressing Arabidopsis γ-tocopherol methyltransferase.

    PubMed

    Zhang, Gui-Yun; Liu, Ru-Ru; Xu, Geng; Zhang, Peng; Li, Yin; Tang, Ke-Xuan; Liang, Guo-Hua; Liu, Qiao-Quan

    2013-02-01

    Vitamin E comprises a group of eight lipid soluble antioxidant compounds that are an essential part of the human diet. The α-isomers of both tocopherol and tocotrienol are generally considered to have the highest antioxidant activities. γ-tocopherol methyltransferase (γ-TMT) catalyzes the final step in vitamin E biosynthesis, the methylation of γ- and δ-isomers to α- and β-isomers. In present study, the Arabidopsis γ-TMT (AtTMT) cDNA was overexpressed constitutively or in the endosperm of the elite japonica rice cultivar Wuyujing 3 (WY3) by Agrobacterium-mediated transformation. HPLC analysis showed that, in brown rice of the wild type or transgenic controls with empty vector, the α-/γ-tocotrienol ratio was only 0.7, much lower than that for tocopherol (~19.0). In transgenic rice overexpressing AtTMT driven by the constitutive Ubi promoter, most of the γ-isomers were converted to α-isomers, especially the γ- and δ-tocotrienol levels were dramatically decreased. As a result, the α-tocotrienol content was greatly increased in the transgenic seeds. Similarly, over-expression of AtTMT in the endosperm also resulted in an increase in the α-tocotrienol content. The results showed that the α-/γ-tocopherol ratio also increased in the transgenic seeds, but there was no significant effect on α-tocopherol level, which may reflect the fact that γ-tocopherol is present in very small amounts in wild type rice seeds. AtTMT overexpression had no effect on the absolute total content of either tocopherols or tocotrienols. Taken together, these results are the first demonstration that the overexpression of a foreign γ-TMT significantly shift the tocotrienol synthesis in rice, which is one of the world's most important food crops.

  9. Overexpression of several Arabidopsis histone genes increases agrobacterium-mediated transformation and transgene expression in plants.

    PubMed

    Tenea, Gabriela N; Spantzel, Joerg; Lee, Lan-Ying; Zhu, Yanmin; Lin, Kui; Johnson, Susan J; Gelvin, Stanton B

    2009-10-01

    The Arabidopsis thaliana histone H2A-1 is important for Agrobacterium tumefaciens-mediated plant transformation. Mutation of HTA1, the gene encoding histone H2A-1, results in decreased T-DNA integration into the genome of Arabidopsis roots, whereas overexpression of HTA1 increases transformation frequency. To understand the mechanism by which HTA1 enhances transformation, we investigated the effects of overexpression of numerous Arabidopsis histones on transformation and transgene expression. Transgenic Arabidopsis containing cDNAs encoding histone H2A (HTA), histone H4 (HFO), and histone H3-11 (HTR11) displayed increased transformation susceptibility, whereas histone H2B (HTB) and most histone H3 (HTR) cDNAs did not increase transformation. A parallel increase in transient gene expression was observed when histone HTA, HFO, or HTR11 overexpression constructs were cotransfected with double- or single-stranded forms of a gusA gene into tobacco (Nicotiana tabacum) protoplasts. However, these cDNAs did not increase expression of a previously integrated transgene. We identified the N-terminal 39 amino acids of H2A-1 as sufficient to increase transient transgene expression in plants. After transfection, transgene DNA accumulates more rapidly in the presence of HTA1 than with a control construction. Our results suggest that certain histones enhance transgene expression, protect incoming transgene DNA during the initial stages of transformation, and subsequently increase the efficiency of Agrobacterium-mediated transformation.

  10. Clavulanic acid increases dopamine release in neuronal cells through a mechanism involving enhanced vesicle trafficking.

    PubMed

    Kost, Gina Chun; Selvaraj, Senthil; Lee, Young Bok; Kim, Deog Joong; Ahn, Chang-Ho; Singh, Brij B

    2011-10-24

    Clavulanic acid is a CNS-modulating compound with exceptional blood-brain barrier permeability and safety profile. Clavulanic acid has been proposed to have anti-depressant activity and is currently entering Phase IIb clinical trials for the treatment of Major Depressive Disorder (MDD). Studies have also shown that clavulanic acid suppresses anxiety and enhances sexual functions in rodent and primate models by a mechanism involving central nervous system (CNS) modulation, although its detailed mechanism of action has yet to be elucidated. To further examine its potential as a CNS modulating agent as well as its mechanism of action, we investigated the effects of clavulanic acid in neuronal cells. Our results indicate that clavulanic acid enhances dopamine release in PC12 and SH-SY5Y cells without affecting dopamine synthesis. Furthermore, using affinity chromatography we were able to identify two proteins, Munc18-1 and Rab4 that potentially bind to clavulanic acid and play a critical role in neurosecretion and the vesicle trafficking process. Consistent with this result, an increase in the translocation of Munc18-1 and Rab4 from the cytoplasm to the plasma membrane was observed in clavulanic acid treated cells. Overall, these data suggest that clavulanic acid enhances dopamine release in a mechanism involving Munc18-1 and Rab4 modulation and warrants further investigation of its therapeutic use in CNS disorders, such as depression.

  11. Clavulanic acid increases dopamine release in neuronal cells through a mechanism involving enhanced vesicle trafficking

    PubMed Central

    Kost, Gina Chun; Selvaraj, Senthil; Lee, Young Bok; Kim, Deog Joong; Ahn, Chang-Ho; Singh, Brij B

    2011-01-01

    Clavulanic acid is a CNS-modulating compound with exceptional blood-brain barrier permeability and safety profile. Clavulanic acid has been proposed to have anti-depressant activity and is currently entering Phase IIb clinical trials for the treatment of Major Depressive Disorder (MDD). Studies have also shown that clavulanic acid suppresses anxiety and enhances sexual functions in rodent and primate models by a mechanism involving central nervous system (CNS) modulation, although its detailed mechanism of action has yet to be elucidated. To further examine its potential as a CNS modulating agent as well as its mechanism of action, we investigated the effects of clavulanic acid in neuronal cells. Our results indicate that clavulanic acid enhances dopamine release in PC12 and SH-SY5Y cells without affecting dopamine synthesis. Furthermore, using affinity chromatography we were able to identify two proteins, Munc18-1 and Rab4 that potentially bind to clavulanic acid and play a critical role in neurosecretion and the vesicle trafficking process. Consistent with this result, an increase in the translocation of Munc18-1 and Rab4 from the cytoplasm to the plasma membrane was observed in clavulanic acid treated cells. Overall, these data suggest that clavulanic acid enhances dopamine release in a mechanism involving Munc18-1 and Rab4 modulation and warrants further investigation of its therapeutic use in CNS disorders, such as depression. PMID:21964384

  12. Mice expressing markedly reduced striatal dopamine transporters exhibit increased locomotor activity, dopamine uptake turnover rate, and cocaine responsiveness.

    PubMed

    Rao, Anjali; Sorkin, Alexander; Zahniser, Nancy R

    2013-10-01

    Variations in the expression levels of the dopamine transporter (DAT) can influence responsiveness to psychostimulant drugs like cocaine. To better understand this relationship, we studied a new DAT-low expresser (DAT-LE) mouse model and performed behavioral and biochemical studies with it. Immunoblotting and [(3) H]WIN 35,428 binding analyses revealed that these mice express ∼35% of wildtype (WT) mouse striatal DAT levels. Compared to WT mice, DAT-LE mice were hyperactive in a novel open-field environment. Despite their higher basal locomotor activity, cocaine (10 or 20 mg/kg, i.p.) induced greater locomotor activation in DAT-LE mice than in WT mice. The maximal velocity (Vmax ) of DAT-mediated [(3) H]DA uptake into striatal synaptosomes was reduced by 46% in DAT-LE mice, as compared to WT. Overall, considering the reduced number of DAT binding sites (Bmax ) along with the reduced Vmax in DAT-LE mice, a 2-fold increase in DA uptake turnover rate (Vmax /Bmax ) was found, relative to WT mice. This suggests that neuroadaptive changes have occurred in the DAT-LE mice that would help to compensate for their low DAT numbers. Interestingly, these changes do not include a reduction in tyrosine hydroxylase levels, as was previously reported in DAT knockout homozygous and heterozygous animals. Further, these changes are not sufficient to prevent elevated novelty- and cocaine-induced locomotor activity. Hence, these mice represent a unique model for studying changes of in vivo DAT function and regulation that result from markedly reduced levels of DAT expression. PMID:23564231

  13. Mice expressing markedly reduced striatal dopamine transporters exhibit increased locomotor activity, dopamine uptake turnover rate, and cocaine responsiveness.

    PubMed

    Rao, Anjali; Sorkin, Alexander; Zahniser, Nancy R

    2013-10-01

    Variations in the expression levels of the dopamine transporter (DAT) can influence responsiveness to psychostimulant drugs like cocaine. To better understand this relationship, we studied a new DAT-low expresser (DAT-LE) mouse model and performed behavioral and biochemical studies with it. Immunoblotting and [(3) H]WIN 35,428 binding analyses revealed that these mice express ∼35% of wildtype (WT) mouse striatal DAT levels. Compared to WT mice, DAT-LE mice were hyperactive in a novel open-field environment. Despite their higher basal locomotor activity, cocaine (10 or 20 mg/kg, i.p.) induced greater locomotor activation in DAT-LE mice than in WT mice. The maximal velocity (Vmax ) of DAT-mediated [(3) H]DA uptake into striatal synaptosomes was reduced by 46% in DAT-LE mice, as compared to WT. Overall, considering the reduced number of DAT binding sites (Bmax ) along with the reduced Vmax in DAT-LE mice, a 2-fold increase in DA uptake turnover rate (Vmax /Bmax ) was found, relative to WT mice. This suggests that neuroadaptive changes have occurred in the DAT-LE mice that would help to compensate for their low DAT numbers. Interestingly, these changes do not include a reduction in tyrosine hydroxylase levels, as was previously reported in DAT knockout homozygous and heterozygous animals. Further, these changes are not sufficient to prevent elevated novelty- and cocaine-induced locomotor activity. Hence, these mice represent a unique model for studying changes of in vivo DAT function and regulation that result from markedly reduced levels of DAT expression.

  14. Dopamine Increases CD14+CD16+ Monocyte Migration and Adhesion in the Context of Substance Abuse and HIV Neuropathogenesis

    PubMed Central

    Coley, Jacqueline S.; Calderon, Tina M.; Gaskill, Peter J.; Eugenin, Eliseo A.; Berman, Joan W.

    2015-01-01

    Drug abuse is a major comorbidity of HIV infection and cognitive disorders are often more severe in the drug abusing HIV infected population. CD14+CD16+ monocytes, a mature subpopulation of peripheral blood monocytes, are key mediators of HIV neuropathogenesis. Infected CD14+CD16+ monocyte transmigration across the blood brain barrier mediates HIV entry into the brain and establishes a viral reservoir within the CNS. Despite successful antiretroviral therapy, continued influx of CD14+CD16+ monocytes, both infected and uninfected, contributes to chronic neuroinflammation and the development of HIV associated neurocognitive disorders (HAND). Drug abuse increases extracellular dopamine in the CNS. Once in the brain, CD14+CD16+ monocytes can be exposed to extracellular dopamine due to drug abuse. The direct effects of dopamine on CD14+CD16+ monocytes and their contribution to HIV neuropathogenesis are not known. In this study, we showed that CD14+CD16+ monocytes express mRNA for all five dopamine receptors by qRT-PCR and D1R, D5R and D4R surface protein by flow cytometry. Dopamine and the D1-like dopamine receptor agonist, SKF38393, increased CD14+CD16+ monocyte migration that was characterized as chemokinesis. To determine whether dopamine affected cell motility and adhesion, live cell imaging was used to monitor the accumulation of CD14+CD16+ monocytes on the surface of a tissue culture dish. Dopamine increased the number and the rate at which CD14+CD16+ monocytes in suspension settled to the dish surface. In a spreading assay, dopamine increased the area of CD14+CD16+ monocytes during the early stages of cell adhesion. In addition, adhesion assays showed that the overall total number of adherent CD14+CD16+ monocytes increased in the presence of dopamine. These data suggest that elevated extracellular dopamine in the CNS of HIV infected drug abusers contributes to HIV neuropathogenesis by increasing the accumulation of CD14+CD16+ monocytes in dopamine rich brain

  15. Chronic administration of cholesterol oximes in mice increases transcription of cytoprotective genes and improves transcriptome alterations induced by alpha-synuclein overexpression in nigrostriatal dopaminergic neurons

    PubMed Central

    Richter, Franziska; Gao, Fuying; Medvedeva, Vera; Lee, Patrick; Bove, Nicholas; Fleming, Sheila M.; Michaud, Magali; Lemesre, Vincent; Patassini, Stefano; De La Rosa, Krystal; Mulligan, Caitlin K.; Sioshansi, Pedrom; Zhu, Chunni; Coppola, Giovanni; Bordet, Thierry; Pruss, Rebecca; Chesselet, Marie-Françoise

    2014-01-01

    Cholesterol-oximes TRO19622 and TRO40303 target outer mitochondrial membrane proteins and have beneficial effects in preclinical models of neurodegenerative diseases leading to their advancement to clinical trials. Dopaminergic neurons degenerate in Parkinson’s disease (PD) and are prone to oxidative stress and mitochondrial dysfunction. In order to provide insights into the neuroprotective potential of TRO19622 and TRO40303 for dopaminergic neurons in vivo, we assessed their effects on gene expression in laser captured nigrostriatal dopaminergic neurons of wildtype mice and of mice that over-express alpha-synuclein, a protein involved in both familial and sporadic forms of PD (Thy1-aSyn mice). Young mice were fed the drugs in food pellets or a control diet from 1 to 4 months of age, approximately 10 months before the appearance of striatal dopamine loss in this model. Unbiased weighted gene co-expression network analysis (WGCNA) of transcriptional changes revealed effects of cholesterol oximes on transcripts related to mitochondria, cytoprotection and anti-oxidant response in wild-type and transgenic mice, including increased transcription of stress defense (e.g. Prdx1, Prdx2, Glrx2, Hspa9, Pink1, Drp1, Trak1) and dopamine-related (Th, Ddc, Gch1, Dat, Vmat2, Drd2, Chnr6a) genes. Even at this young age transgenic mice showed alterations in transcripts implicated in mitochondrial function and oxidative stress (e.g. Bcl-2, Bax, Casp3, Nos2), and both drugs normalized about 20% of these alterations. Young Thy1-aSyn mice exhibit motor deficits that differ from parkinsonism and are established before the onset of treatment; these deficits were not improved by cholesterol oximes. However, high doses of TRO40303 improved olfaction and produced the same effects as dopamine agonists on a challenging beam test, specifically an increase in footslips, an observation congruent with its effects on transcripts involved in dopamine synthesis. High doses of TRO19622 increased

  16. Overexpression of a Drosophila homolog of apolipoprotein D leads to increased stress resistance and extended lifespan.

    PubMed

    Walker, David W; Muffat, Julien; Rundel, Colin; Benzer, Seymour

    2006-04-01

    Increased Apolipoprotein D (ApoD) expression has been reported in various neurological disorders, including Alzheimer's disease, schizophrenia, and stroke, and in the aging brain . However, whether ApoD is toxic or a defense is unknown. In a screen to identify genes that protect Drosophila against acute oxidative stress, we isolated a fly homolog of ApoD, Glial Lazarillo (GLaz). In independent transgenic lines, overexpression of GLaz resulted in increased resistance to hyperoxia (100% O(2)) as well as a 29% extension of lifespan under normoxia. These flies also displayed marked improvements in climbing and walking ability after sublethal exposure to hyperoxia. Overexpression of Glaz also increased resistance to starvation without altering lipid or protein content. To determine whether GLaz might be important in protection against reperfusion injury, we subjected the flies to hypoxia, followed by recovery under normoxia. Overexpression of GLaz was protective against behavioral deficits caused in normal flies by this ischemia/reperfusion paradigm. This and the accompanying paper by Sanchez et al. (in this issue of Current Biology) are the first to manipulate the levels of an ApoD homolog in a model organism. Our data suggest that human ApoD may play a protective role and thus may constitute a therapeutic target to counteract certain neurological diseases.

  17. CTT1 overexpression increases life span of calorie-restricted Saccharomyces cerevisiae deficient in Sod1.

    PubMed

    Rona, Germana; Herdeiro, Ricardo; Mathias, Cristiane Juliano; Torres, Fernando Araripe; Pereira, Marcos Dias; Eleutherio, Elis

    2015-06-01

    Studies using different organisms revealed that reducing calorie intake, without malnutrition, known as calorie restriction (CR), increases life span, but its mechanism is still unkown. Using the yeast Saccharomyces cerevisiae as eukaryotic model, we observed that Cu, Zn-superoxide dismutase (Sod1p) is required to increase longevity, as well as to confer protection against lipid and protein oxidation under CR. Old cells of sod1 strain also presented a premature induction of apoptosis. However, when CTT1 (which codes for cytosolic catalase) was overexpressed, sod1 and WT strains showed similar survival rates. Furthermore, CTT1 overexpression decreased lipid peroxidation and delayed the induction of apoptotic process. Superoxide is rapidly converted to hydrogen peroxide by superoxide dismutase, but it also undergoes spontaneous dismutation albeit at a slower rate. However, the quantity of peroxide produced from superoxide in this way is two-fold higher. Peroxide degradation, catalyzed by catalase, is of vital importance, because in the presence of a reducer transition metal peroxide is reduced to the highly reactive hydroxyl radical, which reacts indiscriminately with most cellular constituents. These findings might explain why overexpression of catalase was able to overcome the deficiency of Sod1p, increasing life span in response to CR. PMID:25573485

  18. Overexpression of Serpinb1 in Chinese hamster ovary cells increases recombinant IgG productivity.

    PubMed

    Lin, Nan; Brooks, Jeanne; Sealover, Natalie; George, Henry J; Kayser, Kevin J

    2015-01-10

    We report the discovery and validation of a novel CHO cell engineering target for improving IgG expression, serpin peptidase inhibitor, clade B, member 1 (Serpinb1). Transcriptomic studies using microarrays revealed that Serpinb1 was up-regulated in cultures with IgG heavy and light chain transcription transiently repressed compared with cultures treated with non-targeting siRNA. As proof of concept, a lentiviral vector was employed to overexpress the Chinese Hamster Serpinb1 in a CHOZN(®) Glutamine Synthetase (-/-) recombinant IgG producing CHO line. The lentiviral stable pool demonstrated 4.2-fold SERPINB1 overexpression compared with the non-transduced control. The peak viable cell density (VCD) and peak IgG volumetric productivity of the lentiviral stable pool increased 1.3 and 2.0 fold, respectively, compared with the non-transduced control. For host cell engineering, a plasmid encoding SERPINB1 was transfected into the CHOZN(®) GS (-/-) host cell line to create several stable pools. Single-cell clones isolated from the pools were characterized for their SERPINB1 expression levels and growth. The clone (SERPINB1_OE_27) with the highest SERPINB1 expression had decreased peak viable cell density and exponential phase growth rate. Selected SERPINB1 OE clones were subsequently evaluated for their IgG expression capabilities using GS selection. Clone SERPINB1_OE_42 with moderate SERPINB1 overexpression demonstrated increased IgG productivity in "bulk" selection. We conclude that manipulating Serpinb1 expression can lead to increased recombinant IgG productivity, but the effect in host cell lines may vary by clone and by overexpression level. This work represents the ongoing effort in applying "-omics" findings to novel CHO host cell line engineering.

  19. Dopamine receptor activation increases glial cell line-derived neurotrophic factor in experimental stroke.

    PubMed

    Kuric, Enida; Wieloch, Tadeusz; Ruscher, Karsten

    2013-09-01

    Treatment with levodopa enhances functional recovery after experimental stroke but its mechanisms of action are elusive. Reactive astrocytes in the ischemic hemisphere are involved in mechanisms promoting recovery and also express dopamine 1 (D1) and dopamine 2 (D2) receptors. Here we investigated if the activation of astrocytic dopamine receptors (D1 and D2) regulates the expression of glial cell line-derived neurotrophic factor (GDNF) after combined in vitro hypoxia/aglycemia (H/A) and studied the expression of GDNF in the ischemic brain after treatment with levodopa/benserazide following transient occlusion of the middle cerebral artery (tMCAO) in the rat. Twenty-four hours after H/A, GDNF levels were upregulated in exposed astrocytes compared to normoxic control cultures and further elevated by the addition of the selective D1 receptor agonist (R)-(+)-SKF-38393 hydrochloride while D1 receptor antagonism by R(+)-SCH-23390 hydrochloride significantly reduced GDNF. No effect on GDNF levels was observed by the application of the D2 receptor agonist R(-)-2,10,11-trihydroxy-N-propyl-noraporphine hydrobromide hydrate or S-(-)-eticlopride hydrochloride (D2 receptor antagonist). After tMCAO, GDNF was upregulated in D1 expressing reactive astrocytes in the peri-infarct area. In addition, treatment with levodopa/benserazide significantly increased GDNF levels in the infarct core and peri-infarct area after tMCAO without affecting the expression of glial fibrillar acidic protein (GFAP), an intermediate filament and marker of reactive gliosis. After stroke, GDNF levels increase in the ischemic hemisphere in rats treated with levodopa, implicating GDNF in the mechanisms of tissue reorganization and plasticity and in l-DOPA enhanced recovery of lost brain function. Our results support levodopa treatment as a potential recovery enhancing therapy in stroke patients.

  20. Melanocortin 3 Receptor Signaling in Midbrain Dopamine Neurons Increases the Motivation for Food Reward.

    PubMed

    Pandit, Rahul; Omrani, Azar; Luijendijk, Mieneke C M; de Vrind, Véronne A J; Van Rozen, Andrea J; Ophuis, Ralph J A Oude; Garner, Keith; Kallo, Imre; Ghanem, Alexander; Liposits, Zsolt; Conzelmann, Karl-Klaus; Vanderschuren, Louk J M J; la Fleur, Susanne E; Adan, Roger A H

    2016-08-01

    The central melanocortin (MC) system mediates its effects on food intake via MC3 (MC3R) and MC4 receptors (MC4R). Although the role of MC4R in meal size determination, satiation, food preference, and motivation is well established, the involvement of MC3R in the modulation of food intake has been less explored. Here, we investigated the role of MC3R on the incentive motivation for food, which is a crucial component of feeding behavior. Dopaminergic neurons within the ventral tegmental area (VTA) have a crucial role in the motivation for food. We here report that MC3Rs are expressed on VTA dopaminergic neurons and that pro-opiomelanocortinergic (POMC) neurons in the arcuate nucleus of the hypothalamus (Arc) innervate these VTA dopaminergic neurons. Our findings show that intracerebroventricular or intra-VTA infusion of the selective MC3R agonist γMSH increases responding for sucrose under a progressive ratio schedule of reinforcement, but not free sucrose consumption in rats. Furthermore, ex vivo electrophysiological recordings show increased VTA dopaminergic neuronal activity upon γMSH application. Consistent with a dopamine-mediated effect of γMSH, the increased motivation for sucrose after intra-VTA infusion of γMSH was blocked by pretreatment with the dopamine receptor antagonist α-flupenthixol. Taken together, we demonstrate an Arc POMC projection onto VTA dopaminergic neurons that modulates motivation for palatable food via activation of MC3R signaling. PMID:26852738

  1. L-Endoglin Overexpression Increases Renal Fibrosis after Unilateral Ureteral Obstruction

    PubMed Central

    Arévalo, Miguel; Núñez-Gómez, Elena; Pérez-Roque, Lucía; Pericacho, Miguel; González-Núñez, María; Langa, Carmen; Martínez-Salgado, Carlos; Perez-Barriocanal, Fernando; Bernabeu, Carmelo; Lopez-Novoa, José M.

    2014-01-01

    Transforming growth factor-β (TGF-β) plays a pivotal role in renal fibrosis. Endoglin, a 180 KDa membrane glycoprotein, is a TGF-β co-receptor overexpressed in several models of chronic kidney disease, but its function in renal fibrosis remains uncertain. Two membrane isoforms generated by alternative splicing have been described, L-Endoglin (long) and S-Endoglin (short) that differ from each other in their cytoplasmic tails, being L-Endoglin the most abundant isoform. The aim of this study was to assess the effect of L-Endoglin overexpression in renal tubulo-interstitial fibrosis. For this purpose, a transgenic mouse which ubiquitously overexpresses human L-Endoglin (L-ENG+) was generated and unilateral ureteral obstruction (UUO) was performed in L-ENG+ mice and their wild type (WT) littermates. Obstructed kidneys from L-ENG+ mice showed higher amounts of type I collagen and fibronectin but similar levels of α-smooth muscle actin (α-SMA) than obstructed kidneys from WT mice. Smad1 and Smad3 phosphorylation were significantly higher in obstructed kidneys from L-ENG+ than in WT mice. Our results suggest that the higher increase of renal fibrosis observed in L-ENG+ mice is not due to a major abundance of myofibroblasts, as similar levels of α-SMA were observed in both L-ENG+ and WT mice, but to the higher collagen and fibronectin synthesis by these fibroblasts. Furthermore, in vivo L-Endoglin overexpression potentiates Smad1 and Smad3 pathways and this effect is associated with higher renal fibrosis development. PMID:25313562

  2. Music and Methamphetamine: Conditioned Cue-induced Increases in Locomotor Activity and Dopamine Release in Rats

    PubMed Central

    Polston, J.E.; Rubbinaccio, H.Y.; Morra, J.T.; Sell, E.M.; Glick, S.D.

    2011-01-01

    Associations between drugs of abuse and cues facilitate the acquisition and maintenance of addictive behaviors. Although significant research has been done to elucidate the role that simple discriminative or discrete conditioned stimuli (e.g., a tone or a light) play in addiction, less is known about complex environmental cues. The purpose of the present study was to examine the role of a musical conditioned stimulus by assessing locomotor activity and in vivo microdialysis. Two groups of rats were given non-contingent injections of methamphetamine (1.0 mg/kg) or vehicle and placed in standard conditioning chambers. During these conditioning sessions both groups were exposed to a continuous conditioned stimulus, in the form of a musical selection (“Four” by Miles Davis) played repeatedly for ninety minutes. After seven consecutive conditioning days subjects were given one day of rest, and subsequently tested for locomotor activity or dopamine release in the absence of drug while the musical conditioned stimulus was continually present. The brain regions examined included the basolateral amygdala, nucleus accumbens, and prefrontal cortex. The results show that music is an effective contextual conditioned stimulus, significantly increasing locomotor activity after repeated association with methamphetamine. Furthermore, this musical conditioned stimulus significantly increased extracellular dopamine levels in the basolateral amygdala and nucleus accumbens. These findings support other evidence showing the importance of these brain regions in conditioned learning paradigms, and demonstrate that music is an effective conditioned stimulus warranting further investigation. PMID:21145911

  3. Human neural stem cells survive long term in the midbrain of dopamine-depleted monkeys after GDNF overexpression and project neurites toward an appropriate target.

    PubMed

    Wakeman, Dustin R; Redmond, D Eugene; Dodiya, Hemraj B; Sladek, John R; Leranth, Csaba; Teng, Yang D; Samulski, R Jude; Snyder, Evan Y

    2014-06-01

    Transplanted multipotent human fetal neural stem cells (hfNSCs) significantly improved the function of parkinsonian monkeys in a prior study primarily by neuroprotection, with only 3%-5% of cells expressing a dopamine (DA) phenotype. In this paper, we sought to determine whether further manipulation of the neural microenvironment by overexpression of a developmentally critical molecule, glial cell-derived neurotrophic factor (GDNF), in the host striatum could enhance DA differentiation of hfNSCs injected into the substantia nigra and elicit growth of their axons to the GDNF-expressing target. hfNSCs were transplanted into the midbrain of 10 green monkeys exposed to 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine. GDNF was delivered concomitantly to the striatum via an adeno-associated virus serotype 5 vector, and the fate of grafted cells was assessed after 11 months. Donor cells remained predominantly within the midbrain at the injection site and sprouted numerous neurofilament-immunoreactive fibers that appeared to course rostrally toward the striatum in parallel with tyrosine hydroxylase-immunoreactive fibers from the host substantia nigra but did not mature into DA neurons. This work suggests that hfNSCs can generate neurons that project long fibers in the adult primate brain. However, in the absence of region-specific signals and despite GDNF overexpression, hfNSCs did not differentiate into mature DA neurons in large numbers. It is encouraging, however, that the adult primate brain appeared to retain axonal guidance cues. We believe that transplantation of stem cells, specifically instructed ex vivo to yield DA neurons, could lead to reconstruction of some portion of the nigrostriatal pathway and prove beneficial for the parkinsonian condition.

  4. Overexpression of α-synuclein simultaneously increases glutamate NMDA receptor phosphorylation and reduces glucocerebrosidase activity.

    PubMed

    Yang, Junfeng; Hertz, Ellen; Zhang, Xiaoqun; Leinartaité, Lina; Lundius, Ebba Gregorsson; Li, Jie; Svenningsson, Per

    2016-01-12

    Progressive accumulation of α-synuclein (α-syn)-containing protein aggregates throughout the nervous system is a pathological hallmark of Parkinson's disease (PD). The mechanisms whereby α-syn exerts neurodegeneration remain to be fully understood. Here we show that overexpression of α-syn in transgenic mice leads to increased phosphorylation of glutamate NMDA receptor (NMDAR) subunits NR1 and NR2B in substantia nigra and striatum as well as reduced glucocerebrosidase (GCase) levels. Similarly, molecular studies performed in mouse N2A cells stably overexpressing human α-syn ((α-syn)N2A) showed that phosphorylation states of the same NMDAR subunits were increased, whereas GCase levels and lysosomal GCase activity were reduced. (α-syn)N2A cells showed an increased sensitivity to neurotoxicity towards 6-hydroxydopamine and NMDA. However, wildtype N2A, but not (α-syn)N2A cells, showed a further reduction in viability when co-incubated with 6-hydroxydopamine and the lysosomal inhibitors NH4Cl and leupeptin, suggesting that α-syn per se perturbs lysosomal functions. NMDA treatment reduced lysosomal GCase activity to the same extent in (α-syn)N2A cells as in wildtype N2A cells, indicating that the α-syn-dependent difference in NMDA neurotoxicity is unrelated to an altered GCase activity. Nevertheless, these data provide molecular evidence that overexpression of α-syn simultaneously induces two potential neurotoxic hits by increasing glutamate NMDA receptor phosphorylation, consistent with increased NMDA receptors functionality, and reducing GCase activity. PMID:26610904

  5. Slit2 overexpression results in increased microvessel density and lesion size in mice with induced endometriosis.

    PubMed

    Guo, Sun-Wei; Zheng, Yu; Lu, Yuan; Liu, Xishi; Geng, Jian-Guo

    2013-03-01

    We recently reported that Slit/Roundabout (ROBO) 1 pathway may be a constituent biomarker for recurrence of endometriosis, likely through promoting angiogenesis. In this study, we sought to determine as whether Slit2 overexpression can facilitate angiogenesis, increase lesion size, and induce hyperalgesia in mice with induced endometriosis. We used 30 Slit2 transgenic (S) and 29 wild-type (W) mice and cross-transplanted endometrial fragments from S to W (group SW) and vice versa (group WS), and also within the S and W (groups SS and WW, respectively), into the peritoneal cavity, inducing endometriosis. We also performed a sham surgery within both S and W mice (groups Sm and Wm, respectively). The size of the ectopic implants, microvessel density (MVD) and immunoreactivity to ROBO1, and vascular endothelial cell growth factor (VEGF) in ectopic and eutopic endometrium, along with hotplate and tail-flick tests in all mice, were then evaluated. We found that the induction of endometriosis resulted in generalized hyperalgesia, which was unaffected by Slit2 overexpression. Slit2 overexpression did increase the lesion size significantly and correlated positively with the MVD in ectopic and eutopic endometrium. Slit2 expression levels appear to correlate with the MVD, but not with VEGF immunoreactivity, in ectopic endometrium. Consequently, we conclude that Slit2 may play an important role in angiogenesis in endometriosis. The increased angiogenesis, as measured by MVD, but not VEGF immunoreactivity, likely resulted in increased lesion size in induced endometriosis. Thus, SLIT2/ROBO1 pathway may be a potential therapeutic target for treating endometriosis.

  6. Overexpression of Fto leads to increased food intake and results in obesity.

    PubMed

    Church, Chris; Moir, Lee; McMurray, Fiona; Girard, Christophe; Banks, Gareth T; Teboul, Lydia; Wells, Sara; Brüning, Jens C; Nolan, Patrick M; Ashcroft, Frances M; Cox, Roger D

    2010-12-01

    Genome-wide association studies have identified SNPs within FTO, the human fat mass and obesity-associated gene, that are strongly associated with obesity. Individuals homozygous for the at-risk rs9939609 A allele weigh, on average, ~3 kg more than individuals with the low-risk T allele. Mice that lack FTO function and/or Fto expression display increased energy expenditure and a lean phenotype. We show here that ubiquitous overexpression of Fto leads to a dose-dependent increase in body and fat mass, irrespective of whether mice are fed a standard or a high-fat diet. Our results suggest that increased body mass results primarily from increased food intake. Mice with increased Fto expression on a high-fat diet develop glucose intolerance. This study provides the first direct evidence that increased Fto expression causes obesity in mice. PMID:21076408

  7. Overexpression of Fto leads to increased food intake and results in obesity.

    PubMed

    Church, Chris; Moir, Lee; McMurray, Fiona; Girard, Christophe; Banks, Gareth T; Teboul, Lydia; Wells, Sara; Brüning, Jens C; Nolan, Patrick M; Ashcroft, Frances M; Cox, Roger D

    2010-12-01

    Genome-wide association studies have identified SNPs within FTO, the human fat mass and obesity-associated gene, that are strongly associated with obesity. Individuals homozygous for the at-risk rs9939609 A allele weigh, on average, ~3 kg more than individuals with the low-risk T allele. Mice that lack FTO function and/or Fto expression display increased energy expenditure and a lean phenotype. We show here that ubiquitous overexpression of Fto leads to a dose-dependent increase in body and fat mass, irrespective of whether mice are fed a standard or a high-fat diet. Our results suggest that increased body mass results primarily from increased food intake. Mice with increased Fto expression on a high-fat diet develop glucose intolerance. This study provides the first direct evidence that increased Fto expression causes obesity in mice.

  8. Overexpression of sweetpotato expansin cDNA (IbEXP1) increases seed yield in Arabidopsis.

    PubMed

    Bae, Jung Myung; Kwak, Man Sup; Noh, Seol Ah; Oh, Mi-Joung; Kim, Youn-Sung; Shin, Jeong Sheop

    2014-08-01

    Results of transcriptome analyses suggest that expansin genes play an active role in seed development and yield, but gain- or loss-of-function studies have not yet elucidated the functional role(s) of the expansin gene(s) in these processes. We have overexpressed a sweetpotato expansin gene (IbEXP1) in Arabidopsis under the control of cauliflower mosaic 35S promoter in an attempt to determine the effect of the expansin gene in seed development and yield in heterologous plants. The growth rate was enhanced in IbEXP1-overexpressing (ox) plants relative to wild-type Col-0 plants during early vegetative growth stage. At the reproductive stage, the number of rosette leaves was higher in IbEXP1-ox plants than that in Col-0 plants, and siliques were thicker. IbEXP1-ox plants produced larger seeds, accumulated more protein and starch in each seed, and produced more inflorescence stems and siliques than Col-0 plants, leading to a 2.1-2.5-fold increase in total seed yield per plant. The transcript level of IbEXP1 was up-regulated in response to brassinosteroid (BR) treatment in sweetpotato, and the transcript levels of three BR-responsive genes, fatty acid elongase 3-ketoacyl-CoA synthase 1, HAIKU1 and MINISEED3, were also increased in IbEXP1-ox Arabidopsis plants, suggesting a possible involvement of IbEXP1 in at least one of the BR signaling pathways. Based on these results, we suggest that overexpression of IbEXP1 gene in heterologous plants is effective in increasing seed size and number and, consequently, seed yield.

  9. Overexpression of CD99 Increases the Migration and Invasiveness of Human Malignant Glioma Cells.

    PubMed

    Seol, Ho Jun; Chang, Jong Hee; Yamamoto, Junkoh; Romagnuolo, Rocco; Suh, Youngchul; Weeks, Adrienne; Agnihotri, Sameer; Smith, Christian A; Rutka, James T

    2012-09-01

    The malignant glioma is the most common primary human brain tumor, and its migration and invasiveness away from the primary tumor mass are considered a leading cause of tumor recurrence and treatment failure. Recently, gene expression profiling revealed that the transmembrane glycoprotein CD99 is more highly expressed in malignant glioma than in normal brain. Although its function is not completely understood, CD99 is implicated in cell adhesion and migration in a variety of different cell types. CD99 has wild-type and splice variant isoforms. Previous studies have shown that wild-type CD99 may be an oncosuppressor in some tumors, distinct from the role of the splice variant isoform. In this study, our data reveal that only wild-type CD99 is expressed in human glioma cells and tissues. Using a tissue microarray, we validated that gliomas demonstrate higher expression of CD99 compared with nonneoplastic brain. To assess the role of CD99 in glioma migration and invasion, we inhibited CD99 expression by siRNA and demonstrated decreased glioma migration and invasion. In contrast, when CD99 was overexpressed in glioma cells, we observed enhancement of cell migration and invasiveness. An orthotopic brain tumor model demonstrates that CD99 overexpression significantly increases invasiveness and decreases survival rate. Interestingly, Rac activity was decreased and Rho activity was increased in CD99 overexpressing glioma cells, and the proportion of amoeboid cells to mesenchymal cells was significantly increased. Taken together, our findings suggest that CD99 may play an important role in the migration and invasion of human gliomas independent of Akt, ERK, or JNK signaling pathways. Moreover, CD99 might be involved in amoeboid-mesenchymal transition in glioma migration. CD99 may be an important future target to inhibit migration and invasion, especially in CD99-expressing gliomas. PMID:23486730

  10. Overexpression of a NAC transcription factor delays leaf senescence and increases grain nitrogen concentration in wheat.

    PubMed

    Zhao, D; Derkx, A P; Liu, D-C; Buchner, P; Hawkesford, M J

    2015-07-01

    Increasing the duration of leaf photosynthesis during grain filling using slow-senescing functional stay-green phenotypes is a possible route for increasing grain yields in wheat (Triticum aestivum L.). However, delayed senescence may negatively affect nutrient remobilisation and hence reduce grain protein concentrations and grain quality. A novel NAC1-type transcription factor (hereafter TaNAC-S) was identified in wheat, with gene expression located primarily in leaf/sheath tissues, which decreased during post-anthesis leaf senescence. Expression of TaNAC-S in the second leaf correlated with delayed senescence in two doubled-haploid lines of an Avalon × Cadenza population (lines 112 and 181), which were distinct for leaf senescence. Transgenic wheat plants overexpressing TaNAC-S resulted in delayed leaf senescence (stay-green phenotype). Grain yield, aboveground biomass, harvest index and total grain N content were unaffected, but NAC over-expressing lines had higher grain N concentrations at similar grain yields compared to non-transgenic controls. These results indicate that TaNAC-S is a negative regulator of leaf senescence, and that delayed leaf senescence may lead not only to increased grain yields but also to increased grain protein concentrations.

  11. H2 relaxin overexpression increases in vivo prostate xenograft tumor growth and angiogenesis.

    PubMed

    Silvertown, Josh D; Ng, Jonathan; Sato, Takeya; Summerlee, Alastair J; Medin, Jeffrey A

    2006-01-01

    Our study reports a preliminary investigation into the role of human H2 relaxin in prostate tumor growth. A luciferase-expressing human prostate cancer cell line, PC-3, was generated and termed PC3-Luc. PC3-Luc cells were transduced with lentiviral vectors engineering the expression of either enhanced green fluorescent protein (eGFP) or both H2 relaxin and eGFP in a bicistronic format. These transduced cells were termed PC3-Luc-eGFP and PC3-Luc-H2/eGFP, respectively. To gauge effects, PC3-Luc-H2/eGFP and PC3-Luc-eGFP cells were injected into NOD/SCID mice and monitored over 6 weeks. PC-3 tumor xenografts overexpressing H2 relaxin exhibited greater tumor volumes compared to control tumors. Circulating H2 relaxin levels in sera increased with the relative size of the tumor, with moderately elevated H2 relaxin levels in mice bearing PC3-Luc-H2/eGFP tumors compared to PC3-Luc-eGFP tumors. Zymographic analysis demonstrated that proMMP-9 enzyme activity was significantly downregulated in H2 relaxin-overexpressing tumors. An advanced angiogenic phenotype was observed in H2 relaxin-overexpressing tumors indicated by greater intratumoral vascularization by immunohistochemical staining of endothelial cells with anti-mouse CD31. Moreover, PC3-Luc-H2/eGFP tumors exhibited increased VEGF transcript by reverse-transcription PCR, compared to basal levels in control animals. Taken together, our study provides the first account of a potential role of H2 relaxin in prostate tumor development.

  12. Overexpression of native Saccharomyces cerevisiae ER-to-Golgi SNARE genes increased heterologous cellulase secretion.

    PubMed

    Van Zyl, John Henry D; Den Haan, Riaan; Van Zyl, Willem H

    2016-01-01

    Soluble N-ethylmaleimide-sensitive factor attachment receptor proteins (SNAREs) are essential components of the yeast protein-trafficking machinery and are required at the majority of membrane fusion events in the cell, where they facilitate SNARE-mediated fusion between the protein transport vesicles, the various membrane-enclosed organelles and, ultimately, the plasma membrane. We have demonstrated an increase in secretory titers for the Talaromyces emersonii Cel7A (Te-Cel7A, a cellobiohydrolase) and the Saccharomycopsis fibuligera Cel3A (Sf-Cel3A, a β-glucosidase) expressed in Saccharomyces cerevisiae through single and co-overexpression of some of the endoplasmic reticulum (ER)-to-Golgi SNAREs (BOS1, BET1, SEC22 and SED5). Overexpression of SED5 yielded the biggest improvements for both of the cellulolytic reporter proteins tested, with maximum increases in extracellular enzyme activity of 22 % for the Sf-Cel3A and 68 % for the Te-Cel7A. Co-overexpression of the ER-to-Golgi SNAREs yielded proportionately smaller increases for the Te-Cel7A (46 %), with the Sf-Cel3A yielding no improvement. Co-overexpression of the most promising exocytic SNARE components identified in literature for secretory enhancement of the cellulolytic proteins tested (SSO1 for Sf-Cel3A and SNC1 for Te-Cel7A) with the most effective ER-to-Golgi SNARE components identified in this study (SED5 for both Sf-Cel3A and Te-Cel7A) yielded variable results, with Sf-Cel3A improved by 131 % and Te-Cel7A yielding no improvement. Improvements were largely independent of gene dosage as all strains only integrated single additional SNARE gene copies, with episomal variance between the most improved strains shown to be insignificant. This study has added further credence to the notion that SNARE proteins fulfil an essential role within a larger cascade of secretory machinery components that could contribute significantly to future improvements to S. cerevisiae as protein production host. PMID:26450509

  13. Over-expression of a putative oxidoreductase (UcpA) for increasing furfural or 5-hydroxymethylfurfural tolerance

    DOEpatents

    Wang, Xuan; Miller, Elliot N.; Yomano, Lorraine P.; Shanmugam, Keelnatham T.; Ingram, Lonnie O'Neal

    2016-05-24

    The subject invention pertains to overexpression of a putative oxidoreductase (ucpA) for increasing furfural tolerance in genetically modified microorganisms. Genetically modified microorganisms capable of overexpressing UcpA are also provided. Increased expression of ucpA was shown to increase furfural tolerance by 50%, and to permit the fermentation of sugars to products in the presence of 15 mM furfural.

  14. Overexpression of YY1 increases the protein production in mammalian cells.

    PubMed

    Tastanova, Aizhan; Schulz, Alexandra; Folcher, Marc; Tolstrup, Anne; Puklowski, Anja; Kaufmann, Hitto; Fussenegger, Martin

    2016-02-10

    The production of therapeutic antibodies using mammalian cells remains a high-priority in the biopharmaceutical manufacturing industry. Bioengineers have targeted different cellular processes, including transcription, translation, secretion and post-translational modifications, to overcome the metabolic bottlenecks limiting production capacity and create high-producing mammalian cell lines. The polycomb group (PcG) proteins belong to a family of chromatin regulators with important roles in multicellular development. By overexpressing and screening genes from the PcG family, we have identified an epigenetic key player for biopharmaceutical manufacturing enhancement: the transcription factor Yin Yang 1 (YY1). The overexpression of YY1 led to an increase in the production of several product genes (SEAP, VEGF165, IgG including Rituximab), provided that human YY1 (hYY1) was expressed in human cells (HeLa, HT-1080, HEK-293T, FreeStyle™ 293-F) and Chinese hamster ovary cell-derived YY1 (cYY1) was expressed in CHO cells (CHO-K1, CHO-easyC, FreeStyle™ CHO-S, CHO-B13-24, CHO-IgG1). Ectopic expression of cYY1 in the stable CHO-derived IgG producer cell lines CHO-B13-24 and CHO-IgG1 increased the antibody titer up to 6-fold, suggesting that epigenetic engineering of mammalian production cell lines could become a new strategy to improve the manufacturing of complex protein pharmaceuticals. PMID:26686315

  15. Induced overexpression of OCT4A in human embryonic stem cells increases cloning efficiency.

    PubMed

    Tsai, Steven C; Chang, David F; Hong, Chang-Mu; Xia, Ping; Senadheera, Dinithi; Trump, Lisa; Mishra, Suparna; Lutzko, Carolyn

    2014-06-15

    Our knowledge of the molecular mechanisms underlying human embryonic stem cell (hESC) self-renewal and differentiation is incomplete. The level of octamer-binding transcription factor 4 (Oct4), a critical regulator of pluripotency, is precisely controlled in mouse embryonic stem cells. However, studies of human OCT4 are often confounded by the presence of three isoforms and six expressed pseudogenes, which has complicated the interpretation of results. Using an inducible lentiviral overexpression and knockdown system to manipulate OCT4A above or below physiological levels, we specifically examine the functional role of the OCT4A isoform in hESC. (We also designed and generated a comparable series of vectors, which were not functional, for the overexpression and knockdown of OCT4B.) We show that specific knockdown of OCT4A results in hESC differentiation, as indicated by morphology changes, cell surface antigen expression, and upregulation of ectodermal genes. In contrast, inducible overexpression of OCT4A in hESC leads to a transient instability of the hESC phenotype, as indicated by changes in morphology, cell surface antigen expression, and transcriptional profile, that returns to baseline within 5 days. Interestingly, sustained expression of OCT4A past 5 days enhances hESC cloning efficiency, suggesting that higher levels of OCT4A can support self-renewal. Overall, our results indicate that high levels of OCT4A increase hESC cloning efficiency and do not induce differentiation (whereas OCT4B expression cannot be induced in hESC), highlighting the importance of isoform-specific studies in a stable and inducible expression system for human OCT4. Additionally, we demonstrate the utility of an efficient method for conditional gene expression in hESC.

  16. Activation of D2 dopamine receptor-expressing neurons in the nucleus accumbens increases motivation

    PubMed Central

    Soares-Cunha, Carina; Coimbra, Barbara; David-Pereira, Ana; Borges, Sonia; Pinto, Luisa; Costa, Patricio; Sousa, Nuno; Rodrigues, Ana J.

    2016-01-01

    Striatal dopamine receptor D1-expressing neurons have been classically associated with positive reinforcement and reward, whereas D2 neurons are associated with negative reinforcement and aversion. Here we demonstrate that the pattern of activation of D1 and D2 neurons in the nucleus accumbens (NAc) predicts motivational drive, and that optogenetic activation of either neuronal population enhances motivation in mice. Using a different approach in rats, we further show that activating NAc D2 neurons increases cue-induced motivational drive in control animals and in a model that presents anhedonia and motivational deficits; conversely, optogenetic inhibition of D2 neurons decreases motivation. Our results suggest that the classic view of D1–D2 functional antagonism does not hold true for all dimensions of reward-related behaviours, and that D2 neurons may play a more prominent pro-motivation role than originally anticipated. PMID:27337658

  17. Activation of D2 dopamine receptor-expressing neurons in the nucleus accumbens increases motivation.

    PubMed

    Soares-Cunha, Carina; Coimbra, Barbara; David-Pereira, Ana; Borges, Sonia; Pinto, Luisa; Costa, Patricio; Sousa, Nuno; Rodrigues, Ana J

    2016-01-01

    Striatal dopamine receptor D1-expressing neurons have been classically associated with positive reinforcement and reward, whereas D2 neurons are associated with negative reinforcement and aversion. Here we demonstrate that the pattern of activation of D1 and D2 neurons in the nucleus accumbens (NAc) predicts motivational drive, and that optogenetic activation of either neuronal population enhances motivation in mice. Using a different approach in rats, we further show that activating NAc D2 neurons increases cue-induced motivational drive in control animals and in a model that presents anhedonia and motivational deficits; conversely, optogenetic inhibition of D2 neurons decreases motivation. Our results suggest that the classic view of D1-D2 functional antagonism does not hold true for all dimensions of reward-related behaviours, and that D2 neurons may play a more prominent pro-motivation role than originally anticipated. PMID:27337658

  18. Tickling increases dopamine release in the nucleus accumbens and 50 kHz ultrasonic vocalizations in adolescent rats.

    PubMed

    Hori, Miyo; Shimoju, Rie; Tokunaga, Ryota; Ohkubo, Masato; Miyabe, Shigeki; Ohnishi, Junji; Murakami, Kazuo; Kurosawa, Mieko

    2013-03-27

    Adolescent rats emit 50 kHz ultrasonic vocalizations, a marker of positive emotion, during rough-and-tumble play or on tickling stimulation. The emission of 50 kHz ultrasonic vocalizations in response to tickling is suggested to be mediated by dopamine release in the nucleus accumbens; however, there is no direct evidence supporting this hypothesis. The present study aimed to elucidate whether play behavior (tickling) in adolescent rats can trigger dopamine release in the nucleus accumbens with hedonic 50 kHz ultrasonic vocalizations. The effect of tickling stimulation was compared with light-touch stimulation, as a discernible stimulus. We examined 35-40-day-old rats, which corresponds to the period of midadolescence. Tickling stimulation for 5 min significantly increased dopamine release in the nucleus accumbens (118±7% of the prestimulus control value). Conversely, light-touch stimulation for 5 min did not significantly change dopamine release. In addition, 50 kHz ultrasonic vocalizations were emitted during tickling stimulation but not during light-touch stimulation. Further, tickling-induced 50 kHz ultrasonic vocalizations were significantly blocked by the direct application of SCH23390 (D1 receptor antagonist) and raclopride (D2/D3 receptor antagonist) into the nucleus accumbens. Our study demonstrates that tickling stimulation in adolescent rats increases dopamine release in the nucleus accumbens, leading to the generation of 50 kHz ultrasonic vocalizations.

  19. Ethanol-induced increases in extracellular dopamine are blunted in brain-derived neurotrophic factor heterozygous mice

    PubMed Central

    Bosse, Kelly E.; Mathews, Tiffany A.

    2010-01-01

    Drugs of abuse like ethanol have the ability to stimulate forebrain dopaminergic pathways. Although the positive reinforcing properties of abused substances are largely attributed to their effects on dopamine transmission, alcohol addiction involves complex interactions between numerous molecular mediators. Brain-derived neurotrophic factor (BDNF) is suggested to have a protective role in regulating the reinforcing effects of ethanol. In the present study, we evaluated the effects of an acute, systemic injection of ethanol (2 g/kg) on BDNF protein levels and extracellular dopamine concentrations, measured by in vivo microdialysis, in the caudate-putamen of wildtype and heterozygous BDNF mice. In both genotypes, the peak increase in extracellular dopamine following ethanol coincided temporally with a decrease in BDNF protein levels following a similar ethanol treatment. Moreover, the effect of ethanol to increase extracellular dopamine was blunted in heterozygous BDNF mice compared to wildtype mice. While the magnitude of decrease in BDNF protein induced by ethanol was similar between genotypes (two-fold), ethanol treatment induced significantly lower BDNF protein levels in heterozygous BDNF mice overall. These findings suggest the effects of ethanol are influenced by an interaction between BDNF and dopamine transmission, which may relate to the pathway through which BDNF regulates ethanol intake. PMID:21163332

  20. The dopamine reuptake inhibitor MRZ-9547 increases progressive ratio responding in rats.

    PubMed

    Sommer, S; Danysz, W; Russ, H; Valastro, B; Flik, G; Hauber, W

    2014-12-01

    Drugs that are able to shift effort-related decision making in intact rats towards high-effort response options are largely unknown. Here, we examined the effects of two candidate drugs, MRZ-9547 and its l-enantiomer MRZ-9546 on progressive ratio (PR) responding using two different tasks, a standard PR task that involves increasing ratio requirements and a PR/chow feeding choice task in which animals can lever press for preferred food pellets under a PR schedule or approach freely available less preferred lab chow. Furthermore, we assessed the mechanisms of action of both drugs using in vitro-assay methods and in vivo-microdialysis. Results reveal that MRZ-9547 is a selective dopamine transporter (DAT) inhibitor that moderately stimulated striatal dopamine release. MRZ-9546 was a much less potent DAT inhibitor. Furthermore, MRZ-9547 dose dependently increased the tendency to work for food reinforcement both in the standard PR task and the PR/chow feeding choice task, MRZ-9546 was considerably less active. Relative to MRZ-9547, other DAT-interfering drugs had only moderate (methylphenidate) or marginal (modafinil, d-amphetamine) stimulant effects on PR responding in either task. Collectively, our data demonstrate that the DAT inhibitor MRZ-9547 can markedly stimulate PR responding and shift effort-related decision making in intact rats towards high-effort response options. An analysis of effort-related decision making in rodents could provide an animal model for motivational dysfunctions related to effort expenditure such as fatigue, e.g. in Parkinson's disease or major depression. Our findings suggest that DAT inhibitors such as MRZ-9547 could be potentially useful for treating energy-related symptoms in neurological or neuropsychiatric disorders. PMID:24964269

  1. Increased lever pressing for amphetamine after pimozide in rats: implications for a dopamine theory of reward.

    PubMed

    Yokel, R A; Wise, R A

    1975-02-14

    Low and high doses of a dopamine blocking agent had effects on lever pressing for intravenous amphetamine reward which resembled the effects of reward reduction and reward termination, respectively. Noradrenaline blockade had no such effects. A role in central mediation of reward perception is suggested for dopamine but not for noradrenaline.

  2. Constitutive overexpression of asm18 increases the production and diversity of maytansinoids in Actinosynnema pretiosum.

    PubMed

    Li, Shanren; Lu, Chunhua; Chang, Xiaoyan; Shen, Yuemao

    2016-03-01

    Ansamitocins isolated from Actinosynnema pretiosum, potent antitumor compounds, belong to the family of maytansinoids, and the antibody-maytansinoid conjugates are currently under different phases of clinical trials. The clinical applications of ansamitocins have stimulated extensive studies to improve their production yields. In this study, we investigated the function of a pathway-specific S treptomyces antibiotic regulatory protein (SARP) family regulator, Asm18, and observed that ectopic overexpression of the asm18 gene increased the production of N-demethyl-4,5-desepoxy-maytansinol (2) to 50 mg/L in the HGF052 + pJTU824-asm18 strain, an increase by 4.7-fold compared to that of the control strain HGF052 + pJTU824. Real-time PCR analysis showed that the overexpression of the asm18 gene selectively increased the transcription levels of the genes involved in the biosynthesis of the starter unit (asm43), polyketide assembly (asmA), post-PKS modification (asm21), as well as the transcription levels of the regulatory gene (asm8), which is a specific LAL-type activator in ansamitocin biosynthesis. With the increase of fermentation titre, seven ansamitocin analogs (1-7) including three new ones (1, 5, and 6) and maytansinol (7) were isolated from the HGF052 + pJTU824-asm18 strain. Our results not only pave the way for further improving the production of ansamitocin analogs but also indicate that the post-PKS modifications of ansamitocin biosynthesis are flexible, which brings a potential of producing maytansinol, the most fascinating intermediate for the synthesis of antibody-maytansinoid conjugates, by optimizing the HGF052 and/or HGF052 + pJTU824-asm18 strains. PMID:26572523

  3. Working memory deficits, increased anxiety-like traits, and seizure susceptibility in BDNF overexpressing mice.

    PubMed

    Papaleo, Francesco; Silverman, Jill L; Aney, Jordan; Tian, Qingjun; Barkan, Charlotte L; Chadman, Kathryn K; Crawley, Jacqueline N

    2011-08-01

    BDNF regulates components of cognitive processes and has been implicated in psychiatric disorders. Here we report that genetic overexpression of the BDNF mature isoform (BDNF-tg) in female mice impaired working memory functions while sparing components of fear conditioning. BDNF-tg mice also displayed reduced breeding efficiency, higher anxiety-like scores, high self-grooming, impaired prepulse inhibition, and higher susceptibility to seizures when placed in a new empty cage, as compared with wild-type (WT) littermate controls. Control measures of general health, locomotor activity, motor coordination, depression-related behaviors, and sociability did not differ between genotypes. The present findings, indicating detrimental effects of life-long increased BDNF in mice, may inform human studies evaluating the role of BDNF functional genetic variations on cognitive abilities and vulnerability to psychiatric disorders. PMID:21791566

  4. Increase in ascorbate content of transgenic tobacco plants overexpressing the acerola (Malpighia glabra) phosphomannomutase gene.

    PubMed

    Badejo, Adebanjo A; Eltelib, Hani A; Fukunaga, Kazunari; Fujikawa, Yukichi; Esaka, Muneharu

    2009-02-01

    Phosphomannomutase (PMM; EC 5.4.2.8) catalyzes the interconversion of mannose-6-phosphate to mannose-1-phosphate in the Smirnoff-Wheeler pathway for the biosynthesis of l-ascorbic acid (AsA). We have cloned the PMM cDNA from acerola (Malpighia glabra), a plant containing an enormous amount of AsA. The AsA contents correlate with the PMM gene expression of the ripening fruits and leaves. The PMM activities in the leaves of acerola, tomato and Arabidopsis correlate with their respective AsA contents. Transgenic tobacco plants overexpressing the acerola PMM gene showed about a 2-fold increase in AsA contents compared with the wild type, with a corresponding correlation with the PMM transcript levels and activities.

  5. Working memory deficits, increased anxiety-like traits, and seizure susceptibility in BDNF overexpressing mice

    PubMed Central

    Papaleo, Francesco; Silverman, Jill L.; Aney, Jordan; Tian, Qingjun; Barkan, Charlotte L.; Chadman, Kathryn K.; Crawley, Jacqueline N.

    2011-01-01

    BDNF regulates components of cognitive processes and has been implicated in psychiatric disorders. Here we report that genetic overexpression of the BDNF mature isoform (BDNF-tg) in female mice impaired working memory functions while sparing components of fear conditioning. BDNF-tg mice also displayed reduced breeding efficiency, higher anxiety-like scores, high self-grooming, impaired prepulse inhibition, and higher susceptibility to seizures when placed in a new empty cage, as compared with wild-type (WT) littermate controls. Control measures of general health, locomotor activity, motor coordination, depression-related behaviors, and sociability did not differ between genotypes. The present findings, indicating detrimental effects of life-long increased BDNF in mice, may inform human studies evaluating the role of BDNF functional genetic variations on cognitive abilities and vulnerability to psychiatric disorders. PMID:21791566

  6. The dopamine antagonist domperidone increases prolactin concentration and enhances milk production in dairy cows.

    PubMed

    Lacasse, P; Ollier, S

    2015-11-01

    In previous studies, our team showed that the inhibition of prolactin (PRL) secretion by the dopamine agonist quinagolide reduces milk production in dairy cows. The objective of this study was to determine the effects of administration of a dopamine antagonist on basal and milking-induced PRL concentrations in blood and on milk production during positive energy balance and feed restriction in dairy cows. Eighteen mid-lactation Holstein cows received daily s.c. injections of either domperidone (300 mg, DOMP, n=9) or the vehicle, canola oil (CTL, n=9), for 5 wk. During wk 5, all cows were fed at 65% of their dry matter intake in the previous week. Blood and milk samples were collected before (for blood) and during (for milk) the a.m. milking thrice weekly from d -9 to 41 (8d after the last injection). In addition, blood samples were collected during the a.m. milking on d -1 (before the first injection), and on d 1, 28, and 34. Basal PRL concentration was similar in both groups before the start of the treatments. Domperidone injections caused a gradual increase in basal PRL concentration. Feed restriction reduced basal PRL concentration in both the CTL and DOMP cows, but PRL concentration remained higher in the DOMP cows. Prolactin concentration remained elevated in the DOMP cows 7d after the last injection. The milk concentration of PRL increased during the DOMP treatment, but the increase was smaller than that observed in serum. In the CTL cows, the milking-induced PRL release above the premilking concentration was similar on d -1, 1, and 28 but was reduced during feed restriction. In the DOMP cows, the milking-induced PRL release was similar on d -1 and 1 but was reduced on d 28 and 34. Milk production was similar for both groups before the treatments started but was greater in the DOMP cows during the treatment period, at 2.9 ± 0.6 and 2.4 ± 0.6 kg/d greater during wk 3 and 4 of treatment, respectively. Milk production declined in both groups during feed

  7. Increased glycogen accumulation in transgenic mice overexpressing glycogen synthase in skeletal muscle.

    PubMed Central

    Manchester, J; Skurat, A V; Roach, P; Hauschka, S D; Lawrence, J C

    1996-01-01

    To investigate the role of glycogen synthase in controlling glycogen accumulation, we generated three lines of transgenic mice in which the enzyme was overexpressed in skeletal muscle by using promoter-enhancer elements derived from the mouse muscle creatine kinase gene. In all three lines, expression was highest in muscles composed primarily of fast-twitch fibers, such as the gastrocnemius and anterior tibialis. In these muscles, glycogen synthase activity was increased by as much as 10-fold, with concomitant increases (up to 5-fold) in the glycogen content. The uridine diphosphoglucose concentrations were markedly decreased, consistent with the increase in glycogen synthase activity. Levels of glycogen phosphorylase in these muscles increased (up to 3-fold), whereas the amount of the insulin-sensitive glucose transporter 4 either remained unchanged or decreased. The observation that increasing glycogen synthase enhances glycogen accumulation supports the conclusion that the activation of glycogen synthase, as well as glucose transport, contributes to the accumulation of glycogen in response to insulin in skeletal muscle. Images Fig. 1 PMID:8855244

  8. Caffeine increases striatal dopamine D2/D3 receptor availability in the human brain

    DOE PAGESBeta

    Volkow, N. D.; Wang, G. -J.; Logan, J.; Alexoff, D.; Fowler, J. S.; Thanos, P. K.; Wong, C.; Casado, V.; Ferre, S.; Tomasi, D.

    2015-04-14

    Caffeine, the most widely consumed psychoactive substance in the world, is used to promote wakefulness and enhance alertness. Like other wake-promoting drugs (stimulants and modafinil), caffeine enhances dopamine (DA) signaling in the brain, which it does predominantly by antagonizing adenosine A2A receptors (A2AR). However, it is unclear if caffeine, at the doses consumed by humans, increases DA release or whether it modulates the functions of postsynaptic DA receptors through its interaction with adenosine receptors, which modulate them. We used positron emission tomography and [11C]raclopride (DA D2/D3 receptor radioligand sensitive to endogenous DA) to assess if caffeine increased DA release inmore » striatum in 20 healthy controls. Caffeine (300mg p.o.) significantly increased the availability of D2/D3 receptors in putamen and ventral striatum, but not in caudate, when compared with placebo. In addition, caffeine-induced increases in D2/D3 receptor availability in the ventral striatum were associated with caffeine-induced increases in alertness. Our findings indicate that in the human brain, caffeine, at doses typically consumed, increases the availability of DA D2/D3 receptors, which indicates that caffeine does not increase DA in the striatum for this would have decreased D2/D3 receptor availability. Instead, we interpret our findings to reflect an increase in D2/D3 receptor levels in striatum with caffeine (or changes in affinity). Furthermore, the association between increases in D2/D3 receptor availability in ventral striatum and alertness suggests that caffeine might enhance arousal, in part, by upregulating D2/D3 receptors.« less

  9. Caffeine increases striatal dopamine D2/D3 receptor availability in the human brain.

    PubMed

    Volkow, N D; Wang, G-J; Logan, J; Alexoff, D; Fowler, J S; Thanos, P K; Wong, C; Casado, V; Ferre, S; Tomasi, D

    2015-01-01

    Caffeine, the most widely consumed psychoactive substance in the world, is used to promote wakefulness and enhance alertness. Like other wake-promoting drugs (stimulants and modafinil), caffeine enhances dopamine (DA) signaling in the brain, which it does predominantly by antagonizing adenosine A2A receptors (A2AR). However, it is unclear if caffeine, at the doses consumed by humans, increases DA release or whether it modulates the functions of postsynaptic DA receptors through its interaction with adenosine receptors, which modulate them. We used positron emission tomography and [(11)C]raclopride (DA D2/D3 receptor radioligand sensitive to endogenous DA) to assess if caffeine increased DA release in striatum in 20 healthy controls. Caffeine (300 mg p.o.) significantly increased the availability of D2/D3 receptors in putamen and ventral striatum, but not in caudate, when compared with placebo. In addition, caffeine-induced increases in D2/D3 receptor availability in the ventral striatum were associated with caffeine-induced increases in alertness. Our findings indicate that in the human brain, caffeine, at doses typically consumed, increases the availability of DA D2/D3 receptors, which indicates that caffeine does not increase DA in the striatum for this would have decreased D2/D3 receptor availability. Instead, we interpret our findings to reflect an increase in D2/D3 receptor levels in striatum with caffeine (or changes in affinity). The association between increases in D2/D3 receptor availability in ventral striatum and alertness suggests that caffeine might enhance arousal, in part, by upregulating D2/D3 receptors. PMID:25871974

  10. Caffeine increases striatal dopamine D2/D3 receptor availability in the human brain.

    PubMed

    Volkow, N D; Wang, G-J; Logan, J; Alexoff, D; Fowler, J S; Thanos, P K; Wong, C; Casado, V; Ferre, S; Tomasi, D

    2015-04-14

    Caffeine, the most widely consumed psychoactive substance in the world, is used to promote wakefulness and enhance alertness. Like other wake-promoting drugs (stimulants and modafinil), caffeine enhances dopamine (DA) signaling in the brain, which it does predominantly by antagonizing adenosine A2A receptors (A2AR). However, it is unclear if caffeine, at the doses consumed by humans, increases DA release or whether it modulates the functions of postsynaptic DA receptors through its interaction with adenosine receptors, which modulate them. We used positron emission tomography and [(11)C]raclopride (DA D2/D3 receptor radioligand sensitive to endogenous DA) to assess if caffeine increased DA release in striatum in 20 healthy controls. Caffeine (300 mg p.o.) significantly increased the availability of D2/D3 receptors in putamen and ventral striatum, but not in caudate, when compared with placebo. In addition, caffeine-induced increases in D2/D3 receptor availability in the ventral striatum were associated with caffeine-induced increases in alertness. Our findings indicate that in the human brain, caffeine, at doses typically consumed, increases the availability of DA D2/D3 receptors, which indicates that caffeine does not increase DA in the striatum for this would have decreased D2/D3 receptor availability. Instead, we interpret our findings to reflect an increase in D2/D3 receptor levels in striatum with caffeine (or changes in affinity). The association between increases in D2/D3 receptor availability in ventral striatum and alertness suggests that caffeine might enhance arousal, in part, by upregulating D2/D3 receptors.

  11. Increased release of norepinephrine and dopamine from canine kidney during bilateral carotid occlusion

    SciTech Connect

    Bradley, T.; Hjemdahl, P.; DiBona, G.F.

    1987-02-01

    The renal overflow of norepinephrine (NE) and dopamine (DA) to plasma from the innervated kidney was studied at rest and during sympathetic nervous system activation by bilateral carotid artery occlusion (BCO) in vagotomized dogs under barbiturate or barbiturate/nitrous oxide anesthesia. BCO elevated arterial pressure and the arterial plasma concentration of NE, DA, and epinephrine (Epi). Renal vascular resistance (renal arterial pressure kept constant) increased by 15 +/- 7% and the net renal venous outflows (renal veno-arterial concentration difference x renal plasma flow) of NE and DA were enhanced. To obtain more correct estimates of the renal contribution to the renal venous catecholamine outflow, they corrected for the renal extraction of arterial catecholamines, assessed as the extractions of (/sup 3/H)NE, (/sup 3/H)DA, or endogenous Epi. The (/sup 3/H)NE corrected renal NE overflow to plasma increased from 144 +/- 40 to 243 +/- 64 pmol-min/sup -1/ during BCO, which, when compared with a previous study of the (/sup 3/H)NE corrected renal NE overflow to plasma evoked by electrical renal nerve stimulation, corresponds to a 40% increase in nerve impulse frequency from approx. 0.6 Hz. If the renal catecholamine extraction was not taken into account the effect of BCO was underestimated. The renal DA overflow to plasma was about one-fifth of the NE overflow both at rest and during BCO, indicating that there was no preferential activation of noradrenergic or putative dopaminergic nerves by BCO.

  12. Increased vulnerability of the brain norepinephrine system of females to corticotropin-releasing factor overexpression.

    PubMed

    Bangasser, D A; Reyes, B A S; Piel, D; Garachh, V; Zhang, X-Y; Plona, Z M; Van Bockstaele, E J; Beck, S G; Valentino, R J

    2013-02-01

    Stress-related psychiatric disorders are more prevalent in women than men. As hypersecretion of the stress neuromediator, corticotropin-releasing factor (CRF) has been implicated in these disorders, sex differences in CRF sensitivity could underlie this disparity. Hyperarousal is a core symptom that is shared by stress-related disorders and this has been attributed to CRF regulation of the locus ceruleus (LC)-norepinephrine arousal system. We recently identified sex differences in CRF(1) receptor (CRF(1)) signaling and trafficking that render LC neurons of female rats more sensitive to CRF and potentially less able to adapt to excess CRF compared with male rats. The present study used a genetic model of CRF overexpression to test the hypothesis that females would be more vulnerable to LC dysregulation by conditions of excess CRF. In both male and female CRF overexpressing (CRF-OE) mice, the LC was more densely innervated by CRF compared with wild-type controls. Despite the equally dense CRF innervation of the LC in male and female CRF-OE mice, LC discharge rates recorded in slices in vitro were selectively elevated in female CRF-OE mice. Immunoelectron microscopy revealed that this sex difference resulted from differential CRF(1) trafficking. In male CRF-OE mice, CRF(1) immunolabeling was prominent in the cytoplasm of LC neurons, indicative of internalization, a process that would protect cells from excessive CRF. However, in female CRF-OE mice, CRF(1) labeling was more prominent on the plasma membrane, suggesting that the compensatory response of internalization was compromised. Together, the findings suggest that the LC-norepinephrine system of females will be particularly affected by conditions resulting in elevated CRF because of differences in receptor trafficking. As excessive LC activation has been implicated in the arousal components of stress-related psychiatric disorders, this may be a cellular mechanism that contributes to the increased incidence of

  13. Overexpression of amyloid precursor protein increases copper content in HEK293 cells

    SciTech Connect

    Suazo, Miriam; Hodar, Christian; Morgan, Carlos; Cerpa, Waldo; Cambiazo, Veronica; Inestrosa, Nibaldo C.; Gonzalez, Mauricio

    2009-05-15

    Amyloid precursor protein (APP) is a transmembrane glycoprotein widely expressed in mammalian tissues and plays a central role in Alzheimer's disease. However, its physiological function remains elusive. Cu{sup 2+} binding and reduction activities have been described in the extracellular APP135-156 region, which might be relevant for cellular copper uptake and homeostasis. Here, we assessed Cu{sup 2+} reduction and {sup 64}Cu uptake in two human HEK293 cell lines overexpressing APP. Our results indicate that Cu{sup 2+} reduction increased and cells accumulated larger levels of copper, maintaining cell viability at supra-physiological levels of Cu{sup 2+} ions. Moreover, wild-type cells exposed to both Cu{sup 2+} ions and APP135-155 synthetic peptides increased copper reduction and uptake. Complementation of function studies in human APP751 transformed Fre1 defective Saccharomyces cerevisiae cells rescued low Cu{sup 2+} reductase activity and increased {sup 64}Cu uptake. We conclude that Cu{sup 2+} reduction activity of APP facilitates copper uptake and may represent an early step in cellular copper homeostasis.

  14. Increased glucose metabolism and insulin sensitivity in transgenic skinny mice overexpressing leptin.

    PubMed

    Ogawa, Y; Masuzaki, H; Hosoda, K; Aizawa-Abe, M; Suga, J; Suda, M; Ebihara, K; Iwai, H; Matsuoka, N; Satoh, N; Odaka, H; Kasuga, H; Fujisawa, Y; Inoue, G; Nishimura, H; Yoshimasa, Y; Nakao, K

    1999-09-01

    Excess of body fat, or obesity, is a major health problem and confers a higher risk of cardiovascular and metabolic disorders such as diabetes, hypertension, and coronary heart disease. Leptin is an adipocyte-derived satiety factor that plays an important role in the regulation of energy homeostasis, and its synthesis and secretion are markedly increased in obese subjects. To explore the metabolic consequences of an increased amount of leptin on a long-term basis in vivo, we generated transgenic skinny mice with elevated plasma leptin concentrations comparable to those in obese subjects. Overexpression of leptin in the liver has resulted in complete disappearance of white and brown adipose tissue for a long period of time in mice. Transgenic skinny mice exhibit increased glucose metabolism accompanied by the activation of insulin signaling in the skeletal muscle and liver. They also show small-sized livers with a marked decrease in glycogen and lipid storage. The phenotypes are in striking contrast to those of recently reported animal models of lipoatrophic diabetes and patients with lipoatrophic diabetes with reduced amount of leptin. The present study provides evidence that leptin is an adipocyte-derived antidiabetic hormone in vivo and suggests its pathophysiologic and therapeutic implications in diabetes.

  15. Harsh corporal punishment is associated with increased T2 relaxation time in dopamine-rich regions.

    PubMed

    Sheu, Yi-Shin; Polcari, Ann; Anderson, Carl M; Teicher, Martin H

    2010-11-01

    Harsh corporal punishment (HCP) was defined as frequent parental administration of corporal punishment (CP) for discipline, with occasional use of objects such as straps, or paddles. CP is linked to increased risk for depression and substance abuse. We examine whether long-term exposure to HCP acts as sub-traumatic stressor that contributes to brain alterations, particularly in dopaminergic pathways, which may mediate their increased vulnerability to drug and alcohol abuse. Nineteen young adults who experienced early HCP but no other forms of maltreatment and twenty-three comparable controls were studied. T2 relaxation time (T2-RT) measurements were performed with an echo planar imaging TE stepping technique and T2 maps were calculated and analyzed voxel-by-voxel to locate regional T2-RT differences between groups. Previous studies indicated that T2-RT provides an indirect index of resting cerebral blood volume. Region of interest (ROI) analyses were also conducted in caudate, putamen, nucleus accumbens, anterior cingulate cortex, dorsolateral prefrontal cortex, thalamus, globus pallidus and cerebellar hemispheres. Voxel-based relaxometry showed that HCP was associated with increased T2-RT in right caudate and putamen. ROI analyses also revealed increased T2-RT in dorsolateral prefrontal cortex, substantia nigra, thalamus and accumbens but not globus pallidus or cerebellum. There were significant associations between T2-RT measures in dopamine target regions and use of drugs and alcohol, and memory performance. Alteration in the paramagnetic or hemodynamic properties of dopaminergic cell body and projection regions were observed in subjects with HCP, and these findings may relate to their increased risk for drug and alcohol abuse.

  16. Early-Life Social Isolation Stress Increases Kappa Opioid Receptor Responsiveness and Downregulates the Dopamine System.

    PubMed

    Karkhanis, Anushree N; Rose, Jamie H; Weiner, Jeffrey L; Jones, Sara R

    2016-08-01

    Chronic early-life stress increases vulnerability to alcoholism and anxiety disorders during adulthood. Similarly, rats reared in social isolation (SI) during adolescence exhibit augmented ethanol intake and anxiety-like behaviors compared with group housed (GH) rats. Prior studies suggest that disruption of dopamine (DA) signaling contributes to SI-associated behaviors, although the mechanisms underlying these alterations are not fully understood. Kappa opioid receptors (KORs) have an important role in regulating mesolimbic DA signaling, and other kinds of stressors have been shown to augment KOR function. Therefore, we tested the hypothesis that SI-induced increases in KOR function contribute to the dysregulation of NAc DA and the escalation in ethanol intake associated with SI. Our ex vivo voltammetry experiments showed that the inhibitory effects of the kappa agonist U50,488 on DA release were significantly enhanced in the NAc core and shell of SI rats. Dynorphin levels in NAc tissue were observed to be lower in SI rats. Microdialysis in freely moving rats revealed that SI was also associated with reduced baseline DA levels, and pretreatment with the KOR antagonist nor-binaltorphimine (nor-BNI) increased DA levels selectively in SI subjects. Acute ethanol elevated DA in SI and GH rats and nor-BNI pretreatment augmented this effect in SI subjects, while having no effect on ethanol-stimulated DA release in GH rats. Together, these data suggest that KORs may have increased responsiveness following SI, which could lead to hypodopaminergia and contribute to an increased drive to consume ethanol. Indeed, SI rats exhibited greater ethanol intake and preference and KOR blockade selectively attenuated ethanol intake in SI rats. Collectively, the findings that nor-BNI reversed SI-mediated hypodopaminergic state and escalated ethanol intake suggest that KOR antagonists may represent a promising therapeutic strategy for the treatment of alcohol use disorders, particularly

  17. Methylphenidate-elicited dopamine increases in ventral striatum are associated with long-term symptom improvement in adults with attention deficit hyperactivity disorder.

    PubMed

    Volkow, Nora D; Wang, Gene-Jack; Tomasi, Dardo; Kollins, Scott H; Wigal, Tim L; Newcorn, Jeffrey H; Telang, Frank W; Fowler, Joanna S; Logan, Jean; Wong, Christopher T; Swanson, James M

    2012-01-18

    Stimulant medications, such as methylphenidate, which are effective treatments for attention deficit hyperactivity disorder (ADHD), enhance brain dopamine signaling. However, the relationship between regional brain dopamine enhancement and treatment response has not been evaluated. Here, we assessed whether the dopamine increases elicited by methylphenidate are associated with long-term clinical response. We used a prospective design to study 20 treatment-naive adults with ADHD who were evaluated before treatment initiation and after 12 months of clinical treatment with a titrated regimen of oral methylphenidate. Methylphenidate-induced dopamine changes were evaluated with positron emission tomography and [(11)C]raclopride (D(2)/D(3) receptor radioligand sensitive to competition with endogenous dopamine). Clinical responses were assessed using the Conners' Adult ADHD Rating Scale and revealed a significant reduction in symptoms of inattention and hyperactivity with long-term methylphenidate treatment. A challenge dose of 0.5 mg/kg intravenous methylphenidate significantly increased dopamine in striatum (assessed as decreases in D(2)/D(3) receptor availability). In the ventral striatum, these dopamine increases were associated with the reductions in ratings of symptoms of inattention with clinical treatment. Statistical parametric mapping additionally showed dopamine increases in prefrontal and temporal cortices with intravenous methylphenidate that were also associated with decreases in symptoms of inattention. Our findings indicate that dopamine enhancement in ventral striatum (the brain region involved with reward and motivation) was associated with therapeutic response to methylphenidate, further corroborating the relevance of the dopamine reward/motivation circuitry in ADHD. It also provides preliminary evidence that methylphenidate-elicited dopamine increases in prefrontal and temporal cortices may also contribute to the clinical response.

  18. Methylphenidate-Elicited Dopamine Increases in Ventral Striatum Are Associated with Long-Term Symptom Improvement in Adults with Attention Deficit Hyperactivity Disorder

    SciTech Connect

    Volkow N. D.; Wang G.; Volkow, N.D.; Wang, G.-J.; Tomasi, D.; Kollins, S.H.; Wigal, T.L.; Newcorn, J.H.; Telang, F.W.; Fowler, J.S.; Logan, J.; Wong, C.T.; Swanson, J.M.

    2012-01-18

    Stimulant medications, such as methylphenidate, which are effective treatments for attention deficit hyperactivity disorder (ADHD), enhance brain dopamine signaling. However, the relationship between regional brain dopamine enhancement and treatment response has not been evaluated. Here, we assessed whether the dopamine increases elicited by methylphenidate are associated with long-term clinical response. We used a prospective design to study 20 treatment-naive adults with ADHD who were evaluated before treatment initiation and after 12 months of clinical treatment with a titrated regimen of oral methylphenidate. Methylphenidate-induced dopamine changes were evaluated with positron emission tomography and [{sup 11}C]raclopride (D{sub 2}/D{sub 3} receptor radioligand sensitive to competition with endogenous dopamine). Clinical responses were assessed using the Conners Adult ADHD Rating Scale and revealed a significant reduction in symptoms of inattention and hyperactivity with long-term methylphenidate treatment. A challenge dose of 0.5 mg/kg intravenous methylphenidate significantly increased dopamine in striatum (assessed as decreases in D{sub 2}/D{sub 3} receptor availability). In the ventral striatum, these dopamine increases were associated with the reductions in ratings of symptoms of inattention with clinical treatment. Statistical parametric mapping additionally showed dopamine increases in prefrontal and temporal cortices with intravenous methylphenidate that were also associated with decreases in symptoms of inattention. Our findings indicate that dopamine enhancement in ventral striatum (the brain region involved with reward and motivation) was associated with therapeutic response to methylphenidate, further corroborating the relevance of the dopamine reward/motivation circuitry in ADHD. It also provides preliminary evidence that methylphenidate-elicited dopamine increases in prefrontal and temporal cortices may also contribute to the clinical response.

  19. Increased mitochondrial DNA deletions in substantia nigra dopamine neurons of the aged rat.

    PubMed

    Parkinson, Gemma M; Dayas, Christopher V; Smith, Doug W

    2014-01-01

    The dopaminergic neurons of the substantia nigra (SN), which constitute the origin of the nigrostriatal system, are vulnerable to age-related degenerative processes. For example, in humans there is a relatively small age-related loss of neurons but a marked decline of the dopaminergic phenotype associated with impaired voluntary motor control. However, the mechanisms responsible for the dysfunction and degeneration of SN dopamine neurons remain poorly understood. One potential contributor is mitochondrial dysfunction, resulting from an increased abundance of mitochondrial DNA (mtDNA) mutations such as deletions. Human studies have identified relatively high levels of mtDNA deletions in these cells in both aging and Parkinson's disease (>35%), with a higher abundance of deletions (>60%) in individual neurons with mitochondrial dysfunction. However, it is unknown whether similar mtDNA mutations occur in other species such as the rat. In the present study, we quantified mtDNA deletion abundance in laser microdissected SN dopaminergic neurons from young and old F344 rats. Our results indicate that mtDNA deletions accumulated with age, with approximately 20% more mtDNA deletions in SN dopaminergic neurons from old compared to young animals. Thus, while rat SN dopaminergic neurons do accumulate mtDNA deletions with aging, this does not reflect the deletion burden in humans, and other mechanisms may be operating to compensate for age-related mtDNA damage in the rat SN dopaminergic neurons. PMID:25612740

  20. Overexpression of Arabidopsis VIT1 increases accumulation of iron in cassava roots and stems.

    PubMed

    Narayanan, Narayanan; Beyene, Getu; Chauhan, Raj Deepika; Gaitán-Solis, Eliana; Grusak, Michael A; Taylor, Nigel; Anderson, Paul

    2015-11-01

    Iron is extremely abundant in the soil, but its uptake in plants is limited due to low solubility in neutral or alkaline soils. Plants can rely on rhizosphere acidification to increase iron solubility. AtVIT1 was previously found to be involved in mediating vacuolar sequestration of iron, which indicates a potential application for iron biofortification in crop plants. Here, we have overexpressed AtVIT1 in the starchy root crop cassava using a patatin promoter. Under greenhouse conditions, iron levels in mature cassava storage roots showed 3-4 times higher values when compared with wild-type plants. Significantly, the expression of AtVIT1 showed a positive correlation with the increase in iron concentration of storage roots. Conversely, young leaves of AtVIT1 transgenic plants exhibit characteristics of iron deficiency such as interveinal chlorosis of leaves (yellowing) and lower iron concentration when compared with the wild type plants. Interestingly, the AtVIT1 transgenic plants showed 4 and 16 times higher values of iron concentration in the young stem and stem base tissues, respectively. AtVIT1 transgenic plants also showed 2-4 times higher values of iron content when compared with wild-type plants, with altered partitioning of iron between source and sink tissues. These results demonstrate vacuolar iron sequestration as a viable transgenic strategy to biofortify crops and to help eliminate micronutrient malnutrition in at-risk human populations.

  1. Overexpression of Arabidopsis VIT1 increases accumulation of iron in cassava roots and stems.

    PubMed

    Narayanan, Narayanan; Beyene, Getu; Chauhan, Raj Deepika; Gaitán-Solis, Eliana; Grusak, Michael A; Taylor, Nigel; Anderson, Paul

    2015-11-01

    Iron is extremely abundant in the soil, but its uptake in plants is limited due to low solubility in neutral or alkaline soils. Plants can rely on rhizosphere acidification to increase iron solubility. AtVIT1 was previously found to be involved in mediating vacuolar sequestration of iron, which indicates a potential application for iron biofortification in crop plants. Here, we have overexpressed AtVIT1 in the starchy root crop cassava using a patatin promoter. Under greenhouse conditions, iron levels in mature cassava storage roots showed 3-4 times higher values when compared with wild-type plants. Significantly, the expression of AtVIT1 showed a positive correlation with the increase in iron concentration of storage roots. Conversely, young leaves of AtVIT1 transgenic plants exhibit characteristics of iron deficiency such as interveinal chlorosis of leaves (yellowing) and lower iron concentration when compared with the wild type plants. Interestingly, the AtVIT1 transgenic plants showed 4 and 16 times higher values of iron concentration in the young stem and stem base tissues, respectively. AtVIT1 transgenic plants also showed 2-4 times higher values of iron content when compared with wild-type plants, with altered partitioning of iron between source and sink tissues. These results demonstrate vacuolar iron sequestration as a viable transgenic strategy to biofortify crops and to help eliminate micronutrient malnutrition in at-risk human populations. PMID:26475197

  2. IL-35 over-expression increases apoptosis sensitivity and suppresses cell growth in human cancer cells.

    PubMed

    Long, Jun; Zhang, Xulong; Wen, Mingjie; Kong, Qingli; Lv, Zhe; An, Yunqing; Wei, Xiao-Qing

    2013-01-01

    Interleukin (IL)-35 is a novel heterodimeric cytokine in the IL-12 family and is composed of two subunits: Epstein-Barr virus-induced gene 3 (EBI3) and IL-12p35. IL-35 is expressed in T regulatory (Treg) cells and contributes to the immune suppression function of these cells. In contrast, we found that both IL-35 subunits were expressed concurrently in most human cancer cell lines compared to normal cell lines. In addition, we found that TNF-α and IFN-γ stimulation led to increased IL-35 expression in human cancer cells. Furthermore, over-expression of IL-35 in human cancer cells suppressed cell growth in vitro, induced cell cycle arrest at the G1 phase, and mediated robust apoptosis induced by serum starvation, TNF-α, and IFN-γ stimulation through the up-regulation of Fas and concurrent down-regulation of cyclinD1, survivin, and Bcl-2 expression. In conclusion, our results reveal a novel functional role for IL-35 in suppressing cancer activity, inhibiting cancer cell growth, and increasing the apoptosis sensitivity of human cancer cells through the regulation of genes related to the cell cycle and apoptosis. Thus, this research provides new insights into IL-35 function and presents a possible target for the development of novel cancer therapies.

  3. Overexpression of 12/15-lipoxygenase increases anxiety behavior in female mice.

    PubMed

    Joshi, Yash B; Di Meco, Antonio; Praticò, Domenico

    2014-05-01

    The 12/15-lipoxygenase (12/15-LO) is an enzyme widely distributed in the central nervous system, and it has been involved in the neurobiology of Alzheimer's disease. However, whether this pathway is also involved in neuropsychiatry disorders including anxiety remain to be investigated. In this study we investigated whether genetic over-expression of 12/15-LO (H12/15-LO) modulates some elevated plus maze and Y-maze behaviors. While we observed that H12/15LO mice at the age of 12 months did not differ from wild type in the elevated plus maze paradigm, when they reached the age of 15 months, they manifested an increased anxiety-like behavior compared with controls. By contrast, no differences between the 2 groups at both ages when they were tested for working memory in the Y-maze paradigm. Additionally, we found that the change in anxiety was associated with a reduction in phosphorylation of the transcription factor CREB (cAMP response element-binding protein), and a significant increase in the synaptic protein synaptophysin. Taken together our findings suggest a novel role for 12/15-LO in the pathogenesis of anxiety-like behavior.

  4. Chronic mild stress increases alcohol intake in mice with low dopamine D2 receptor levels.

    PubMed

    Delis, Foteini; Thanos, Panayotis K; Rombola, Christina; Rosko, Lauren; Grandy, David; Wang, Gene-Jack; Volkow, Nora D

    2013-02-01

    Alcohol use disorders emerge from a complex interaction between environmental and genetic factors. Stress and dopamine D2 receptor levels (DRD2) have been shown to play a central role in alcoholism. To better understand the interactions between DRD2 and stress in ethanol intake behavior, we subjected Drd2 wild-type (+/+), heterozygous (+/-), and knockout (-/-) mice to 4 weeks of chronic mild stress (CMS) and to an ethanol two-bottle choice during CMS weeks 2-4. Prior to and at the end of the experiment, the animals were tested in the forced swim and open field tests. We measured ethanol intake and preference, immobility in the force swim test, and activity in the open field. We show that under no CMS, Drd2+/- and Drd2-/- mice had lower ethanol intake and preference compared with Drd2+/+. Exposure to CMS decreased ethanol intake and preference in Drd2+/+ and increased them in Drd2+/- and Drd2-/- mice. At baseline, Drd2+/- and Drd2-/- mice had significantly lower activity in the open field than Drd2+/+, whereas no genotype differences were observed in the forced swim test. Exposure to CMS increased immobility during the forced swim test in Drd2+/- mice, but not in Drd2+/+ or Drd2-/- mice, and ethanol intake reversed this behavior. No changes were observed in open field test measures. These findings suggest that in the presence of a stressful environment, low DRD2 levels are associated with increased ethanol intake and preference and that under this condition, increased ethanol consumption could be used as a strategy to alleviate negative mood. PMID:23148856

  5. Increased dopamine D2 receptor activity in the striatum alters the firing pattern of dopamine neurons in the ventral tegmental area.

    PubMed

    Krabbe, Sabine; Duda, Johanna; Schiemann, Julia; Poetschke, Christina; Schneider, Gaby; Kandel, Eric R; Liss, Birgit; Roeper, Jochen; Simpson, Eleanor H

    2015-03-24

    There is strong evidence that the core deficits of schizophrenia result from dysfunction of the dopamine (DA) system, but details of this dysfunction remain unclear. We previously reported a model of transgenic mice that selectively and reversibly overexpress DA D2 receptors (D2Rs) in the striatum (D2R-OE mice). D2R-OE mice display deficits in cognition and motivation that are strikingly similar to the deficits in cognition and motivation observed in patients with schizophrenia. Here, we show that in vivo, both the firing rate (tonic activity) and burst firing (phasic activity) of identified midbrain DA neurons are impaired in the ventral tegmental area (VTA), but not in the substantia nigra (SN), of D2R-OE mice. Normalizing striatal D2R activity by switching off the transgene in adulthood recovered the reduction in tonic activity of VTA DA neurons, which is concordant with the rescue in motivation that we previously reported in our model. On the other hand, the reduction in burst activity was not rescued, which may be reflected in the observed persistence of cognitive deficits in D2R-OE mice. We have identified a potential molecular mechanism for the altered activity of DA VTA neurons in D2R-OE mice: a reduction in the expression of distinct NMDA receptor subunits selectively in identified mesolimbic DA VTA, but not nigrostriatal DA SN, neurons. These results suggest that functional deficits relevant for schizophrenia symptoms may involve differential regulation of selective DA pathways. PMID:25675529

  6. Increased dopamine transporter function as a mechanism for dopamine hypoactivity in the adult infralimbic medial prefrontal cortex following adolescent social stress.

    PubMed

    Novick, Andrew M; Forster, Gina L; Hassell, James E; Davies, Daniel R; Scholl, Jamie L; Renner, Kenneth J; Watt, Michael J

    2015-10-01

    Being bullied during adolescence is associated with later mental illnesses characterized by deficits in cognitive tasks mediated by prefrontal cortex (PFC) dopamine (DA). Social defeat of adolescent male rats, as a model of teenage bullying victimization, results in medial PFC (mPFC) dopamine (DA) hypofunction in adulthood that is associated with increased drug seeking and working memory deficits. Increased expression of the DA transporter (DAT) is also seen in the adult infralimbic mPFC following adolescent defeat. We propose the functional consequence of this increased DAT expression is enhanced DA clearance and subsequently decreased infralimbic mPFC DA availability. To test this, in vivo chronoamperometry was used to measure changes in accumulation of the DA signal following DAT blockade, with increased DAT-mediated clearance being reflected by lower DA signal accumulation. Previously defeated rats and controls were pre-treated with the norepinephrine transporter (NET) inhibitor desipramine (20 mg/kg, ip.) to isolate infralimbic mPFC DA clearance to DAT, then administered the selective DAT inhibitor GBR-12909 (20 or 40 mg/kg, sc.). Sole NET inhibition with desipramine produced no differences in DA signal accumulation between defeated rats and controls. However, rats exposed to adolescent social defeat demonstrated decreased DA signal accumulation compared to controls in response to both doses of GBR-12909, indicating greater DAT-mediated clearance of infralimbic mPFC DA. These results suggest that protracted increases in infralimbic mPFC DAT function represent a mechanism by which adolescent social defeat stress produces deficits in adult mPFC DA activity and corresponding behavioral and cognitive dysfunction.

  7. Norepinephrine and dopamine increase motility, biofilm formation, and virulence of Vibrio harveyi

    PubMed Central

    Yang, Qian; Anh, Nguyen D. Q.; Bossier, Peter; Defoirdt, Tom

    2014-01-01

    Vibrio harveyi is one of the major pathogens of aquatic organisms, affecting both vertebrates and invertebrates, and causes important losses in the aquaculture industry. In order to develop novel methods to control disease caused by this pathogen, we need to obtain a better understanding of pathogenicity mechanisms. Sensing of catecholamines increases both growth and production of virulence-related factors in pathogens of terrestrial animals and humans. However, at this moment, knowledge on the impact of catecholamines on the virulence of pathogens of aquatic organisms is lacking. In the present study, we report that in V. harveyi, norepinephrine (NE) and dopamine (Dopa) increased growth in serum-supplemented medium, siderophore production, swimming motility, and expression of genes involved in flagellar motility, biofilm formation, and exopolysaccharide production. Consistent with this, pretreatment of V. harveyi with catecholamines prior to inoculation into the rearing water resulted in significantly decreased survival of gnotobiotic brine shrimp larvae, when compared to larvae challenged with untreated V. harveyi. Further, NE-induced effects could be neutralized by α-adrenergic antagonists or by the bacterial catecholamine receptor antagonist LED209, but not by β-adrenergic or dopaminergic antagonists. Dopa-induced effects could be neutralized by dopaminergic antagonists or LED209, but not by adrenergic antagonists. Together, our results indicate that catecholamine sensing increases the success of transmission of V. harveyi and that interfering with catecholamine sensing might be an interesting strategy to control vibriosis in aquaculture. We hypothesize that upon tissue and/or hemocyte damage during infection, pathogens come into contact with elevated catecholamine levels, and that this stimulates the expression of virulence factors that are required to colonize a new host. PMID:25414697

  8. Overexpression of CREB in the nucleus accumbens shell increases cocaine reinforcement in self-administering rats.

    PubMed

    Larson, Erin B; Graham, Danielle L; Arzaga, Rose R; Buzin, Nicole; Webb, Joseph; Green, Thomas A; Bass, Caroline E; Neve, Rachael L; Terwilliger, Ernest F; Nestler, Eric J; Self, David W

    2011-11-01

    Chronic exposure to addictive drugs enhances cAMP response element binding protein (CREB)-regulated gene expression in nucleus accumbens (NAc), and these effects are thought to reduce the positive hedonic effects of passive cocaine administration. Here, we used viral-mediated gene transfer to produce short- and long-term regulation of CREB activity in NAc shell of rats engaging in volitional cocaine self-administration. Increasing CREB expression in NAc shell markedly enhanced cocaine reinforcement of self-administration behavior, as indicated by leftward (long-term) and upward (short-term) shifts in fixed ratio dose-response curves. CREB also increased the effort exerted by rats to obtain cocaine on more demanding progressive ratio schedules, an effect highly correlated with viral-induced modulation of BDNF protein in the NAc shell. CREB enhanced cocaine reinforcement when expressed either throughout acquisition of self-administration or when expression was limited to postacquisition tests, indicating a direct effect of CREB independent of reinforcement-related learning. Downregulating endogenous CREB in NAc shell by expressing a short hairpin RNA reduced cocaine reinforcement in similar tests, while overexpression of a dominant-negative CREB(S133A) mutant had no significant effect on cocaine self-administration. Finally, increasing CREB expression after withdrawal from self-administration enhanced cocaine-primed relapse, while reducing CREB levels facilitated extinction of cocaine seeking, but neither altered relapse induced by cocaine cues or footshock stress. Together, these findings indicate that CREB activity in NAc shell increases the motivation for cocaine during active self-administration or after withdrawal from cocaine. Our results also highlight that volitional and passive drug administration can lead to substantially different behavioral outcomes.

  9. Increasing Dopamine Levels in the Brain Improves Feedback-Based Procedural Learning in Healthy Participants: An Artificial-Grammar-Learning Experiment

    ERIC Educational Resources Information Center

    de Vries, Meinou H.; Ulte, Catrin; Zwitserlood, Pienie; Szymanski, Barbara; Knecht, Stefan

    2010-01-01

    Recently, an increasing number of studies have suggested a role for the basal ganglia and related dopamine inputs in procedural learning, specifically when learning occurs through trial-by-trial feedback (Shohamy, Myers, Kalanithi, & Gluck. (2008). "Basal ganglia and dopamine contributions to probabilistic category learning." "Neuroscience and…

  10. Overexpression of UDP-glucose pyrophosphorylase gene could increase cellulose content in Jute (Corchorus capsularis L.).

    PubMed

    Zhang, Gaoyang; Qi, Jianmin; Xu, Jiantang; Niu, Xiaoping; Zhang, Yujia; Tao, Aifen; Zhang, Liwu; Fang, Pingping; Lin, Lihui

    2013-12-13

    In this study, the full-length cDNA of the UDP-glucose pyrophosphorylase gene was isolated from jute by homologous cloning (primers were designed according to the sequence of UGPase gene of other plants) and modified RACE techniques; the cloned gene was designated CcUGPase. Using bioinformatic analysis, the gene was identified as a member of the UGPase gene family. Real-time PCR analysis revealed differential spatial and temporal expression of the CcUGPase gene, with the highest expression levels at 40 and 120d. PCR and Southern hybridization results indicate that the gene was integrated into the jute genome. Overexpression of CcUGPase gene in jute revealed increased height and cellulose content compared with control lines, although the lignin content remained unchanged. The results indicate that the jute UGPase gene participates in cellulose biosynthesis. These data provide an important basis for the application of the CcUGPase gene in the improvement of jute fiber quality.

  11. In1-ghrelin splicing variant is overexpressed in pituitary adenomas and increases their aggressive features.

    PubMed

    Ibáñez-Costa, Alejandro; Gahete, Manuel D; Rivero-Cortés, Esther; Rincón-Fernández, David; Nelson, Richard; Beltrán, Manuel; de la Riva, Andrés; Japón, Miguel A; Venegas-Moreno, Eva; Gálvez, Ma Ángeles; García-Arnés, Juan A; Soto-Moreno, Alfonso; Morgan, Jennifer; Tsomaia, Natia; Culler, Michael D; Dieguez, Carlos; Castaño, Justo P; Luque, Raúl M

    2015-03-04

    Pituitary adenomas comprise a heterogeneous subset of pathologies causing serious comorbidities, which would benefit from identification of novel, common molecular/cellular biomarkers and therapeutic targets. The ghrelin system has been linked to development of certain endocrine-related cancers. Systematic analysis of the presence and functional implications of some components of the ghrelin system, including native ghrelin, receptors and the recently discovered splicing variant In1-ghrelin, in human normal pituitaries (n = 11) and pituitary adenomas (n = 169) revealed that expression pattern of ghrelin system suffers a clear alteration in pituitary adenomasas compared with normal pituitary, where In1-ghrelin is markedly overexpressed. Interestingly, in cultured pituitary adenoma cells In1-ghrelin treatment (acylated peptides at 100 nM; 24-72 h) increased GH and ACTH secretion, Ca(2+) and ERK1/2 signaling and cell viability, whereas In1-ghrelin silencing (using a specific siRNA; 100 nM) reduced cell viability. These results indicate that an alteration of the ghrelin system, specially its In1-ghrelin variant, could contribute to pathogenesis of different pituitary adenomas types, and suggest that this variant and its related ghrelin system could provide new tools to identify novel, more general diagnostic, prognostic and potential therapeutic targets in pituitary tumors.

  12. Overexpression of UDP-glucose pyrophosphorylase gene could increase cellulose content in Jute (Corchorus capsularis L.).

    PubMed

    Zhang, Gaoyang; Qi, Jianmin; Xu, Jiantang; Niu, Xiaoping; Zhang, Yujia; Tao, Aifen; Zhang, Liwu; Fang, Pingping; Lin, Lihui

    2013-12-13

    In this study, the full-length cDNA of the UDP-glucose pyrophosphorylase gene was isolated from jute by homologous cloning (primers were designed according to the sequence of UGPase gene of other plants) and modified RACE techniques; the cloned gene was designated CcUGPase. Using bioinformatic analysis, the gene was identified as a member of the UGPase gene family. Real-time PCR analysis revealed differential spatial and temporal expression of the CcUGPase gene, with the highest expression levels at 40 and 120d. PCR and Southern hybridization results indicate that the gene was integrated into the jute genome. Overexpression of CcUGPase gene in jute revealed increased height and cellulose content compared with control lines, although the lignin content remained unchanged. The results indicate that the jute UGPase gene participates in cellulose biosynthesis. These data provide an important basis for the application of the CcUGPase gene in the improvement of jute fiber quality. PMID:24269810

  13. In1-ghrelin splicing variant is overexpressed in pituitary adenomas and increases their aggressive features

    PubMed Central

    Ibáñez-Costa, Alejandro; Gahete, Manuel D.; Rivero-Cortés, Esther; Rincón-Fernández, David; Nelson, Richard; Beltrán, Manuel; de la Riva, Andrés; Japón, Miguel A.; Venegas-Moreno, Eva; Gálvez, Ma Ángeles; García-Arnés, Juan A.; Soto-Moreno, Alfonso; Morgan, Jennifer; Tsomaia, Natia; Culler, Michael D.; Dieguez, Carlos; Castaño, Justo P.; Luque, Raúl M.

    2015-01-01

    Pituitary adenomas comprise a heterogeneous subset of pathologies causing serious comorbidities, which would benefit from identification of novel, common molecular/cellular biomarkers and therapeutic targets. The ghrelin system has been linked to development of certain endocrine-related cancers. Systematic analysis of the presence and functional implications of some components of the ghrelin system, including native ghrelin, receptors and the recently discovered splicing variant In1-ghrelin, in human normal pituitaries (n = 11) and pituitary adenomas (n = 169) revealed that expression pattern of ghrelin system suffers a clear alteration in pituitary adenomasas comparedwith normal pituitary, where In1-ghrelin is markedly overexpressed. Interestingly, in cultured pituitary adenoma cells In1-ghrelin treatment (acylated peptides at 100 nM; 24–72 h) increased GH and ACTH secretion, Ca2+ and ERK1/2 signaling and cell viability, whereas In1-ghrelin silencing (using a specific siRNA; 100 nM) reduced cell viability. These results indicate that an alteration of the ghrelin system, specially its In1-ghrelin variant, could contribute to pathogenesis of different pituitary adenomas types, and suggest that this variant and its related ghrelin system could provide new tools to identify novel, more general diagnostic, prognostic and potential therapeutic targets in pituitary tumors. PMID:25737012

  14. Increased consumption of ethanol and sugar water in mice lacking the dopamine D2 long receptor.

    PubMed

    Bulwa, Zachary B; Sharlin, Jordan A; Clark, Peter J; Bhattacharya, Tushar K; Kilby, Chessa N; Wang, Yanyan; Rhodes, Justin S

    2011-11-01

    Individual differences in dopamine D2 receptor (D2R) expression in the brain are thought to influence motivation and reinforcement for ethanol and other rewards. D2R exists in two isoforms, D2 long (D2LR) and D2 short (D2SR), produced by alternative splicing of the same gene. The relative contributions of D2LR versus D2SR to ethanol and sugar water drinking are not known. Genetic engineering was used to produce a line of knockout (KO) mice that lack D2LR and consequently have increased expression of D2SR. KO and wild-type (WT) mice of both sexes were tested for intake of 20% ethanol, 10% sugar water and plain tap water using established drinking-in-the-dark procedures. Mice were also tested for effects of the D2 antagonist eticlopride on intake of ethanol to determine whether KO responses were caused by lack of D2LR or overrepresentation of D2SR. Locomotor activity on running wheels and in cages without wheels was also measured for comparison. D2L KO mice drank significantly more ethanol than WT in both sexes. KO mice drank more sugar water than WT in females but not in males. Eticlopride dose dependently decreased ethanol intake in all groups except male KO. KO mice were less physically active than WT in cages with or without running wheels. Results suggest that overrepresentation of D2SR contributes to increased intake of ethanol in the KO mice. Decreasing wheel running and general levels of physical activity in the KO mice rules out the possibility that higher intake results from higher motor activity. Results extend the literature implicating altered expression of D2R in risk for addiction by delineating the contribution of individual D2R isoforms. These findings suggest that D2LR and D2SR play differential roles in consumption of alcohol and sugar rewards.

  15. Increased consumption of ethanol and sugar water in mice lacking the dopamine D2 long receptor.

    PubMed

    Bulwa, Zachary B; Sharlin, Jordan A; Clark, Peter J; Bhattacharya, Tushar K; Kilby, Chessa N; Wang, Yanyan; Rhodes, Justin S

    2011-11-01

    Individual differences in dopamine D2 receptor (D2R) expression in the brain are thought to influence motivation and reinforcement for ethanol and other rewards. D2R exists in two isoforms, D2 long (D2LR) and D2 short (D2SR), produced by alternative splicing of the same gene. The relative contributions of D2LR versus D2SR to ethanol and sugar water drinking are not known. Genetic engineering was used to produce a line of knockout (KO) mice that lack D2LR and consequently have increased expression of D2SR. KO and wild-type (WT) mice of both sexes were tested for intake of 20% ethanol, 10% sugar water and plain tap water using established drinking-in-the-dark procedures. Mice were also tested for effects of the D2 antagonist eticlopride on intake of ethanol to determine whether KO responses were caused by lack of D2LR or overrepresentation of D2SR. Locomotor activity on running wheels and in cages without wheels was also measured for comparison. D2L KO mice drank significantly more ethanol than WT in both sexes. KO mice drank more sugar water than WT in females but not in males. Eticlopride dose dependently decreased ethanol intake in all groups except male KO. KO mice were less physically active than WT in cages with or without running wheels. Results suggest that overrepresentation of D2SR contributes to increased intake of ethanol in the KO mice. Decreasing wheel running and general levels of physical activity in the KO mice rules out the possibility that higher intake results from higher motor activity. Results extend the literature implicating altered expression of D2R in risk for addiction by delineating the contribution of individual D2R isoforms. These findings suggest that D2LR and D2SR play differential roles in consumption of alcohol and sugar rewards. PMID:21803530

  16. Overexpression of TFAM or Twinkle Increases mtDNA Copy Number and Facilitates Cardioprotection Associated with Limited Mitochondrial Oxidative Stress

    PubMed Central

    Ikeda, Masataka; Ide, Tomomi; Fujino, Takeo; Arai, Shinobu; Saku, Keita; Kakino, Takamori; Tyynismaa, Henna; Yamasaki, Toshihide; Yamada, Ken-ichi; Kang, Dongchon; Suomalainen, Anu; Sunagawa, Kenji

    2015-01-01

    Background Mitochondrial DNA (mtDNA) copy number decreases in animal and human heart failure (HF), yet its role in cardiomyocytes remains to be elucidated. Thus, we investigated the cardioprotective function of increased mtDNA copy number resulting from the overexpression of human transcription factor A of mitochondria (TFAM) or Twinkle helicase in volume overload (VO)-induced HF. Methods and Results Two strains of transgenic (TG) mice, one overexpressing TFAM and the other overexpressing Twinkle helicase, exhibit an approximately 2-fold equivalent increase in mtDNA copy number in heart. These TG mice display similar attenuations in eccentric hypertrophy and improved cardiac function compared to wild-type (WT) mice without any deterioration of mitochondrial enzymatic activities in response to VO, which was accompanied by a reduction in matrix-metalloproteinase (MMP) activity and reactive oxygen species after 8 weeks of VO. Moreover, acute VO-induced MMP-2 and MMP-9 upregulation was also suppressed at 24 h in both TG mice. In isolated rat cardiomyocytes, mitochondrial reactive oxygen species (mitoROS) upregulated MMP-2 and MMP-9 expression, and human TFAM (hTFAM) overexpression suppressed mitoROS and their upregulation. Additionally, mitoROS were equally suppressed in H9c2 rat cardiomyoblasts that overexpress hTFAM or rat Twinkle, both of which exhibit increased mtDNA copy number. Furthermore, mitoROS and mitochondrial protein oxidation from both TG mice were suppressed compared to WT mice. Conclusions The overexpression of TFAM or Twinkle results in increased mtDNA copy number and facilitates cardioprotection associated with limited mitochondrial oxidative stress. Our findings suggest that increasing mtDNA copy number could be a useful therapeutic strategy to target mitoROS in HF. PMID:25822152

  17. sCD44 overexpression increases intraocular pressure and aqueous outflow resistance

    PubMed Central

    Giovingo, Michael; Nolan, Michael; McCarty, Ryan; Pang, Iok-Hou; Clark, Abbot F.; Beverley, Rachel M.; Schwartz, Steven; Stamer, W. Daniel; Walker, Loyal; Grybauskas, Algis; Skuran, Kevin; Kuprys, Paulius V.; Yue, Beatrice Y.J.T.

    2013-01-01

    Purpose CD44 plays major roles in multiple physiologic processes. The ectodomain concentration of the CD44 receptor, soluble CD44 (sCD44), is significantly increased in the aqueous humor of primary open-angle glaucoma (POAG). The purpose of this study was to determine if adenoviral constructs of CD44 and isolated 32-kDa sCD44 change intraocular pressure (IOP) in vivo and aqueous outflow resistance in vitro. Methods Adenoviral constructs of human standard CD44 (Ad-CD44S), soluble CD44 (Ad-sCD44), and empty viral cDNA were injected into the vitreous of BALB/cJ mice, followed by serial IOP measurements. Overexpression of CD44S and sCD44 was verified in vitro by enzyme-linked immunosorbent assay (ELISA) and western blot analysis. Anterior segments of porcine eyes were perfused with the isolated sCD44. sCD44-treated human trabecular meshwork (TM) cells and microdissected porcine TM were examined by confocal microscopy and Optiprep density gradient with western blot analysis to determine changes in lipid raft components. Results Intravitreous injection of adenoviral constructs with either Ad-CD44S or Ad-sCD44 vectors caused prolonged ocular hypertension in mice. Eight days after vector injection, Ad-CD44S significantly elevated IOP to 28.3±1.2 mmHg (mean±SEM, n=8; p<0.001); Ad-sCD44 increased IOP to 18.5±2.6 mmHg (n=8; p<0.01), whereas the IOP of uninjected eyes was 12.7±0.2 mmHg (n=16). The IOP elevation lasted more than 50 days. Topical administration of a γ-secretase inhibitor normalized Ad-sCD44-induced elevated IOP. sCD44 levels were significantly elevated in the aqueous humor of Ad-CD44S and Ad-sCD44 eyes versus contralateral uninjected eyes (p<0.01). Anterior segment perfusion of isolated 32-kDa sCD44 significantly decreased aqueous outflow rates. Co-administration of isolated sCD44 and CD44 neutralizing antibody or of γ-secretase inhibitor significantly enhanced flow rates. sCD44-treated human TM cells displayed cross-linked actin network formation

  18. Loss of Dopamine D2 Receptors Increases Parvalbumin-Positive Interneurons in the Anterior Cingulate Cortex

    PubMed Central

    2015-01-01

    Disruption to dopamine homeostasis during brain development has been implicated in a variety of neuropsychiatric disorders, including depression and schizophrenia. Inappropriate expression or activity of GABAergic interneurons are common features of many of these disorders. We discovered a persistent upregulation of GAD67+ and parvalbumin+ neurons within the anterior cingulate cortex of dopamine D2 receptor knockout mice, while other GABAergic interneuron markers were unaffected. Interneuron distribution and number were not altered in the striatum or in the dopamine-poor somatosensory cortex. The changes were already present by postnatal day 14, indicating a developmental etiology. D2eGFP BAC transgenic mice demonstrated the presence of D2 receptor expression within a subset of parvalbumin-expressing cortical interneurons, suggesting the possibility of a direct cellular mechanism through which D2 receptor stimulation regulates interneuron differentiation or survival. D2 receptor knockout mice also exhibited decreased depressive-like behavior compared with wild-type controls in the tail suspension test. These data indicate that dopamine signaling modulates interneuron number and emotional behavior and that developmental D2 receptor loss or blockade could reveal a potential mechanism for the prodromal basis of neuropsychiatric disorders. PMID:25393953

  19. Increased sensitivity of estrogen receptor alpha overexpressing antral follicles to methoxychlor and its metabolites.

    PubMed

    Paulose, Tessie; Hernández-Ochoa, Isabel; Basavarajappa, Mallikarjuna S; Peretz, Jackye; Flaws, Jodi A

    2011-04-01

    Methoxychlor (MXC), an organochlorine pesticide, and its metabolites, mono-hydroxy MXC (MOH) and bis-hydroxy MXC (HPTE) are known ovarian toxicants and can cause inhibition of antral follicle growth. Since these chemicals bind to estrogen receptor alpha (ESR1), we hypothesized that ovaries overexpressing ESR1 (ESR1 OE) would be more susceptible to toxicity induced by MXC and its metabolites because the chemicals can bind to more ESR1 in the antral follicles. We cultured antral follicles from controls and ESR1 OE mouse ovaries with either the vehicle dimethylsulfoxide (DMSO), MXC, MOH, or HPTE. The data show that at 96 h, the cultured antral follicles from ESR1 OE antral follicles are more susceptible to toxicity induced by MXC, MOH, and HPTE because low doses of these chemicals cause follicle growth inhibition in ESR1 OE mice but not in control mice. On comparing gene expression levels of nuclear receptors in the cultured antral follicles of ESR1 OE and control follicles, we found differential messenger RNA (mRNA) expression of Esr1, estrogen receptor beta (Esr2), androgen receptor (Ar), progesterone receptor (Pr), and aryl hydrocarbon receptor (Ahr) between the genotypes. We also analyzed mRNA levels of Cyp3a41a, the enzyme metabolizing MOH and HPTE, in the cultured follicles and found that Cyp3a41a was significantly lower in DMSO-treated ESR1 OE follicles compared with controls. In ESR1 OE livers, we found that Cyp3a41a levels were significantly lower compared with control livers. Collectively, these data suggest that MXC and its metabolites cause differential gene expression in ESR1 OE mice compared with controls. The results also suggest that the increased sensitivity of ESR1 OE mouse ovaries to toxicity induced by MXC and its metabolites is due to low clearance of the metabolites by the liver and ovary.

  20. BH4 treatment in BH4-responsive PKU patients: preliminary data on blood prolactin concentrations suggest increased cerebral dopamine concentrations.

    PubMed

    van Vliet, Danique; Anjema, Karen; Jahja, Rianne; de Groot, Martijn J; Liemburg, Geertje B; Heiner-Fokkema, M Rebecca; van der Zee, Eddy A; Derks, Terry G J; Kema, Ido P; van Spronsen, Francjan J

    2015-01-01

    In phenylketonuria (PKU), cerebral neurotransmitter deficiencies have been suggested to contribute to brain dysfunction. Present treatment aims to reduce blood phenylalanine concentrations by a phenylalanine-restricted diet, while in some patients blood phenylalanine concentrations also respond to cofactor treatment with tetrahydrobiopterin (BH4). Recently, a repurposing approach of BH4 was suggested to increase cerebral neurotransmitter synthesis. To investigate whether BH4 may improve cerebral dopamine concentrations in PKU patients beyond its effect through lowering blood phenylalanine concentrations, we investigated blood prolactin concentrations-as a parameter of brain dopamine availability. We retrospectively compared blood prolactin in relation to blood phenylalanine concentrations of nine (male) BH4-responsive PKU patients, when being treated without and with BH4. Blood prolactin concentrations positively correlated to blood phenylalanine concentrations (p=0.002), being significantly lower with than without BH4 treatment (p=0.047). In addition, even in this small number of male patients, blood prolactin concentrations tended to be lower at increasing BH4 dose (p=0.054), while taking blood phenylalanine concentrations into account (p=0.002). In individual BH4-responsive patients, median blood prolactin concentrations were significantly lower while using BH4 than before using BH4 treatment (p=0.024), whereas median blood phenylalanine concentrations tended to be lower, but this did not reach statistical significance (p=0.107). Therefore, these data show that high blood phenylalanine in BH4-responsive PKU male patients seems to be associated with increased blood prolactin concentrations, suggesting reduced cerebral dopamine availability. Moreover, these data suggest that BH4 treatment in itself could decrease blood prolactin concentrations in a dose-responsive way, independent of blood phenylalanine concentrations. We conclude that these preliminary data

  1. Overexpression of {alpha}-catenin increases osteoblastic differentiation in mouse mesenchymal C3H10T1/2 cells

    SciTech Connect

    Kim, Dohee; Yang, Jae-Yeon; Shin, Chan Soo

    2009-05-15

    {alpha}- and {beta}-Catenin link cadherins to the actin-based cytoskeleton at adherens junctions and regulate cell-cell adhesion. Although roles of cadherins and canonical Wnt-/{beta}-catenin-signaling in osteoblastic differentiation have been extensively studied, the role of {alpha}-catenin is not known. Murine embryonic mesenchymal stem cells, C3H10T1/2 cells, were transduced with retrovirus encoding {alpha}-catenin (MSCV-{alpha}-catenin-HA-GFP). In the presence of Wnt-3A conditioned medium or osteogenic medium ({beta}-glycerol phosphate and ascorbic acid), cells overexpressing {alpha}-catenin showed enhanced osteoblastic differentiation as measured by alkaline phosphatase (ALP) staining and ALP activity assay compared to cells transduced with empty virus (MSCV-GFP). In addition, mRNA expression of osteocalcin and Runx2 was significantly increased compared to control. Cell aggregation assay revealed that {alpha}-catenin overexpression has significantly increased cell-cell aggregation. However, cellular {beta}-catenin levels (total, cytoplasmic-nuclear ratio) and {beta}-catenin-TCF/LEF transcriptional activity did not change by overexpression of {alpha}-catenin. Knock-down of {alpha}-catenin using siRNA decreased osteoblastic differentiation as measured by ALP assay. These results suggest that {alpha}-catenin overexpression increases osteoblastic differentiation by increasing cell-cell adhesion rather than Wnt-/{beta}-catenin-signaling.

  2. 5-(2-Aminopropyl)benzofuran and phenazepam demonstrate the possibility of dependence by increasing dopamine levels in the brain.

    PubMed

    Cha, Hye Jin; Lee, Kwang-Wook; Eom, Jang-Hyeon; Kim, Young-Hoon; Shin, Jisoon; Yun, Jaesuk; Han, Kyoungmoon; Kim, Hyung Soo

    2016-10-01

    Although 5-(2-aminopropyl)benzofuran (5-APB) and 7-bromo-5-(2-chlorophenyl)-1,3-dihydro-2H-1,4-benzodiazepin-2-one (phenazepam) are being used as recreational drugs, research on their dependence liability or mechanisms of action is lacking. The present study aimed to evaluate the behavioral effects and dependence liability of these drugs using conditioned place preference and self-administration paradigms in rodents. Additionally, biochemical techniques were used to assess the substance-induced alterations in synaptosome-released dopamine. While both of the tested substances elicited increases in conditioned place preference and dopamine, neither of them facilitated self-administration, suggesting that 5-APB and phenazepam have rewarding effects, rather than reinforcing effects. PMID:27502147

  3. Working Memory Deficits, Increased Anxiety-Like Traits, and Seizure Susceptibility in BDNF Overexpressing Mice

    ERIC Educational Resources Information Center

    Papaleo, Francesco; Silverman, Jill L.; Aney, Jordan; Tian, Qingjun; Barkan, Charlotte L.; Chadman, Kathryn K.; Crawley, Jacqueline N.

    2011-01-01

    BDNF regulates components of cognitive processes and has been implicated in psychiatric disorders. Here we report that genetic overexpression of the BDNF mature isoform (BDNF-tg) in female mice impaired working memory functions while sparing components of fear conditioning. BDNF-tg mice also displayed reduced breeding efficiency, higher…

  4. Chronic social defeat stress increases dopamine D2 receptor dimerization in the prefrontal cortex of adult mice.

    PubMed

    Bagalkot, T R; Jin, H-M; Prabhu, V V; Muna, S S; Cui, Y; Yadav, B K; Chae, H-J; Chung, Y-C

    2015-12-17

    The present study aimed to examine the effects of chronic social defeat stress on the dopamine receptors and proteins involved in post-endocytic trafficking pathways. Adult mice were divided into susceptible and unsusceptible groups after 10 days of social defeat stress. Western blot analysis was used to measure the protein expression levels of dopamine D2 receptors (D2Rs), a short (D2S) and a long form (D2L) and, D2R monomers and dimers, dopamine D1 receptors (D1Rs), neuronal calcium sensor-1 (NCS-1) and G protein-coupled receptor-associated sorting protein-1 (GASP-1), and reverse transcription-polymerase chain reaction (RT-PCR) was used to measure the mRNA expression levels of D2S, D2L, D2R monomers and dimers, and D1Rs in different brain areas. We observed increased expression of D2S, D2L and D2Rs dimers in the prefrontal cortex (PFC) of susceptible and/or unsusceptible mice compared with controls. The only significant findings with regard to mRNA expression levels were lower expression of D2S mRNA in the amygdala (AMYG) of susceptible and unsusceptible mice compared with controls. The present study demonstrated that chronic social defeat stress induced increased expression of D2S, D2L, and D2R dimers in the PFC of susceptible and/or unsusceptible mice. PMID:26484605

  5. Dopamine D2-Receptor-Mediated Increase in Vascular and Endothelial NOS Activity Ameliorates Cerebral Vasospasm After Subarachnoid Hemorrhage In Vitro

    PubMed Central

    Caudell, Danielle N.; Cooper, Matthew; Clark, Joseph F.; Shutter, Lori A.

    2008-01-01

    Introduction Cerebral vasospasm after subarachnoid hemorrhage (SAH) is a serious complication resulting in delayed neurological deficit, increased morbidity, mortality, longer hospital stays, and rehabilitation time. It afflicts approximately 35 per 100,000 Americans per year, and there is currently no effective therapy. We present in vitro data suggesting that increasing intrinsic nitric oxide relaxation pathways in vascular smooth muscle via dopaminergic agonism ameliorates cerebral vasospasm after SAH. Methods Cerebrospinal fluid (CSF) from patients with cerebral vasospasm after SAH (CSFV) was used to induce vasospasm in porcine carotid artery in vitro. Dopamine was added to test its ability to reverse spasm, and specific dopamine receptor antagonists were used to determine which receptor mediated the protection. Immunohistochemical techniques confirmed the presence of dopamine receptor subtypes and the involvement of NOS in the mechanism of dopamine protection. Results Dopamine receptor 1, 2, and 3 subtypes are all present in porcine carotid artery. Dopamine significantly reversed spasm in vitro (67% relaxation), and this relaxation was prevented by Haloperidol, a D2R antagonist (10% relaxation, P < 0.05), but not by D1 or D3-receptor antagonism. Both eNOS and iNOS expression were increased significantly in response to CSFV alone, and this was significantly enhanced by addition of dopamine, and blocked by Haloperidol. Conclusion Cerebral vasospasm is significantly reversed in a functional measure of vasospasm in vitro by dopamine, via a D2R-mediated pathway. The increase in NOS protein seen in both the endothelium and vascular smooth muscle in response to CSFV is enhanced by dopamine, also in a D2R-dependent mechanism. PMID:18807216

  6. Overexpression of the trichodiene synthase gene tri5 increases trichodermin production and antimicrobial activity in Trichoderma brevicompactum.

    PubMed

    Tijerino, Anamariela; Cardoza, R Elena; Moraga, Javier; Malmierca, Mónica G; Vicente, Francisca; Aleu, Josefina; Collado, Isidro G; Gutiérrez, Santiago; Monte, Enrique; Hermosa, Rosa

    2011-03-01

    Trichoderma brevicompactum produces trichodermin, a simple trichothecene-type toxin that shares the first steps of the sesquiterpene biosynthetic pathway with other phytotoxic trichothecenes from Fusarium spp. Trichodiene synthase catalyses the conversion of farnesyl pyrophosphate to trichodiene and it is encoded by the tri5 gene that was cloned and analysed functionally by homologous overexpression in T. brevicompactum. tri5 expression was up-regulated in media with glucose, H(2)O(2) or glycerol. tri5 repression was observed in cultures supplemented with the antioxidants ferulic acid and tyrosol. Acetone extracts of tri5-overexpressing transformants displayed higher antifungal activity than those from the wild-type. Chromatographic and spectroscopic analyses revealed that tri5 overexpression led to an increased production of trichodermin and tyrosol. Agar diffusion assays with these two purified metabolites from the tri5-overexpressing transformant T. brevicompactum Tb41tri5 showed that only trichodermin had antifungal activity against Saccharomyces cerevisiae, Kluyveromyces marxianus, Candida albicans, Candida glabrata, Candida tropicalis and Aspergillus fumigatus, in most cases such activity being higher than that observed for amphotericin B and hygromycin. Our results point to the significant role of tri5 in the production of trichodermin and in the antifungal activity of T. brevicompactum. PMID:21145409

  7. Overexpression of a CYP94 family gene CYP94C2b increases internode length and plant height in rice

    PubMed Central

    Kurotani, Ken-Ich; Hattori, Tsukaho; Takeda, Shin

    2015-01-01

    Plant growth is controlled by intrinsic developmental programmes and environmental cues. Jasmonate (JA) has important roles in both processes, by regulating cell division and differentiation, as well as in defense responses and senescence. We report an increase in rice plant height caused by overexpression of a gene encoding a cytochrome P450 enzyme, CYP94C2b, which promoted deactivation of JA-Ile. The height increase occurred through enhanced elongation of internodes in the absence of concomitant cell elongation, unlike previous findings with coi1 knock-down plants. Thus, modulating JA metabolism can increase the number of elongated cells in an internode. Based on these and previous findings, we discuss the difference in the effects of CYP94C2b overexpression vs. coi1 knock-down. PMID:26251886

  8. Increasing dopamine levels in the brain improves feedback-based procedural learning in healthy participants: an artificial-grammar-learning experiment.

    PubMed

    de Vries, Meinou H; Ulte, Catrin; Zwitserlood, Pienie; Szymanski, Barbara; Knecht, Stefan

    2010-09-01

    Recently, an increasing number of studies have suggested a role for the basal ganglia and related dopamine inputs in procedural learning, specifically when learning occurs through trial-by-trial feedback (Shohamy, Myers, Kalanithi, & Gluck. (2008). Basal ganglia and dopamine contributions to probabilistic category learning. Neuroscience and Biobehavioral Reviews, 32, 219-236). A necessary relationship has however only been demonstrated in patient studies. In the present study, we show for the first time that increasing dopamine levels in the brain improves the gradual acquisition of complex information in healthy participants. We implemented two artificial-grammar-learning tasks, one with and one without performance feedback. Learning was improved after levodopa intake for the feedback-based learning task only, suggesting that dopamine plays a specific role in trial-by-trial feedback-based learning. This provides promising directions for future studies on dopaminergic modulation of cognitive functioning.

  9. Over-expression of cytosolic glutamine synthetase increases photosynthesis and growth at low nitrogen concentrations.

    PubMed

    Fuentes, S I; Allen, D J; Ortiz-Lopez, A; Hernández, G

    2001-05-01

    Nitrogen, which is a major limiting nutrient for plant growth, is assimilated as ammonium by the concerted action of glutamine synthetase (GS) and glutamate synthase (GOGAT). GS catalyses the critical incorporation of inorganic ammonium into the amino acid glutamine. Two types of GS isozymes, located in the cytosol (GS1) and in the chloroplast (GS2) have been identified in plants. Tobacco (Nicotiana tabacum) transformants, over-expressing GS1 driven by the constitutive CaMV 35S promoter were analysed. GS in leaves of GS-5 and GS-8 plants was up-regulated, at the level of RNA and proteins. These transgenic plants had six times higher leaf GS activity than controls. Under optimum nitrogen fertilization conditions there was no effect of GS over-expression on photosynthesis or growth. However, under nitrogen starvation the GS transgenics had c. 70% higher shoot and c. 100% greater root dry weight as well as 50% more leaf area than low nitrogen controls. This was achieved by the maintenance of photosynthesis at rates indistinguishable from plants under high nitrogen, while photosynthesis in control plants was inhibited by 40-50% by nitrogen deprivation. It was demonstrated that manipulation of GS activity has the potential to maintain crop photosynthetic productivity while reducing nitrogen fertilization and the concomitant pollution. PMID:11432923

  10. The catecholamine stress hormones norepinephrine and dopamine increase the virulence of pathogenic Vibrio anguillarum and Vibrio campbellii.

    PubMed

    Pande, Gde Sasmita J; Suong, Nguyen Thao; Bossier, Peter; Defoirdt, Tom

    2014-12-01

    Obtaining a better understanding of mechanisms involved in bacterial infections is of paramount importance for the development of novel agents to control disease caused by (antibiotic resistant) pathogens in aquaculture. In this study, we investigated the impact of catecholamine stress hormones on growth and virulence factor production of pathogenic vibrios (i.e. two Vibrio campbellii strains and two Vibrio anguillarum strains). Both norepinephrine and dopamine (at 100 μM) significantly induced growth in media containing serum. The compounds also increased swimming motility of the tested strains, whereas they had no effect on caseinase, chitinase, and hemolysin activities. Further, antagonists for eukaryotic catecholamine receptors were able to neutralize some of the effects of the catecholamines. Indeed, the dopaminergic receptor antagonist chlorpromazine neutralized the effect of dopamine, and the α-adrenergic receptor antagonists phentolamine and phenoxybenzamine neutralized the effect of norepinephrine, whereas the β-adrenergic receptor antagonist propranolol had limited to no effect. Finally, pretreatment of pathogenic V. campbellii with catecholamines significantly increased its virulence toward giant freshwater prawn larvae. However, the impact of catecholamine receptor antagonists on in vivo virulence was less clear-cut when compared to the in vitro experiments. In summary, our results show that—similar to enteric pathogens—catecholamines also increase the virulence of vibrios that are pathogenic to aquatic organisms by increasing motility and growth in media containing serum.

  11. Over-expression of Trxo1 increases the viability of tobacco BY-2 cells under H2O2 treatment

    PubMed Central

    Ortiz-Espín, Ana; Locato, Vittoria; Camejo, Daymi; Schiermeyer, Andreas; De Gara, Laura; Sevilla, Francisca; Jiménez, Ana

    2015-01-01

    Background and Aims Reactive oxygen species (ROS), especially hydrogen peroxide, play a critical role in the regulation of plant development and in the induction of plant defence responses during stress adaptation, as well as in plant cell death. The antioxidant system is responsible for controlling ROS levels in these processes but redox homeostasis is also a key factor in plant cell metabolism under normal and stress situations. Thioredoxins (Trxs) are ubiquitous small proteins found in different cell compartments, including mitochondria and nuclei (Trxo1), and are involved in the regulation of target proteins through reduction of disulphide bonds, although their role under oxidative stress has been less well studied. This study describes over-expression of a Trxo1 for the first time, using a cell-culture model subjected to an oxidative treatment provoked by H2O2. Methods Control and over-expressing PsTrxo1 tobacco (Nicotiana tabacum) BY-2 cells were treated with 35 mm H2O2 and the effects were analysed by studying the growth dynamics of the cultures together with oxidative stress parameters, as well as several components of the antioxidant systems involved in the metabolism of H2O2. Analysis of different hallmarks of programmed cell death was also carried out. Key Results Over-expression of PsTrxo1 caused significant differences in the response of TBY-2 cells to high concentrations of H2O2, namely higher and maintained viability in over-expressing cells, whilst the control line presented a severe decrease in viability and marked indications of oxidative stress, with generalized cell death after 3 d of treatment. In over-expressing cells, an increase in catalase activity, decreases in H2O2 and nitric oxide contents and maintenance of the glutathione redox state were observed. Conclusions A decreased content of endogenous H2O2 may be responsible in part for the delayed cell death found in over-expressing cells, in which changes in oxidative parameters and

  12. Nicotine enhancement of dopamine release by a calcium-dependent increase in the size of the readily releasable pool of synaptic vesicles.

    PubMed

    Turner, Timothy J

    2004-12-15

    A major factor underlying compulsive tobacco use is nicotine-induced modulation of dopamine release in the mesolimbic reward pathway (Wise and Rompre, 1989). An established biochemical mechanism for nicotine-enhanced dopamine release is by activating presynaptic nicotinic acetylcholine receptors (nAChRs) (Wonnacott, 1997). Prolonged application of 10(-7) to 10(-5) m nicotine to striatal synaptosomes promoted a sustained efflux of [3H]dopamine. This nicotine effect was mediated by non-alpha7 nAChRs, because it was blocked by 5 mum mecamylamine but was resistant to 100 nm alpha-bungarotoxin (alphaBgTx). Dopamine release was diminished by omitting Na+ or by applying peptide calcium channel blockers, indicating that nAChRs trigger release by depolarizing the nerve terminals. However, because alpha7 receptors rapidly desensitize in the continuous presence of agonists, a repetitive stimulation protocol was used to evaluate the possible significance of desensitization. This protocol produced a transient increase in [3H]dopamine released by depolarization and a significant increase in the response to hypertonic solutions that measure the size of the readily releasable pool (RRP) of synaptic vesicles. The nicotine-induced increase in the size of the readily releasable pool was blocked by alphaBgTx and by the calmodulin antagonist calmidazolium, suggesting that Ca2+ entry through alpha7 nAChRs specifically enhances synaptic vesicle mobilization at dopamine terminals. Thus, nicotine enhances dopamine release by two complementary actions mediated by discrete nAChR subtypes and suggest that the alpha7 nAChR-mediated pathway is tightly and specifically coupled to refilling of the RRP of vesicles in dopamine terminals.

  13. Exposure to nicotine increases dopamine receptor content in the mesocorticolimbic pathway of rat dams and offspring during lactation.

    PubMed

    Pinheiro, C R; Oliveira, E; Manhães, A C; Fraga, M C; Claudio-Neto, S; Younes-Rapozo, V; Lotufo, B M; Moura, E G; Lisboa, P C

    2015-09-01

    Nicotine exposure causes the release of dopamine from the ventral tegmental area (VTA) to the nucleus accumbens (NAc). We have previously shown that maternal exposure to nicotine during lactation causes hyperleptinemia in dams and pups, and leptin is known to decrease dopamine release from the VTA. Here we evaluated whether maternal exposure to nicotine during lactation causes changes in dopamine and leptin signaling pathways at the end of exposure and after 5days of withdrawal in the: VTA, NAc, arcuate nucleus (ARC) and dorsal striatum (DS). On postnatal day (PN) 2, lactating Wistar rats were implanted with minipumps releasing nicotine (NIC; 6mg/kg/day, s.c.) or saline (C) for 14days. Offspring were tested in the elevated plus maze (EPM) and open field on PN14 or PN20, and euthanized on PN15 or PN21. Entries into the open arms and head dips in the EPM were reduced in NIC pups at P20. At weaning (PN21), NIC dams had: lower tyrosine hydroxylase (TH), higher OBRb and SOCS3 contents in VTA; lower TH, higher D1R, D2R and DAT contents in NAc; higher TH content in DS; and higher D2R and SOCS3 contents in ARC. On PN15, NIC offspring had higher D1R, D2R and lower DAT contents in NAc, while on PN21, they had lower DAT in DS, and lower pSTAT3 content in ARC. We evidenced that postnatal nicotine exposure induces relevant changes in the brain reward system of dams and pups, possibly associated with changes in leptinemia and increased offspring anxiety-like behavior.

  14. Amphetamine induced dopamine release increases anxiety in individuals recovered from anorexia nervosa

    PubMed Central

    Bailer, Ursula F.; Narendran, Rajesh; Frankle, W. Gordon; Himes, Michael L; Duvvuri, Vikas; Mathis, Chester A; Kaye, W.H.

    2011-01-01

    Objective Genetic, pharmacologic, and physiological data suggest that individuals with anorexia nervosa (AN) have altered striatal dopamine (DA) function. Method We used an amphetamine challenge and positron emission tomography [11C]raclopride paradigm to explore DA striatal transmission in 10 recovered (REC) AN compared to 9 control women (CW). Results REC AN and CW were similar for baseline, post-amphetamine [11C]raclopride binding potential (BPND) and change (Δ) in BPND for all regions. In CW, ventral striatum Δ BPND was associated with euphoria (r = − .76; p = .03), which was not found for REC AN. Instead, REC AN showed a significant relationship between anxiety and Δ BPND in the pre-commissural dorsal caudate (r = −.62, p = .05). Discussion REC AN have a positive association between endogenous DA release and anxiety in the dorsal caudate. This finding could explain why food-related DA release produces anxiety in AN, whereas feeding is pleasurable in healthy participants. PMID:21541980

  15. Overexpression of the laeA gene leads to increased production of cyclopiazonic acid in Aspergillus fumisynnematus.

    PubMed

    Hong, Eun Jin; Kim, Na Kyeong; Lee, Doyup; Kim, Won Gon; Lee, Inhyung

    2015-11-01

    To explore novel bioactive compounds produced via activation of secondary metabolite (SM) gene clusters, we overexpressed an ortholog of laeA, a gene that encodes a global positive regulator of secondary metabolism in Aspergillus fumisynnematus F746. Overexpression of the laeA gene under the alcA promoter resulted in the production of less pigment, shorter conidial head chains, and fewer conidia. Thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) analysis revealed that SM production in OE::laeA was significantly increased, and included new metabolites that were not detected in the wild type. Among them, a compound named F1 was selected on the basis of its high production levels and antibacterial effects. F1 was purified by column chromatography and preparative TLC and identified as cyclopiazonic acid (CPA) by LC/MS, which had been previously known as mycotoxin. As A. fumisynnematus was not known to produce CPA, these results suggest that overexpression of the laeA gene can be used to explore the synthesis of useful bioactive compounds, even in a fungus for which the genome sequence is unavailable.

  16. Over-expression of a novel JAZ family gene from Glycine soja, increases salt and alkali stress tolerance

    SciTech Connect

    Zhu, Dan; Cai, Hua; Luo, Xiao; Bai, Xi; Deyholos, Michael K.; Chen, Qin; Chen, Chao; Ji, Wei; Zhu, Yanming

    2012-09-21

    Highlights: Black-Right-Pointing-Pointer We isolated and characterized a novel JAZ family gene, GsJAZ2, from Glycine soja. Black-Right-Pointing-Pointer Overexpression of GsJAZ2 enhanced plant tolerance to salt and alkali stress. Black-Right-Pointing-Pointer The transcriptions of stress marker genes were higher in GsJAZ2 overexpression lines. Black-Right-Pointing-Pointer GsJAZ2 was localized to nucleus. -- Abstract: Salt and alkali stress are two of the main environmental factors limiting crop production. Recent discoveries show that the JAZ family encodes plant-specific genes involved in jasmonate signaling. However, there is only limited information about this gene family in abiotic stress response, and in wild soybean (Glycine soja), which is a species noted for its tolerance to alkali and salinity. Here, we isolated and characterized a novel JAZ family gene, GsJAZ2, from G. soja. Transcript abundance of GsJAZ2 increased following exposure to salt, alkali, cold and drought. Over-expression of GsJAZ2 in Arabidopsis resulted in enhanced plant tolerance to salt and alkali stress. The expression levels of some alkali stress response and stress-inducible marker genes were significantly higher in the GsJAZ2 overexpression lines as compared to wild-type plants. Subcellular localization studies using a GFP fusion protein showed that GsJAZ2 was localized to the nucleus. These results suggest that the newly isolated wild soybean GsJAZ2 is a positive regulator of plant salt and alkali stress tolerance.

  17. Overexpression of SOD in Retina; Need for Increase in H202-Detoxifying Enzyme in Same Cellular Compartment

    PubMed Central

    Usui, Shinichi; Oveson, Brian C; Iwase, Takeshi; Lu, Lili; Lee, Sun Young; Jo, Young-Joon; Wu, Zhihao; Choi, Eun-Young; Samulski, Richard J.; Campochiaro, Peter A

    2011-01-01

    In retinitis pigmentosa (RP), various mutations cause rod photoreceptor cell death leading to increased oxygen levels in the outer retina, progressive oxidative damage to cones, and gradual loss of cone cell function. We have been exploring the potential of overexpressing components of the endogenous antioxidant defense system to preserve cone cell function in rd10+/+ mice, a model of RP. Rd10+/+ mice deficient in superoxide dismutase 1 (SOD1) showed increased levels of superoxide radicals and carbonyl adducts (a marker of oxidative damage) in the retina, and more rapid loss of cone function than rd10+/+ mice with normal levels of SOD1. This suggests that SOD1 is an important component of the antioxidant defense system of cones, but increased expression of SOD1 in rd10+/+ mice increased oxidative damage and accelerated the loss of cone function. Co-expression of SOD1 with glutathione peroxidase 4 (Gpx4), which like SOD1 is localized in the cytoplasm, but not with catalase targeted to the mitochondria, reduced oxidative damage in the retina and significantly slowed the loss of cone cell function in rd10+/+ mice. Gene transfer resulting in increased expression of SOD2, but not co-expression of SOD2 and mitochondrial Gpx4, resulted in high levels of H2O2 in the retina. These data suggest that in order to provide benefit in RP, over-expression of a SOD must be combined with expression of a peroxide detoxifying enzyme in the same cellular compartment. PMID:21736939

  18. Increased tau phosphorylation and aggregation in the hippocampus of mice overexpressing corticotropin-releasing factor.

    PubMed

    Campbell, Shannon N; Zhang, Cheng; Monte, Louise; Roe, Allyson D; Rice, Kenner C; Taché, Yvette; Masliah, Eliezer; Rissman, Robert A

    2015-01-01

    Clinical and basic science research suggests that stress and/or changes in central stress signaling intermediates may be involved in Alzheimer's disease (AD) pathogenesis. Although the links between stress and AD remain unsettled, data from our group and others have established that stress exposure in rodents may confer susceptibility to AD pathology by inducing hippocampal tau phosphorylation (tau-P). Work in our laboratory has shown that stress-induced tau-P requires activation of the type-1 corticotropin-releasing factor receptor (CRFR1). CRF overexpressing (CRF-OE) mice are a model of chronic stress that display cognitive impairment at 9-10 month of age. In this study we used 6-7 month old CRF-OE mice to examine whether sustained exposure to CRF and stress steroids would impact hippocampal tau-P and kinase activity in the presence or absence of the CRFR1-specific antagonist, R121919, given daily for 30 days. CRF-OE mice had significantly elevated tau-P compared to wild type (WT) mice at the AT8 (S202/T204), PHF-1 (S396/404), S262, and S422 sites. Treating CRF-OE mice with R121919 blocked phosphorylation at the AT8 (S202/T204) and PHF-1 (S396/404) sites, but not at the S262 and S422 sites and reduced phosphorylation of c-Jun N Terminal Kinase (JNK). Examination of hippocampal extracts from CRF-OE mice at the ultrastructural level revealed negatively stained round/globular aggregates that were positively labeled by PHF-1. These data suggest critical roles for CRF and CRFR1 in tau-P and aggregation and may have implications for the development of AD cognitive decline.

  19. Differential behavioral reinforcement effects of dopamine receptor agonists in the rat with bilateral lesion of the posterior ventral tegmental area.

    PubMed

    Ouachikh, Omar; Dieb, Wisam; Durif, Franck; Hafidi, Aziz

    2013-09-01

    Dopamine dysregulation syndrome in Parkinson's disease has been attributed to dopamine replacement therapies and/or a lesion of the dopaminergic system. The dopaminergic neuronal loss targets the substantia nigra and the ventral tegmental area (VTA). We hypothesize that dopamine replacement therapy is responsible for the potential reinforcement effect in Parkinson's disease by acting on the neuronal reward circuitry. Therefore this study was designed to explore the potential motivational effect of dopamine replacement therapy in bilateral VTA-lesioned animals. The posterior (p)VTA, which project to the nucleus accumbens (NAc) constitutes the major dopamine neuronal circuitry implicated in addictive disorders. Using the conditioned place preference (CPP) behavioral paradigm, we investigated the motivational effects of dopamine receptor agonists, and cocaine in rat with a 6-OHDA bilateral lesion of the pVTA. Amongst the dopamine receptor agonists used in this study only the D2R and D3R agonists (bromocriptine, PD128907 and pramipexole), induced a significant CPP in pVTA-lesioned animals. Dopamine receptor agonists did not induce behavioral sensitization in sham animals. Moreover, confocal D2R immunostaining analysis showed a significant increase in the number of D2R per cell body in the NAc shell of pVTA lesioned rats compared to sham. This result correlated, for the first time, the dopamine receptor agonists effect with DR2 overexpression in the NAc shell of pVTA-lesioned rats. In addition, cocaine, which is known to increase dopamine release, induced behavioral sensitization in sham group but not in dopamine deprived group. Thus, the later result highlighted the importance of pVTA-NAc dopaminergic pathway in positive reinforcements. Altogether these data suggested that the implication of the dopamine replacement therapy in the appearance of dopamine dysregulation syndrome in Parkinson's disease is probably due to both neuronal degeneration in the posterior VTA and

  20. 3,4-Dihydroxyphenylethanol (Hydroxytyrosol) Mitigates the Increase in Spontaneous Oxidation of Dopamine During Monoamine Oxidase Inhibition in PC12 Cells.

    PubMed

    Goldstein, David S; Jinsmaa, Yunden; Sullivan, Patti; Holmes, Courtney; Kopin, Irwin J; Sharabi, Yehonatan

    2016-09-01

    The catecholaldehyde hypothesis predicts that monoamine oxidase (MAO) inhibition should slow the progression of Parkinson's disease, by decreasing production of the autotoxic dopamine metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL). Inhibiting MAO, however, diverts the fate of cytoplasmic dopamine toward potentially harmful spontaneous oxidation products, indicated by increased 5-S-cysteinyl-dopamine (Cys-DA) levels. 3,4-Dihydroxyphenylethanol (hydroxytyrosol) is an abundant anti-oxidant phenol in constituents of the Mediterranean diet. Whether hydroxytyrosol alters enzymatic or spontaneous oxidation of dopamine has been unknown. Rat pheochromocytoma PC12 cells were incubated with hydroxytyrosol (10 µM, 180 min) alone or with the MAO-A inhibitor clorgyline (1 nM) or the MAO-B inhibitors rasagiline or selegiline (0.5 µM). Hydroxytyrosol decreased levels of DOPAL by 30 % and Cys-DA by 49 % (p < 0.0001 each). Co-incubation with hydroxytyrosol prevented the increases in Cys-DA seen with all 3 MAO inhibitors. Hydroxytyrosol therefore inhibits both enzymatic and spontaneous oxidation of endogenous dopamine and mitigates the increase in spontaneous oxidation during MAO inhibition. PMID:27220335

  1. Dopamine and opioid gene variants are associated with increased smoking reward and reinforcement owing to negative mood.

    PubMed

    Perkins, Kenneth A; Lerman, Caryn; Grottenthaler, Amy; Ciccocioppo, Melinda M; Milanak, Melissa; Conklin, Cynthia A; Bergen, Andrew W; Benowitz, Neal L

    2008-09-01

    Negative mood increases smoking reinforcement and risk of relapse. We explored associations of gene variants in the dopamine, opioid, and serotonin pathways with smoking reward ('liking') and reinforcement (latency to first puff and total puffs) as a function of negative mood and expected versus actual nicotine content of the cigarette. Smokers of European ancestry (n=72) were randomized to one of four groups in a 2x2 balanced placebo design, corresponding with manipulation of actual (0.6 vs. 0.05 mg) and expected (told nicotine and told denicotinized) nicotine 'dose' in cigarettes during each of two sessions (negative vs. positive mood induction). Following mood induction and expectancy instructions, they sampled and rated the assigned cigarette, and then smoked additional cigarettes ad lib during continued mood induction. The increase in smoking amount owing to negative mood was associated with: dopamine D2 receptor (DRD2) C957T (CC>TT or CT), SLC6A3 (presence of 9 repeat>absence of 9), and among those given a nicotine cigarette, DRD4 (presence of 7 repeat>absence of 7) and DRD2/ANKK1 TaqIA (TT or CT>CC). SLC6A3, and DRD2/ANKK1 TaqIA were also associated with smoking reward and smoking latency. OPRM1 (AA>AG or GG) was associated with smoking reward, but SLC6A4 variable number tandem repeat was unrelated to any of these measures. These results warrant replication but provide the first evidence for genetic associations with the acute increase in smoking reward and reinforcement owing to negative mood.

  2. Dopamine and opioid gene variants are associated with increased smoking reward and reinforcement owing to negative mood.

    PubMed

    Perkins, Kenneth A; Lerman, Caryn; Grottenthaler, Amy; Ciccocioppo, Melinda M; Milanak, Melissa; Conklin, Cynthia A; Bergen, Andrew W; Benowitz, Neal L

    2008-09-01

    Negative mood increases smoking reinforcement and risk of relapse. We explored associations of gene variants in the dopamine, opioid, and serotonin pathways with smoking reward ('liking') and reinforcement (latency to first puff and total puffs) as a function of negative mood and expected versus actual nicotine content of the cigarette. Smokers of European ancestry (n=72) were randomized to one of four groups in a 2x2 balanced placebo design, corresponding with manipulation of actual (0.6 vs. 0.05 mg) and expected (told nicotine and told denicotinized) nicotine 'dose' in cigarettes during each of two sessions (negative vs. positive mood induction). Following mood induction and expectancy instructions, they sampled and rated the assigned cigarette, and then smoked additional cigarettes ad lib during continued mood induction. The increase in smoking amount owing to negative mood was associated with: dopamine D2 receptor (DRD2) C957T (CC>TT or CT), SLC6A3 (presence of 9 repeat>absence of 9), and among those given a nicotine cigarette, DRD4 (presence of 7 repeat>absence of 7) and DRD2/ANKK1 TaqIA (TT or CT>CC). SLC6A3, and DRD2/ANKK1 TaqIA were also associated with smoking reward and smoking latency. OPRM1 (AA>AG or GG) was associated with smoking reward, but SLC6A4 variable number tandem repeat was unrelated to any of these measures. These results warrant replication but provide the first evidence for genetic associations with the acute increase in smoking reward and reinforcement owing to negative mood. PMID:18690118

  3. The relation between dopamine oxidation currents in the nucleus accumbens and conditioned increases in motor activity in rats following repeated administration of d-amphetamine or cocaine.

    PubMed

    Di Ciano, P; Blaha, C D; Phillips, A G

    1998-03-01

    Chronoamperometric recording techniques were used to monitor extracellular dopamine efflux in the nucleus accumbens associated with unconditioned and conditioned increases in motor activity in rats, following the intravenous administration of either d-amphetamine (0.63 mg/kg) or cocaine (3 mg/kg), or the presentation of a conditioned stimulus paired repeatedly with one of these psychostimulants. Each drug was administered daily for 7 days, either in the home cage or an environment in which a compound stimulus (light offset, odour) was presented. Rats in control groups received saline instead of drug in the distinctive test environment. On day 7 of training, significant increases in unconditioned motor activity were observed in the 30 min session following infusions of either d-amphetamine or cocaine. Associated dopamine oxidation currents in the nucleus accumbens increased immediately following administration of either drug and remained significantly elevated above baseline during the entire 30 min recording period. On the test day, presentation of the conditioned stimulus with vehicle infusions, in the distinct environment, was accompanied by an increase in dopamine oxidation currents and a conditioned increase in motor activity, only in the groups in which these stimuli had been paired with d-amphetamine or cocaine. Neither the magnitude or duration of the conditioned motor activity matched the corresponding change in extracellular dopamine efflux in the nucleus accumbens. Accordingly, it is argued that the increase in dopamine concentration serves as a neurochemical correlate of the unconditioned and conditioned stimuli. The change in motor activity constitutes the unconditioned and conditioned responses that are subserved by the neural systems activated by the initial rise in extracellular dopamine. PMID:9753179

  4. Anosmin-1 over-expression increases adult neurogenesis in the subventricular zone and neuroblast migration to the olfactory bulb.

    PubMed

    García-González, Diego; Murcia-Belmonte, Verónica; Esteban, Pedro F; Ortega, Felipe; Díaz, David; Sánchez-Vera, Irene; Lebrón-Galán, Rafael; Escobar-Castañondo, Laura; Martínez-Millán, Luis; Weruaga, Eduardo; García-Verdugo, José Manuel; Berninger, Benedikt; de Castro, Fernando

    2016-01-01

    New subventricular zone (SVZ)-derived neuroblasts that migrate via the rostral migratory stream are continuously added to the olfactory bulb (OB) of the adult rodent brain. Anosmin-1 (A1) is an extracellular matrix protein that binds to FGF receptor 1 (FGFR1) to exert its biological effects. When mutated as in Kallmann syndrome patients, A1 is associated with severe OB morphogenesis defects leading to anosmia and hypogonadotropic hypogonadism. Here, we show that A1 over-expression in adult mice strongly increases proliferation in the SVZ, mainly with symmetrical divisions, and produces substantial morphological changes in the normal SVZ architecture, where we also report the presence of FGFR1 in almost all SVZ cells. Interestingly, for the first time we show FGFR1 expression in the basal body of primary cilia in neural progenitor cells. Additionally, we have found that A1 over-expression also enhances neuroblast motility, mainly through FGFR1 activity. Together, these changes lead to a selective increase in several GABAergic interneuron populations in different OB layers. These specific alterations in the OB would be sufficient to disrupt the normal processing of sensory information and consequently alter olfactory memory. In summary, this work shows that FGFR1-mediated A1 activity plays a crucial role in the continuous remodelling of the adult OB. PMID:25300351

  5. Overexpression of Striated Muscle Activator of Rho Signaling (STARS) Increases C2C12 Skeletal Muscle Cell Differentiation

    PubMed Central

    Wallace, Marita A.; Della Gatta, Paul A.; Ahmad Mir, Bilal; Kowalski, Greg M.; Kloehn, Joachim; McConville, Malcom J.; Russell, Aaron P.; Lamon, Séverine

    2016-01-01

    Background: Skeletal muscle growth and regeneration depend on the activation of satellite cells, which leads to myocyte proliferation, differentiation and fusion with existing muscle fibers. Skeletal muscle cell proliferation and differentiation are tightly coordinated by a continuum of molecular signaling pathways. The striated muscle activator of Rho signaling (STARS) is an actin binding protein that regulates the transcription of genes involved in muscle cell growth, structure and function via the stimulation of actin polymerization and activation of serum-response factor (SRF) signaling. STARS mediates cell proliferation in smooth and cardiac muscle models; however, whether STARS overexpression enhances cell proliferation and differentiation has not been investigated in skeletal muscle cells. Results: We demonstrate for the first time that STARS overexpression enhances differentiation but not proliferation in C2C12 mouse skeletal muscle cells. Increased differentiation was associated with an increase in the gene levels of the myogenic differentiation markers Ckm, Ckmt2 and Myh4, the differentiation factor Igf2 and the myogenic regulatory factors (MRFs) Myf5 and Myf6. Exposing C2C12 cells to CCG-1423, a pharmacological inhibitor of SRF preventing the nuclear translocation of its co-factor MRTF-A, had no effect on myotube differentiation rate, suggesting that STARS regulates differentiation via a MRTF-A independent mechanism. Conclusion: These findings position STARS as an important regulator of skeletal muscle growth and regeneration. PMID:26903873

  6. Kappa Opioid Receptor Activation Potentiates the Cocaine-Induced Increase in Evoked Dopamine Release Recorded In Vivo in the Mouse Nucleus Accumbens

    PubMed Central

    Ehrich, Jonathan M; Phillips, Paul E M; Chavkin, Charles

    2014-01-01

    Behavioral stressors increase addiction risk in humans and increase the rewarding valence of drugs of abuse including cocaine, nicotine and ethanol in animal models. Prior studies have established that this potentiation of drug reward was mediated by stress-induced release of the endogenous dynorphin opioids and subsequent kappa opioid receptor (KOR) activation. In this study, we used in vivo fast scan cyclic voltammetry to test the hypothesis that KOR activation before cocaine administration might potentiate the evoked release of dopamine from ventral tegmental (VTA) synaptic inputs to the nucleus accumbens (NAc) and thereby increase the rewarding valence of cocaine. The KOR agonist U50488 inhibited dopamine release evoked by either medial forebrain bundle (MFB) or pedunculopontine tegmental nucleus (PPTg) activation of VTA inputs to the shell or core of the mouse NAc. Cocaine administration increased the dopamine response recorded in either the shell or core evoked by either MFB or PPTg stimulation. Administration of U50488 15 min before cocaine blocked the conditioned place preference (CPP) to cocaine, but only significantly reduced the effect of cocaine on the dopamine response evoked by PPTg stimulation to NAc core. In contrast, administration of U50488 60 min before cocaine significantly potentiated cocaine CPP and significantly increased the effects of cocaine on the dopamine response evoked by either MFB or PPTg stimulation, recorded in either NAc shell or core. Results of this study support the concept that stress-induced activation of KOR by endogenous dynorphin opioids may enhance the rewarding valence of drugs of abuse by potentiating the evoked dopamine response. PMID:24971603

  7. Kappa opioid receptor activation potentiates the cocaine-induced increase in evoked dopamine release recorded in vivo in the mouse nucleus accumbens.

    PubMed

    Ehrich, Jonathan M; Phillips, Paul E M; Chavkin, Charles

    2014-12-01

    Behavioral stressors increase addiction risk in humans and increase the rewarding valence of drugs of abuse including cocaine, nicotine and ethanol in animal models. Prior studies have established that this potentiation of drug reward was mediated by stress-induced release of the endogenous dynorphin opioids and subsequent kappa opioid receptor (KOR) activation. In this study, we used in vivo fast scan cyclic voltammetry to test the hypothesis that KOR activation before cocaine administration might potentiate the evoked release of dopamine from ventral tegmental (VTA) synaptic inputs to the nucleus accumbens (NAc) and thereby increase the rewarding valence of cocaine. The KOR agonist U50488 inhibited dopamine release evoked by either medial forebrain bundle (MFB) or pedunculopontine tegmental nucleus (PPTg) activation of VTA inputs to the shell or core of the mouse NAc. Cocaine administration increased the dopamine response recorded in either the shell or core evoked by either MFB or PPTg stimulation. Administration of U50488 15 min before cocaine blocked the conditioned place preference (CPP) to cocaine, but only significantly reduced the effect of cocaine on the dopamine response evoked by PPTg stimulation to NAc core. In contrast, administration of U50488 60 min before cocaine significantly potentiated cocaine CPP and significantly increased the effects of cocaine on the dopamine response evoked by either MFB or PPTg stimulation, recorded in either NAc shell or core. Results of this study support the concept that stress-induced activation of KOR by endogenous dynorphin opioids may enhance the rewarding valence of drugs of abuse by potentiating the evoked dopamine response. PMID:24971603

  8. Overexpression of a phosphatidic acid phosphatase type 2 leads to an increase in triacylglycerol production in oleaginous Rhodococcus strains.

    PubMed

    Hernández, Martín A; Comba, Santiago; Arabolaza, Ana; Gramajo, Hugo; Alvarez, Héctor M

    2015-03-01

    Oleaginous Rhodococcus strains are able to accumulate large amounts of triacylglycerol (TAG). Phosphatidic acid phosphatase (PAP) enzyme catalyzes the dephosphorylation of phosphatidic acid (PA) to yield diacylglycerol (DAG), a key precursor for TAG biosynthesis. Studies to establish its role in lipid metabolism have been mainly focused in eukaryotes but not in bacteria. In this work, we identified and characterized a putative PAP type 2 (PAP2) encoded by the ro00075 gene in Rhodococcus jostii RHA1. Heterologous expression of ro00075 in Escherichia coli resulted in a fourfold increase in PAP activity and twofold in DAG content. The conditional deletion of ro00075 in RHA1 led to a decrease in the content of DAG and TAG, whereas its overexpression in both RHA1 and Rhodococcus opacus PD630 promoted an increase up to 10 to 15 % by cellular dry weight in TAG content. On the other hand, expression of ro00075 in the non-oleaginous strain Rhodococcus fascians F7 promoted an increase in total fatty acid content up to 7 % at the expense of free fatty acid (FFA), DAG, and TAG fractions. Moreover, co-expression of ro00075/atf2 genes resulted in a fourfold increase in total fatty acid content by a further increase of the FFA and TAG fractions. The results of this study suggest that ro00075 encodes for a PAP2 enzyme actively involved in TAG biosynthesis. Overexpression of this gene, as single one or with an atf gene, provides an alternative approach to increase the biosynthesis and accumulation of bacterial oils as a potential source of raw material for biofuel production.

  9. Over-expression of a novel JAZ family gene from Glycine soja, increases salt and alkali stress tolerance.

    PubMed

    Zhu, Dan; Cai, Hua; Luo, Xiao; Bai, Xi; Deyholos, Michael K; Chen, Qin; Chen, Chao; Ji, Wei; Zhu, Yanming

    2012-09-21

    Salt and alkali stress are two of the main environmental factors limiting crop production. Recent discoveries show that the JAZ family encodes plant-specific genes involved in jasmonate signaling. However, there is only limited information about this gene family in abiotic stress response, and in wild soybean (Glycine soja), which is a species noted for its tolerance to alkali and salinity. Here, we isolated and characterized a novel JAZ family gene, GsJAZ2, from G. soja. Transcript abundance of GsJAZ2 increased following exposure to salt, alkali, cold and drought. Over-expression of GsJAZ2 in Arabidopsis resulted in enhanced plant tolerance to salt and alkali stress. The expression levels of some alkali stress response and stress-inducible marker genes were significantly higher in the GsJAZ2 overexpression lines as compared to wild-type plants. Subcellular localization studies using a GFP fusion protein showed that GsJAZ2 was localized to the nucleus. These results suggest that the newly isolated wild soybean GsJAZ2 is a positive regulator of plant salt and alkali stress tolerance.

  10. Peroxisome Proliferator–Activated Receptor-γ Coactivator-1α Overexpression Increases Lipid Oxidation in Myocytes From Extremely Obese Individuals

    PubMed Central

    Consitt, Leslie A.; Bell, Jill A.; Koves, Timothy R.; Muoio, Deborah M.; Hulver, Matthew W.; Haynie, Kimberly R.; Dohm, G. Lynis; Houmard, Joseph A.

    2010-01-01

    OBJECTIVE To determine whether the obesity-related decrement in fatty acid oxidation (FAO) in primary human skeletal muscle cells (HSkMC) is linked with lower mitochondrial content and whether this deficit could be corrected via overexpression of peroxisome proliferator–activated receptor-γ coactivator-1α (PGC-1α). RESEARCH DESIGN AND METHODS FAO was studied in HSkMC from lean (BMI 22.4 ± 0.9 kg/m2; N = 12) and extremely obese (45.3 ± 1.4 kg/m2; N = 9) subjects. Recombinant adenovirus was used to increase HSkMC PGC-1α expression (3.5- and 8.0-fold), followed by assessment of mitochondrial content (mtDNA and cytochrome C oxidase IV [COXIV]), complete (14CO2 production from labeled oleate), and incomplete (acid soluble metabolites [ASM]) FAO, and glycerolipid synthesis. RESULTS Obesity was associated with a 30% decrease (P < 0.05) in complete FAO, which was accompanied by higher relative rates of incomplete FAO ([14C]ASM production/14CO2), increased partitioning of fatty acid toward storage, and lower (P < 0.05) mtDNA (−27%), COXIV (−35%), and mitochondrial transcription factor (mtTFA) (−43%) protein levels. PGC-1α overexpression increased (P < 0.05) FAO, mtDNA, COXIV, mtTFA, and fatty acid incorporation into triacylglycerol in both lean and obese groups. Perturbations in FAO, triacylglycerol synthesis, mtDNA, COXIV, and mtTFA in obese compared with lean HSkMC persisted despite PGC-1α overexpression. When adjusted for mtDNA and COXIV content, FAO was equivalent between lean and obese groups. CONCLUSION Reduced mitochondrial content is related to impaired FAO in HSkMC derived from obese individuals. Increasing PGC-1α protein levels did not correct the obesity-related absolute reduction in FAO or mtDNA content, implicating mechanisms other than PGC-1α abundance. PMID:20200320

  11. Mas receptor overexpression increased Ang-(1-7) relaxation response in renovascular hypertensive rat carotid.

    PubMed

    Olivon, V C; Aires, R D; Santiago, L B; Ramalho, L Z N; Cortes, S F; Lemos, V S

    2015-09-01

    Renin-angiotensin system (RAS) is an important factor in the pathophysiology of hypertension. Mas receptor, Angiotensin-(1-7) [Ang-(1-7)]-activated receptor, is an important RAS component and exerts protective effects in the vasculature. Ang-(1-7) vascular effects and Mas receptor expression in carotid from renovascular hypertensive (2K-1C) rats is not clear. In the present study we investigated Mas receptor vasodilator response activated by Ang-(1-7) in the carotid rings from sham and 2K-1C rats. Changes in isometric tension were recorded on organ chamber. Mas receptors expression was investigated in carotid by Western blot. Nitric oxide production was evaluated by 2,3-diaminonaphthalene (DAN) and eNOS expression and activity by immunofluoresce and western blot, respectively. Ang-(1-7) induced concentration-dependent vasodilator effect in carotid rings from sham and 2K-1C, which the hypertension increased vasodilatation response. In the 2K-1C carotid rings, A-779 (Mas receptor antagonist) reduced but not abolish the vasodilator effect of Ang-(1-7). Corroborating, Mas receptor protein expression was significantly increased in the 2K-1C rats. L-NAME and ibuprofen decreased Ang-(1-7) vasodilator response and L-NAME plus ibuprofen practically abolish the remaining vasodilatation response. Nitric oxide production is increased due increased of eNOS expression and pSer(1177) activity. Our results demonstrated that renovascular hypertension increased Mas receptors expression and nitric oxide production in the rats carotid which, consequently increased Ang-(1-7)-vasorelaxant response. PMID:26256416

  12. Overexpression of a citrus NDR1 ortholog increases disease resistance in Arabidopsis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Emerging devastating diseases, such as Huanglongbing (HLB) and citrus canker, have caused tremendous losses to the citrus industry worldwide. Genetic engineering is a powerful approach that could allow us to increase citrus resistance against these diseases. The key to the success of this approach r...

  13. Overexpression of Arabidopsis VIT1 increases accumulation of iron in cassava roots and stems

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Iron is extremely abundant in the soil, but its uptake in plants is limited due to low solubility in neutral or alkaline soils. Plants can rely on rhizosphere acidification to increase iron solubility. AtVIT1 was previously found to be involved in mediating vacuolar sequestration of iron, which indi...

  14. Overexpression of Poplar Xylem Sucrose Synthase in Tobacco Leads to a Thickened Cell Wall and Increased Height

    PubMed Central

    Wei, Zhigang; Qu, Zanshuang; Zhang, Lijie; Zhao, Shuanjing; Bi, Zhihong; Ji, Xiaohui; Wang, Xiaowen; Wei, Hairong

    2015-01-01

    Sucrose synthase (SuSy) is considered the first key enzyme for secondary growth because it is a highly regulated cytosolic enzyme that catalyzes the reversible conversion of sucrose and UDP into UDP-glucose and fructose. Although SuSy enzymes preferentially functions in the direction of sucrose cleavage at most cellular condition, they also catalyze the synthetic reaction. We isolated a gene that encodes a SuSy from Populus simonii×Populus nigra and named it PsnSuSy2 because it shares high similarity to SuSy2 in Populus trichocarpa. RT-PCR revealed that PsnSuSy2 was highly expressed in xylem, but lowly expressed in young leaves. To characterize its functions in secondary growth, multiple tobacco overexpression transgenic lines of PnsSuSy2 were generated via Agrobacterium-mediated transformation. The PsnSuSy2 expression levels and altered wood properties in stem segments from the different transgenic lines were carefully characterized. The results demonstrated that the levels of PsnSuSy2 enzyme activity, chlorophyll content, total soluble sugars, fructose and glucose increased significantly, while the sucrose level decreased significantly. Consequently, the cellulose content and fiber length increased, whereas the lignin content decreased, suggesting that PsnSuSy2 plays a significant role in cleaving sucrose into UDP-glucose and fructose to facilitate cellulose biosynthesis and that promotion of cellulose biosynthesis suppresses lignin biosynthesis. Additionally, the noticeable increase in the lodging resistance in transgenic tobacco stem suggested that the cell wall characteristics were altered by PsnSuSy2 overexpression. Scanning electron microscopy was performed to study the cell wall morphology of stem, and surprisingly, we found that the secondary cell wall was significantly thicker in transgenic tobacco. However, the thickened secondary cell wall did not negatively affect the height of the plants because the PsnSuSy2- overexpressing lines grew taller than the

  15. Transgenic tobacco overexpressing Brassica juncea HMG-CoA synthase 1 shows increased plant growth, pod size and seed yield.

    PubMed

    Liao, Pan; Wang, Hui; Wang, Mingfu; Hsiao, An-Shan; Bach, Thomas J; Chye, Mee-Len

    2014-01-01

    Seeds are very important not only in the life cycle of the plant but they represent food sources for man and animals. We report herein a mutant of 3-hydroxy-3-methylglutaryl-coenzyme A synthase (HMGS), the second enzyme in the mevalonate (MVA) pathway that can improve seed yield when overexpressed in a phylogenetically distant species. In Brassica juncea, the characterisation of four isogenes encoding HMGS has been previously reported. Enzyme kinetics on recombinant wild-type (wt) and mutant BjHMGS1 had revealed that S359A displayed a 10-fold higher enzyme activity. The overexpression of wt and mutant (S359A) BjHMGS1 in Arabidopsis had up-regulated several genes in sterol biosynthesis, increasing sterol content. To quickly assess the effects of BjHMGS1 overexpression in a phylogenetically more distant species beyond the Brassicaceae, wt and mutant (S359A) BjHMGS1 were expressed in tobacco (Nicotiana tabacum L. cv. Xanthi) of the family Solanaceae. New observations on tobacco OEs not previously reported for Arabidopsis OEs included: (i) phenotypic changes in enhanced plant growth, pod size and seed yield (more significant in OE-S359A than OE-wtBjHMGS1) in comparison to vector-transformed tobacco, (ii) higher NtSQS expression and sterol content in OE-S359A than OE-wtBjHMGS1 corresponding to greater increase in growth and seed yield, and (iii) induction of NtIPPI2 and NtGGPPS2 and downregulation of NtIPPI1, NtGGPPS1, NtGGPPS3 and NtGGPPS4. Resembling Arabidopsis HMGS-OEs, tobacco HMGS-OEs displayed an enhanced expression of NtHMGR1, NtSMT1-2, NtSMT2-1, NtSMT2-2 and NtCYP85A1. Overall, increased growth, pod size and seed yield in tobacco HMGS-OEs were attributed to the up-regulation of native NtHMGR1, NtIPPI2, NtSQS, NtSMT1-2, NtSMT2-1, NtSMT2-2 and NtCYP85A1. Hence, S359A has potential in agriculture not only in improving phytosterol content but also seed yield, which may be desirable in food crops. This work further demonstrates HMGS function in plant reproduction

  16. Transgenic Tobacco Overexpressing Brassica juncea HMG-CoA Synthase 1 Shows Increased Plant Growth, Pod Size and Seed Yield

    PubMed Central

    Liao, Pan; Wang, Hui; Wang, Mingfu; Hsiao, An-Shan; Bach, Thomas J.; Chye, Mee-Len

    2014-01-01

    Seeds are very important not only in the life cycle of the plant but they represent food sources for man and animals. We report herein a mutant of 3-hydroxy-3-methylglutaryl-coenzyme A synthase (HMGS), the second enzyme in the mevalonate (MVA) pathway that can improve seed yield when overexpressed in a phylogenetically distant species. In Brassica juncea, the characterisation of four isogenes encoding HMGS has been previously reported. Enzyme kinetics on recombinant wild-type (wt) and mutant BjHMGS1 had revealed that S359A displayed a 10-fold higher enzyme activity. The overexpression of wt and mutant (S359A) BjHMGS1 in Arabidopsis had up-regulated several genes in sterol biosynthesis, increasing sterol content. To quickly assess the effects of BjHMGS1 overexpression in a phylogenetically more distant species beyond the Brassicaceae, wt and mutant (S359A) BjHMGS1 were expressed in tobacco (Nicotiana tabacum L. cv. Xanthi) of the family Solanaceae. New observations on tobacco OEs not previously reported for Arabidopsis OEs included: (i) phenotypic changes in enhanced plant growth, pod size and seed yield (more significant in OE-S359A than OE-wtBjHMGS1) in comparison to vector-transformed tobacco, (ii) higher NtSQS expression and sterol content in OE-S359A than OE-wtBjHMGS1 corresponding to greater increase in growth and seed yield, and (iii) induction of NtIPPI2 and NtGGPPS2 and downregulation of NtIPPI1, NtGGPPS1, NtGGPPS3 and NtGGPPS4. Resembling Arabidopsis HMGS-OEs, tobacco HMGS-OEs displayed an enhanced expression of NtHMGR1, NtSMT1-2, NtSMT2-1, NtSMT2-2 and NtCYP85A1. Overall, increased growth, pod size and seed yield in tobacco HMGS-OEs were attributed to the up-regulation of native NtHMGR1, NtIPPI2, NtSQS, NtSMT1-2, NtSMT2-1, NtSMT2-2 and NtCYP85A1. Hence, S359A has potential in agriculture not only in improving phytosterol content but also seed yield, which may be desirable in food crops. This work further demonstrates HMGS function in plant reproduction

  17. Overexpression of petunia chalcone isomerase in tomato results in fruit containing increased levels of flavonols.

    PubMed

    Muir, S R; Collins, G J; Robinson, S; Hughes, S; Bovy, A; Ric De Vos, C H; van Tunen, A J; Verhoeyen, M E

    2001-05-01

    Tomatoes are an excellent source of the carotenoid lycopene, a compound that is thought to be protective against prostate cancer. They also contain small amounts of flavonoids in their peel ( approximately 5-10 mg/kg fresh weight), mainly naringenin chalcone and the flavonol rutin, a quercetin glycoside. Flavonols are very potent antioxidants, and an increasing body of epidemiological data suggests that high flavonoid intake is correlated with a decreased risk for cardiovascular disease. We have upregulated flavonol biosynthesis in the tomato in order to generate fruit with increased antioxidant capacity and a wider range of potential health benefit properties. This involved transformation of tomato with the Petunia chi-a gene encoding chalcone isomerase. Resulting transgenic tomato lines produced an increase of up to 78 fold in fruit peel flavonols, mainly due to an accumulation of rutin. No gross phenotypical differences were observed between high-flavonol transgenic and control lines. The phenotype segregated with the transgene and demonstrated a stable inheritance pattern over four subsequent generations tested thus far. Whole-fruit flavonol levels in the best of these lines are similar to those found in onions, a crop with naturally high levels of flavonol compounds. Processing of high-flavonol tomatoes demonstrated that 65% of flavonols present in the fresh fruit were retained in the processed paste, supporting their potential as raw materials for tomato-based functional food products. PMID:11329019

  18. Overexpression of petunia chalcone isomerase in tomato results in fruit containing increased levels of flavonols.

    PubMed

    Muir, S R; Collins, G J; Robinson, S; Hughes, S; Bovy, A; Ric De Vos, C H; van Tunen, A J; Verhoeyen, M E

    2001-05-01

    Tomatoes are an excellent source of the carotenoid lycopene, a compound that is thought to be protective against prostate cancer. They also contain small amounts of flavonoids in their peel ( approximately 5-10 mg/kg fresh weight), mainly naringenin chalcone and the flavonol rutin, a quercetin glycoside. Flavonols are very potent antioxidants, and an increasing body of epidemiological data suggests that high flavonoid intake is correlated with a decreased risk for cardiovascular disease. We have upregulated flavonol biosynthesis in the tomato in order to generate fruit with increased antioxidant capacity and a wider range of potential health benefit properties. This involved transformation of tomato with the Petunia chi-a gene encoding chalcone isomerase. Resulting transgenic tomato lines produced an increase of up to 78 fold in fruit peel flavonols, mainly due to an accumulation of rutin. No gross phenotypical differences were observed between high-flavonol transgenic and control lines. The phenotype segregated with the transgene and demonstrated a stable inheritance pattern over four subsequent generations tested thus far. Whole-fruit flavonol levels in the best of these lines are similar to those found in onions, a crop with naturally high levels of flavonol compounds. Processing of high-flavonol tomatoes demonstrated that 65% of flavonols present in the fresh fruit were retained in the processed paste, supporting their potential as raw materials for tomato-based functional food products.

  19. Dopamine D3 receptor deletion increases tissue plasminogen activator (tPA) activity in prefrontal cortex and hippocampus.

    PubMed

    Castorina, A; D'Amico, A G; Scuderi, S; Leggio, G M; Drago, F; D'Agata, V

    2013-10-10

    Considerable evidence indicates that dopamine (DA) influences tissue plasminogen activator (tPA)-mediated proteolytic processing of the precursor of brain-derived neurotrophic factor (proBDNF) into mature BDNF (mBDNF). However, specific roles in this process for the dopamine D3 receptor (D3R) and the underlying molecular mechanisms are yet to be fully characterized. In the present study, we hypothesized that D3R deletion could influence tPA activity in the prefrontal cortex and hippocampus. Using D3R knockout (D3(-/-)) mice, we show that receptor inactivation is associated with increased tPA expression/activity both in the prefrontal cortex and, to a greater extent, in the hippocampus. Augmented tPA expression in D3(-/-) mice correlated with increased BDNF mRNA levels, plasmin/plasminogen protein ratio and the conversion of proBDNF into mBDNF, as well as enhanced tPA and mBDNF immunoreactivity, as determined by quantitative real time polymerase chain reaction (qRT-PCR), immunoblot and immunohistochemistry. In addition, when compared to wild-type controls, D3(-/-) mice exhibited increased basal activation of the canonical cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)-driven Akt/cAMP-response element-binding protein (CREB) signaling cascade, as determined by the increased Akt phosphorylation both at Thr304 and Ser473 residues, of DA and cAMP-regulated protein of 32kDa (DARPP-32) at Thr34 and a phosphorylation state-dependent inhibition of glycogen synthetase kinase-3β (GSK-3β) at Ser9, a substrate of Akt whose constitutive function impairs normal CREB transcriptional activity through phosphorylation at its Ser129 residue. Accordingly, CREB phosphorylation at Ser133 was significantly increased in D3(-/-) mice, whereas the GSK-3β-dependent phosphorylation at Ser129 was diminished. Altogether, our finding reveals that mice lacking D3Rs show enhanced tPA proteolytic activity on BDNF which may involve, at least in part, a potentiated Akt/CREB signaling

  20. The absence of VGLUT3 predisposes to cocaine abuse by increasing dopamine and glutamate signaling in the nucleus accumbens.

    PubMed

    Sakae, D Y; Marti, F; Lecca, S; Vorspan, F; Martín-García, E; Morel, L J; Henrion, A; Gutiérrez-Cuesta, J; Besnard, A; Heck, N; Herzog, E; Bolte, S; Prado, V F; Prado, M A M; Bellivier, F; Eap, C B; Crettol, S; Vanhoutte, P; Caboche, J; Gratton, A; Moquin, L; Giros, B; Maldonado, R; Daumas, S; Mameli, M; Jamain, S; El Mestikawy, S

    2015-11-01

    Tonically active cholinergic interneurons (TANs) from the nucleus accumbens (NAc) are centrally involved in reward behavior. TANs express a vesicular glutamate transporter referred to as VGLUT3 and thus use both acetylcholine and glutamate as neurotransmitters. The respective roles of each transmitter in the regulation of reward and addiction are still unknown. In this study, we showed that disruption of the gene that encodes VGLUT3 (Slc17a8) markedly increased cocaine self-administration in mice. Concomitantly, the amount of dopamine (DA) release was strongly augmented in the NAc of VGLUT3(-/-) mice because of a lack of signaling by metabotropic glutamate receptors. Furthermore, dendritic spines and glutamatergic synaptic transmission on medium spiny neurons were increased in the NAc of VGLUT3(-/-) mice. Increased DA and glutamate signaling in the NAc are hallmarks of addiction. Our study shows that TANs use glutamate to reduce DA release and decrease reinforcing properties of cocaine in mice. Interestingly, we also observed an increased frequency of rare variations in SLC17A8 in a cohort of severe drug abusers compared with controls. Our findings identify VGLUT3 as an unexpected regulator of drug abuse. PMID:26239290

  1. Overexpression of a pH-sensitive nitrate transporter in rice increases crop yields

    PubMed Central

    Fan, Xiaorong; Tang, Zhong; Tan, Yawen; Zhang, Yong; Luo, Bingbing; Yang, Meng; Lian, Xingming; Shen, Qirong; Miller, Anthony John; Xu, Guohua

    2016-01-01

    Cellular pH homeostasis is fundamental for life, and all cells adapt to maintain this balance. In plants, the chemical form of nitrogen supply, nitrate and ammonium, is one of the cellular pH dominators. We report that the rice nitrate transporter OsNRT2.3 is transcribed into two spliced isoforms with a natural variation in expression ratio. One splice form, OsNRT2.3b is located on the plasma membrane, is expressed mainly in the phloem, and has a regulatory motif on the cytosolic side that acts to switch nitrate transport activity on or off by a pH-sensing mechanism. High OsNRT2.3b expression in rice enhances the pH-buffering capacity of the plant, increasing N, Fe, and P uptake. In field trials, increased expression of OsNRT2.3b improved grain yield and nitrogen use efficiency (NUE) by 40%. These results indicate that pH sensing by the rice nitrate transporter OsNRT2.3b is important for plant adaption to varied N supply forms and can provide a target for improving NUE. PMID:27274069

  2. Increasing morphinan alkaloid production by over-expressing codeinone reductase in transgenic Papaver somniferum.

    PubMed

    Larkin, Philip J; Miller, James A C; Allen, Robert S; Chitty, Julie A; Gerlach, Wayne L; Frick, Susanne; Kutchan, Toni M; Fist, Anthony J

    2007-01-01

    Only plants of the Papaver genus (poppies) are able to synthesize morphinan alkaloids, and cultivation of P. somniferum, opium poppy, remains critical for the production and supply of morphine, codeine and various semi-synthetic analgesics. Opium poppy was transformed with constitutively expressed cDNA of codeinone reductase (PsCor1.1), the penultimate step in morphine synthesis. Most transgenic lines showed significant increases in capsule alkaloid content in replicated glasshouse and field trials over 4 years. The morphinan alkaloid contents on a dry weight basis were between 15% and 30% greater than those in control high-yielding genotypes and control non-transgenic segregants. Transgenic leaves had approximately 10-fold greater levels of Cor transcript compared with non-transgenic controls. Two cycles of crossing of the best transgenic line into an elite high-morphine genotype resulted in significant increases in morphine and total alkaloids relative to the elite recurrent parent. No significant changes in alkaloid profiles or quantities were observed in leaf, roots, pollen and seed.

  3. Overexpression of a pH-sensitive nitrate transporter in rice increases crop yields.

    PubMed

    Fan, Xiaorong; Tang, Zhong; Tan, Yawen; Zhang, Yong; Luo, Bingbing; Yang, Meng; Lian, Xingming; Shen, Qirong; Miller, Anthony John; Xu, Guohua

    2016-06-28

    Cellular pH homeostasis is fundamental for life, and all cells adapt to maintain this balance. In plants, the chemical form of nitrogen supply, nitrate and ammonium, is one of the cellular pH dominators. We report that the rice nitrate transporter OsNRT2.3 is transcribed into two spliced isoforms with a natural variation in expression ratio. One splice form, OsNRT2.3b is located on the plasma membrane, is expressed mainly in the phloem, and has a regulatory motif on the cytosolic side that acts to switch nitrate transport activity on or off by a pH-sensing mechanism. High OsNRT2.3b expression in rice enhances the pH-buffering capacity of the plant, increasing N, Fe, and P uptake. In field trials, increased expression of OsNRT2.3b improved grain yield and nitrogen use efficiency (NUE) by 40%. These results indicate that pH sensing by the rice nitrate transporter OsNRT2.3b is important for plant adaption to varied N supply forms and can provide a target for improving NUE. PMID:27274069

  4. Overexpression of laminin alpha1 chain in colonic cancer cells induces an increase in tumor growth.

    PubMed

    De Arcangelis, A; Lefebvre, O; Méchine-Neuville, A; Arnold, C; Klein, A; Rémy, L; Kedinger, M; Simon-Assmann, P

    2001-10-01

    Laminins represent a growing family of glycoproteins constituting the basement membrane. They are known to direct many biological processes. With respect to carcinogenesis, laminins play an important role in cell adhesion, mitogenesis, differentiation and even metastasis. To further study the biological significance of laminin-1 (composed of alpha1, beta1 and gamma1 chains) in intestinal cell differentiation or tumorigenesis, an alpha1-laminin expression vector was introduced into the HT29 colonic cancer cells, in which laminin alpha1 chain is not expressed. Upon transfection of the alpha1 chain, the alpha1beta1gamma1 trimer was found secreted in the media along with free alpha1 chain as assessed by immunoprecipitation. The presence of the laminin alpha1 chain did not significantly modify the levels of the other laminin chains nor the integrins expressed by the HT29 cells. In spite of similar growth properties with the control cells in vitro (plastic dish, soft agar), the laminin alpha1 transfectants showed a significantly increased tumor growth when injected in nude mice. Histologic and immunohistochemic examination of the laminin alpha1-expressing tumors points to an increased recruitment of the host stromal and vascular cells, without modification in the differentiation profile and invasion potential. In parallel, a clear accumulation of laminin-10 (alpha5beta1gamma1) at the carcinoma/stromal interface and a segregation of the integrin beta4 subunit at the basal pole of the cancer cells occurred, compared to control tumors. Overall, our observations emphasize the importance of laminin-1 as a chemoattractant of both stromal and vascular cells and in epithelial/stromal cell interactions for the organization of the basement membrane and segregation of integrins leading to an epithelial cell growth signal. Such a sequence of events is reminiscent of what occurs during development.

  5. Osteoblast-specific overexpression of amphiregulin leads to transient increase in femoral cancellous bone mass in mice.

    PubMed

    Vaidya, Mithila; Lehner, Diana; Handschuh, Stephan; Jay, Freya F; Erben, Reinhold G; Schneider, Marlon R

    2015-12-01

    The epidermal growth factor receptor ligand amphiregulin (AREG) has been implicated in bone physiology and in bone anabolism mediated by intermittent parathyroid hormone treatment. However, the functions of AREG in bone have been only incipiently evaluated in vivo. Here, we generated transgenic mice overexpressing AREG specifically in osteoblasts (Col1-Areg). pQCT analysis of the femoral metaphysis revealed increased trabecular bone mass at 4, 8, and 10weeks of age in Col1-Areg mice compared to control littermates. However, the high bone mass phenotype was transient and disappeared in older animals. Micro-CT analysis of the secondary spongiosa confirmed increased trabecular bone volume and trabecular number in the distal femur of 4-week-old AREG-tg mice compared to control littermates. Furthermore, μ-CT analysis of the primary spongiosa revealed unaltered production of new bone trabeculae in distal femora of Col1-Areg mice. Histomorphometric analysis revealed a reduced number of osteoclasts in 4-week-old Col1-Areg mice, but not at later time points. Cancellous bone formation rate remained unchanged in Col1-Areg mice at all time points. In addition, bone mass and bone turnover in lumbar vertebral bodies were similar in Col1-Areg and control mice at all ages examined. Proliferation and differentiation of osteoblasts isolated from neonatal calvariae did not differ between Col1-Areg and control mice. Taken together, these data suggest that AREG overexpression in osteoblasts induces a transient high bone mass phenotype in the trabecular compartment of the appendicular skeleton by a growth-related, non-cell autonomous mechanism, leading to a positive bone balance with unchanged bone formation and lowered bone resorption.

  6. Caffeine increases striatal dopamine D2/D3 receptor availability in the human brain

    SciTech Connect

    Volkow, N. D.; Wang, G. -J.; Logan, J.; Alexoff, D.; Fowler, J. S.; Thanos, P. K.; Wong, C.; Casado, V.; Ferre, S.; Tomasi, D.

    2015-04-14

    Caffeine, the most widely consumed psychoactive substance in the world, is used to promote wakefulness and enhance alertness. Like other wake-promoting drugs (stimulants and modafinil), caffeine enhances dopamine (DA) signaling in the brain, which it does predominantly by antagonizing adenosine A2A receptors (A2AR). However, it is unclear if caffeine, at the doses consumed by humans, increases DA release or whether it modulates the functions of postsynaptic DA receptors through its interaction with adenosine receptors, which modulate them. We used positron emission tomography and [11C]raclopride (DA D2/D3 receptor radioligand sensitive to endogenous DA) to assess if caffeine increased DA release in striatum in 20 healthy controls. Caffeine (300mg p.o.) significantly increased the availability of D2/D3 receptors in putamen and ventral striatum, but not in caudate, when compared with placebo. In addition, caffeine-induced increases in D2/D3 receptor availability in the ventral striatum were associated with caffeine-induced increases in alertness. Our findings indicate that in the human brain, caffeine, at doses typically consumed, increases the availability of DA D2/D3 receptors, which indicates that caffeine does not increase DA in the striatum for this would have decreased D2/D3 receptor availability. Instead, we interpret our findings to reflect an increase in D2/D3 receptor levels in striatum with caffeine (or changes in affinity). Furthermore, the association between increases in D2/D3 receptor availability in ventral striatum and alertness suggests that caffeine might enhance arousal, in part, by upregulating D2/D3 receptors.

  7. Increased Infectivity of Anchorless Mouse Scrapie Prions in Transgenic Mice Overexpressing Human Prion Protein

    PubMed Central

    Phillips, Katie; Meade-White, Kimberly; Striebel, James; Chesebro, Bruce

    2015-01-01

    prions produced by mice expressing only anchorless PrP were more infectious than prions produced in mice expressing anchored PrP. Thus, the lack of the GPI anchor on prions reduced the effect of the mouse-human species barrier. Our results suggest that prion diseases that produce higher levels of anchorless PrP may pose an increased risk for cross-species infection. PMID:25810548

  8. Leptin Increases Striatal Dopamine D2 Receptor Binding in Leptin-Deficient Obese (ob/ob) Mice

    SciTech Connect

    Pfaffly, J.; Michaelides, M.; Wang, G-J.; Pessin, J.E.; Volkow, N.D.; Thanos, P.K.

    2010-06-01

    Peripheral and central leptin administration have been shown to mediate central dopamine (DA) signaling. Leptin-receptor deficient rodents show decreased DA D2 receptor (D2R) binding in striatum and unique DA profiles compared to controls. Leptin-deficient mice show increased DA activity in reward-related brain regions. The objective of this study was to examine whether basal D2R-binding differences contribute to the phenotypic behaviors of leptin-deficient ob/ob mice, and whether D2R binding is altered in response to peripheral leptin treatment in these mice. Leptin decreased body weight, food intake, and plasma insulin concentration in ob/ob mice but not in wild-type mice. Basal striatal D2R binding (measured with autoradiography [{sup 3}H] spiperone) did not differ between ob/ob and wild-type mice but the response to leptin did. In wild-type mice, leptin decreased striatal D2R binding, whereas, in ob/ob mice, leptin increased D2R binding. Our findings provide further evidence that leptin modulates D2R expression in striatum and that these effects are genotype/phenotype dependent.

  9. Brief intermittent cocaine self-administration and abstinence sensitizes cocaine effects on the dopamine transporter and increases drug seeking.

    PubMed

    Calipari, Erin S; Siciliano, Cody A; Zimmer, Benjamin A; Jones, Sara R

    2015-02-01

    Although traditional sensitization paradigms, which result in an augmentation of cocaine-induced locomotor behavior and dopamine (DA) overflow following repeated experimenter-delivered cocaine injections, are often used as a model to study drug addiction, similar effects have been difficult to demonstrate following cocaine self-administration. We have recently shown that intermittent access (IntA) to cocaine can result in increased cocaine potency at the DA transporter (DAT); however, traditional sensitization paradigms often show enhanced effects following withdrawal/abstinence periods. Therefore, we determined a time course of IntA-induced sensitization by examining the effects of 1 or 3 days of IntA, as well as a 7-day abstinence period on DA function, cocaine potency, and reinforcement. Here we show that cocaine potency is increased following as little as 3 days of IntA and further augmented following an abstinence period. In addition, IntA plus abstinence produced greater evoked DA release in the presence of cocaine as compared with all other groups, demonstrating that following abstinence, both cocaine's ability to increase DA release and inhibit uptake at the DAT, two separate mechanisms for increasing DA levels, are enhanced. Finally, we found that IntA-induced sensitization of the DA system resulted in an increased reinforcing efficacy of cocaine, an effect that was augmented after the 7-day abstinence period. These results suggest that sensitization of the DA system may have an important role in the early stages of drug abuse and may drive the increased drug seeking and taking that characterize the transition to uncontrolled drug use. Human data suggest that intermittency, sensitization, and periods of abstinence have an integral role in the process of addiction, highlighting the importance of utilizing pre-clinical models that integrate these phenomena, and suggesting that IntA paradigms may serve as novel models of human addiction. PMID:25212486

  10. Exposure to repeated immobilization stress inhibits cocaine-induced increase in dopamine extracellular levels in the rat ventral tegmental area.

    PubMed

    Sotomayor-Zárate, Ramón; Abarca, Jorge; Araya, Katherine A; Renard, Georgina M; Andrés, María E; Gysling, Katia

    2015-11-01

    A higher vulnerability to drug abuse has been observed in human studies of individuals exposed to chronic or persistent stress, as well as in animal models of drug abuse. Here, we explored the effect of repeated immobilization stress on cocaine-induced increase in dopamine extracellular levels in VTA and its regulation by corticotropin-releasing factor (CRF) and GABA systems. Cocaine (10mg/Kg i.p.) induced an increase of VTA DA extracellular levels in control rats. However, this effect was not observed in repeated stress rats. Considering the evidence relating stress with CRF, we decided to perfuse CRF and CP-154526 (selective antagonist of CRF1 receptor) in the VTA of control and repeated stress rats, respectively. We observed that perfusion of 20μM CRF inhibited the increase of VTA DA extracellular levels induced by cocaine in control rats. Interestingly, we observed that in the presence of 10μM CP-154526, cocaine induced a significant increase of VTA DA extracellular levels in repeated stress rats. Regarding the role of VTA GABA neurotransmission, cocaine administration induced a significant increase in VTA GABA extracellular levels only in repeated stress rats. Consistently, cocaine was able to increase VTA DA extracellular levels in repeated stress rats when 100μM bicuculline, an antagonist of GABAA receptor, was perfused intra VTA. Thus, both CRF and GABA systems are involved in the lack of response to cocaine in the VTA of repeated stress rats. It is tempting to suggest that the loss of response in VTA dopaminergic neurons to cocaine, after repeated stress, is due to an interaction between CRF and GABA systems. PMID:26318765

  11. Brief Intermittent Cocaine Self-Administration and Abstinence Sensitizes Cocaine Effects on the Dopamine Transporter and Increases Drug Seeking

    PubMed Central

    Calipari, Erin S; Siciliano, Cody A; Zimmer, Benjamin A; Jones, Sara R

    2015-01-01

    Although traditional sensitization paradigms, which result in an augmentation of cocaine-induced locomotor behavior and dopamine (DA) overflow following repeated experimenter-delivered cocaine injections, are often used as a model to study drug addiction, similar effects have been difficult to demonstrate following cocaine self-administration. We have recently shown that intermittent access (IntA) to cocaine can result in increased cocaine potency at the DA transporter (DAT); however, traditional sensitization paradigms often show enhanced effects following withdrawal/abstinence periods. Therefore, we determined a time course of IntA-induced sensitization by examining the effects of 1 or 3 days of IntA, as well as a 7-day abstinence period on DA function, cocaine potency, and reinforcement. Here we show that cocaine potency is increased following as little as 3 days of IntA and further augmented following an abstinence period. In addition, IntA plus abstinence produced greater evoked DA release in the presence of cocaine as compared with all other groups, demonstrating that following abstinence, both cocaine's ability to increase DA release and inhibit uptake at the DAT, two separate mechanisms for increasing DA levels, are enhanced. Finally, we found that IntA-induced sensitization of the DA system resulted in an increased reinforcing efficacy of cocaine, an effect that was augmented after the 7-day abstinence period. These results suggest that sensitization of the DA system may have an important role in the early stages of drug abuse and may drive the increased drug seeking and taking that characterize the transition to uncontrolled drug use. Human data suggest that intermittency, sensitization, and periods of abstinence have an integral role in the process of addiction, highlighting the importance of utilizing pre-clinical models that integrate these phenomena, and suggesting that IntA paradigms may serve as novel models of human addiction. PMID:25212486

  12. Exposure to repeated immobilization stress inhibits cocaine-induced increase in dopamine extracellular levels in the rat ventral tegmental area.

    PubMed

    Sotomayor-Zárate, Ramón; Abarca, Jorge; Araya, Katherine A; Renard, Georgina M; Andrés, María E; Gysling, Katia

    2015-11-01

    A higher vulnerability to drug abuse has been observed in human studies of individuals exposed to chronic or persistent stress, as well as in animal models of drug abuse. Here, we explored the effect of repeated immobilization stress on cocaine-induced increase in dopamine extracellular levels in VTA and its regulation by corticotropin-releasing factor (CRF) and GABA systems. Cocaine (10mg/Kg i.p.) induced an increase of VTA DA extracellular levels in control rats. However, this effect was not observed in repeated stress rats. Considering the evidence relating stress with CRF, we decided to perfuse CRF and CP-154526 (selective antagonist of CRF1 receptor) in the VTA of control and repeated stress rats, respectively. We observed that perfusion of 20μM CRF inhibited the increase of VTA DA extracellular levels induced by cocaine in control rats. Interestingly, we observed that in the presence of 10μM CP-154526, cocaine induced a significant increase of VTA DA extracellular levels in repeated stress rats. Regarding the role of VTA GABA neurotransmission, cocaine administration induced a significant increase in VTA GABA extracellular levels only in repeated stress rats. Consistently, cocaine was able to increase VTA DA extracellular levels in repeated stress rats when 100μM bicuculline, an antagonist of GABAA receptor, was perfused intra VTA. Thus, both CRF and GABA systems are involved in the lack of response to cocaine in the VTA of repeated stress rats. It is tempting to suggest that the loss of response in VTA dopaminergic neurons to cocaine, after repeated stress, is due to an interaction between CRF and GABA systems.

  13. Overexpression of the brassinosteroid biosynthetic gene DWF4 in Brassica napus simultaneously increases seed yield and stress tolerance.

    PubMed

    Sahni, Sangita; Prasad, Bishun D; Liu, Qing; Grbic, Vojislava; Sharpe, Andrew; Singh, Surinder P; Krishna, Priti

    2016-01-01

    As a resource allocation strategy, plant growth and defense responses are generally mutually antagonistic. Brassinosteroid (BR) regulates many aspects of plant development and stress responses, however, genetic evidence of its integrated effects on plant growth and stress tolerance is lacking. We overexpressed the Arabidopsis BR biosynthetic gene AtDWF4 in the oilseed plant Brassica napus and scored growth and stress response phenotypes. The transgenic B. napus plants, in comparison to wild type, displayed increased seed yield leading to increased overall oil content per plant, higher root biomass and root length, significantly better tolerance to dehydration and heat stress, and enhanced resistance to necrotrophic fungal pathogens Leptosphaeria maculans and Sclerotinia sclerotiorum. Transcriptome analysis supported the integrated effects of BR on growth and stress responses; in addition to BR responses associated with growth, a predominant plant defense signature, likely mediated by BES1/BZR1, was evident in the transgenic plants. These results establish that BR can interactively and simultaneously enhance abiotic and biotic stress tolerance and plant productivity. The ability to confer pleiotropic beneficial effects that are associated with different agronomic traits suggests that BR-related genes may be important targets for simultaneously increasing plant productivity and performance under stress conditions. PMID:27324083

  14. Overexpression of the brassinosteroid biosynthetic gene DWF4 in Brassica napus simultaneously increases seed yield and stress tolerance

    PubMed Central

    Sahni, Sangita; Prasad, Bishun D.; Liu, Qing; Grbic, Vojislava; Sharpe, Andrew; Singh, Surinder P.; Krishna, Priti

    2016-01-01

    As a resource allocation strategy, plant growth and defense responses are generally mutually antagonistic. Brassinosteroid (BR) regulates many aspects of plant development and stress responses, however, genetic evidence of its integrated effects on plant growth and stress tolerance is lacking. We overexpressed the Arabidopsis BR biosynthetic gene AtDWF4 in the oilseed plant Brassica napus and scored growth and stress response phenotypes. The transgenic B. napus plants, in comparison to wild type, displayed increased seed yield leading to increased overall oil content per plant, higher root biomass and root length, significantly better tolerance to dehydration and heat stress, and enhanced resistance to necrotrophic fungal pathogens Leptosphaeria maculans and Sclerotinia sclerotiorum. Transcriptome analysis supported the integrated effects of BR on growth and stress responses; in addition to BR responses associated with growth, a predominant plant defense signature, likely mediated by BES1/BZR1, was evident in the transgenic plants. These results establish that BR can interactively and simultaneously enhance abiotic and biotic stress tolerance and plant productivity. The ability to confer pleiotropic beneficial effects that are associated with different agronomic traits suggests that BR–related genes may be important targets for simultaneously increasing plant productivity and performance under stress conditions. PMID:27324083

  15. Selective disruption of dopamine D2 receptors in pituitary lactotropes increases body weight and adiposity in female mice.

    PubMed

    Perez Millan, Maria Ines; Luque, Guillermina Maria; Ramirez, Maria Cecilia; Noain, Daniela; Ornstein, Ana Maria; Rubinstein, Marcelo; Becu-Villalobos, Damasia

    2014-03-01

    Prolactin, a pleiotropic hormone secreted by lactotropes, has reproductive and metabolic functions. Chronically elevated prolactin levels increase food intake, but in some hyperprolactinemic states such as in the global dopamine D2 receptor (D2R) knockout mouse, food intake is not increased. Here, we conduct a cell-specific genetic dissection study using conditional mutant mice that selectively lack D2Rs from pituitary lactotropes (lacDrd2KO) to evaluate the role of elevated prolactin levels without any confounding effect of central D2Rs on motor and reward mechanisms related to food intake. LacDrd2KO female mice exhibited chronic hyperprolactinemia, pituitary hyperplasia, and a preserved GH axis. In addition, lacDrd2KO female but not male mice showed increased food intake by 3 months of age, and from 5 months onward their body weights were heavier. Marked increments in fat depots, adipocyte size, serum triglycerides, and nonesterified fatty acid levels and a decrease in lipolytic enzymes in adipose tissue were seen. Furthermore, lacDrd2KO female mice had glucose intolerance but a preserved response to insulin. In the hypothalamus, Npy mRNA expression was increased, and Pomc and Ppo mRNA levels were unaltered (in contrast to results in global D2R knockout mice). Thus, the orexigenic effect of prolactin and its action on hypothalamic Npy expression were fully evidenced, leading to increased food intake and adiposity. Our results highlight the metabolic role of prolactin and illustrate the value of studying cell-specific mutant mice to disentangle the pathophysiological mechanisms otherwise masked in null allele mutants or in animals treated with pervasive pharmacological agents.

  16. Fetal Alcohol Exposure Reduces Dopamine Receptor D2 and Increases Pituitary Weight and Prolactin Production via Epigenetic Mechanisms

    PubMed Central

    Gangisetty, Omkaram; Wynne, Olivia; Jabbar, Shaima; Nasello, Cara; Sarkar, Dipak K.

    2015-01-01

    Recent evidence indicated that alcohol exposure during the fetal period increases the susceptibility to tumor development in mammary and prostate tissues. Whether fetal alcohol exposure increases the susceptibility to prolactin-producing tumor (prolactinoma) development in the pituitary was studied by employing the animal model of estradiol-induced prolactinomas in Fischer 344 female rats. We employed an animal model of fetal alcohol exposure that simulates binge alcohol drinking during the first two trimesters of human pregnancy and involves feeding pregnant rats with a liquid diet containing 6.7% alcohol during gestational day 7 to day 21. Control rats were pair-fed with isocaloric liquid diet or fed ad libitum with rat chow diet. Adult alcohol exposed and control female offspring rats were used in this study on the day of estrus or after estrogen treatment. Results show that fetal alcohol-exposed rats had increased levels of pituitary weight, pituitary prolactin (PRL) protein and mRNA, and plasma PRL. However, these rats show decreased pituitary levels of dopamine D2 receptor (D2R) mRNA and protein and increased pituitary levels of D2R promoter methylation. Also, they show elevated pituitary mRNA levels of DNA methylating genes (DNMT1, DNMT3b, MeCP2) and histone modifying genes (HDAC2, HDAC4, G9a). When fetal alcohol exposed rats were treated neonatally with a DNA methylation inhibitor 5-Aza deoxycytidine and/or a HDAC inhibitor trichostatin-A their pituitary D2R mRNA, pituitary weights and plasma PRL levels were normalized. These data suggest that fetal alcohol exposure programs the pituitary to increase the susceptibility to the development of prolactinomas possibly by enhancing the methylation of the D2R gene promoter and repressing the synthesis and control of D2R on PRL-producing cells. PMID:26509893

  17. Methamphetamine-induced short-term increase and long-term decrease in spatial working memory affects protein Kinase M zeta (PKMζ), dopamine, and glutamate receptors.

    PubMed

    Braren, Stephen H; Drapala, Damian; Tulloch, Ingrid K; Serrano, Peter A

    2014-01-01

    Methamphetamine (MA) is a toxic, addictive drug shown to modulate learning and memory, yet the neural mechanisms are not fully understood. We investigated the effects of 2 weekly injections of MA (30 mg/kg) on working memory using the radial 8-arm maze (RAM) across 5 weeks in adolescent-age mice. MA-treated mice show a significant improvement in working memory performance 1 week following the first MA injection compared to saline-injected controls. Following 5 weeks of MA abstinence mice were re-trained on a reference and working memory version of the RAM to assess cognitive flexibility. MA-treated mice show significantly more working memory errors without effects on reference memory performance. The hippocampus and dorsal striatum were assessed for expression of glutamate receptors subunits, GluA2 and GluN2B; dopamine markers, dopamine 1 receptor (D1), dopamine transporter (DAT) and tyrosine hydroxylase (TH); and memory markers, protein kinase M zeta (PKMζ) and protein kinase C zeta (PKCζ). Within the hippocampus, PKMζ and GluA2 are both significantly reduced after MA supporting the poor memory performance. Additionally, a significant increase in GluN2B and decrease in D1 identifies dysregulated synaptic function. In the striatum, MA treatment increased cytosolic DAT and TH levels associated with dopamine hyperfunction. MA treatment significantly reduced GluN2B while increasing both PKMζ and PKCζ within the striatum. We discuss the potential role of PKMζ/PKCζ in modulating dopamine and glutamate receptors after MA treatment. These results identify potential underlying mechanisms for working memory deficits induced by MA. PMID:25566006

  18. Neuroinflammation mediated by IL-1β increases susceptibility of dopamine neurons to degeneration in an animal model of Parkinson's disease

    PubMed Central

    Koprich, James B; Reske-Nielsen, Casper; Mithal, Prabhakar; Isacson, Ole

    2008-01-01

    Background The etiology of Parkinson's disease (PD) remains elusive despite identification of several genetic mutations. It is more likely that multiple factors converge to give rise to PD than any single cause. Here we report that inflammation can trigger degeneration of dopamine (DA) neurons in an animal model of Parkinson's disease. Methods We examined the effects of inflammation on the progressive 6-OHDA rat model of Parkinson's disease using immunohistochemistry, multiplex ELISA, and cell counting stereology. Results We show that a non-toxic dose of lipopolysaccharide (LPS) induced secretion of cytokines and predisposed DA neurons to be more vulnerable to a subsequent low dose of 6-hydroxydopamine. Alterations in cytokines, prominently an increase in interleukin-1beta (IL-1β), were identified as being potential mediators of this effect that was associated with activation of microglia. Administration of an interleukin-1 receptor antagonist resulted in significant reductions in tumor necrosis factor-α and interferon-γ and attenuated the augmented loss of DA neurons caused by the LPS-induced sensitization to dopaminergic degeneration. Conclusion These data provide insight into the etiology of PD and support a role for inflammation as a risk factor for the development of neurodegenerative disease. PMID:18304357

  19. Acute intraperitoneal injection of caffeine improves endurance exercise performance in association with increasing brain dopamine release during exercise.

    PubMed

    Zheng, Xinyan; Takatsu, Satomi; Wang, Hongli; Hasegawa, Hiroshi

    2014-07-01

    The purpose of this study was to examine changes of thermoregulation, neurotransmitters in the preoptic area and anterior hypothalamus (PO/AH), which is the thermoregulatory center, and endurance exercise performance after the intraperitoneal injection of caffeine in rats. Core body temperature (Tcore), oxygen consumption (VO₂) and tail skin temperature (Ttail) were measured. A microdialysis probe was inserted in the PO/AH, and samples for the measurements of extracellular dopamine (DA), noradrenaline (NA) and serotonin (5-HT) levels were collected. During the rest experiment, 1 h after baseline collections in the chamber (23 °C), the rats were intraperitoneally injected with saline, or 3 mg kg(-1) or 10 mg kg(-1) caffeine. The duration of the test was 4 h. During the exercise experiment, baseline collections on the treadmill were obtained for 1 h. One hour before the start of exercise, rats were intraperitoneally injected with either 10 mg kg(-1) caffeine (CAF) or saline (SAL). Animals ran until fatigue at a speed of 18 m min(-1), at a 5% grade, on the treadmill in a normal environment (23 °C). At rest, 3 mg kg(-1) caffeine did not influence Tcore, Ttail, VO₂, extracellular DA, NA and 5-HT. 10 mg kg(-1) caffeine caused significant increases in Tcore, VO₂, Ttail and extracellular DA in the PO/AH. In addition, 10 mg kg(-1) caffeine increased the run time to fatigue (SAL: 104.4 ± 30.9 min, CAF: 134.0 ± 31.1 min, p<0.05). The combination of caffeine and exercise increased Tcore, VO₂, Ttail and extracellular DA in the PO/AH. NA increased during exercise, while neither caffeine nor exercise changed 5-HT. These results indicate that caffeine has ergogenic and hyperthermic effects, and these effects may be related to changes of DA release in the brain. PMID:24726708

  20. Acute intraperitoneal injection of caffeine improves endurance exercise performance in association with increasing brain dopamine release during exercise.

    PubMed

    Zheng, Xinyan; Takatsu, Satomi; Wang, Hongli; Hasegawa, Hiroshi

    2014-07-01

    The purpose of this study was to examine changes of thermoregulation, neurotransmitters in the preoptic area and anterior hypothalamus (PO/AH), which is the thermoregulatory center, and endurance exercise performance after the intraperitoneal injection of caffeine in rats. Core body temperature (Tcore), oxygen consumption (VO₂) and tail skin temperature (Ttail) were measured. A microdialysis probe was inserted in the PO/AH, and samples for the measurements of extracellular dopamine (DA), noradrenaline (NA) and serotonin (5-HT) levels were collected. During the rest experiment, 1 h after baseline collections in the chamber (23 °C), the rats were intraperitoneally injected with saline, or 3 mg kg(-1) or 10 mg kg(-1) caffeine. The duration of the test was 4 h. During the exercise experiment, baseline collections on the treadmill were obtained for 1 h. One hour before the start of exercise, rats were intraperitoneally injected with either 10 mg kg(-1) caffeine (CAF) or saline (SAL). Animals ran until fatigue at a speed of 18 m min(-1), at a 5% grade, on the treadmill in a normal environment (23 °C). At rest, 3 mg kg(-1) caffeine did not influence Tcore, Ttail, VO₂, extracellular DA, NA and 5-HT. 10 mg kg(-1) caffeine caused significant increases in Tcore, VO₂, Ttail and extracellular DA in the PO/AH. In addition, 10 mg kg(-1) caffeine increased the run time to fatigue (SAL: 104.4 ± 30.9 min, CAF: 134.0 ± 31.1 min, p<0.05). The combination of caffeine and exercise increased Tcore, VO₂, Ttail and extracellular DA in the PO/AH. NA increased during exercise, while neither caffeine nor exercise changed 5-HT. These results indicate that caffeine has ergogenic and hyperthermic effects, and these effects may be related to changes of DA release in the brain.

  1. Overexpression of the Brassica napus BnLAS gene in Arabidopsis affects plant development and increases drought tolerance.

    PubMed

    Yang, Minggui; Yang, Qingyong; Fu, Tingdong; Zhou, Yongming

    2011-03-01

    The GRAS proteins are a family of transcription regulators found in plants and play diverse roles in plant growth and development. To study the biological roles of GRAS family genes in Brassica napus, an Arabidopsis LAS homologous gene, BnLAS and its two homologs were cloned from B. napus and its two progenitor species, Brassica rapa and Brassica oleracea. Relatively high levels of BnLAS were observed in roots, shoot tips, lateral meristems and flower organs based on the analysis of the transcripts by quantitative RT-PCR and promoter-reporter assays. Constitutive overexpression of BnLAS in Arabidopsis resulted in inhibition of growth, and delays in leaf senescence and flowering time. A large portion of transgenic lines had darker leaf color and higher chlorophyll content than in wild type plants. Interestingly, water lose rates in transgenic leaves were reduced, and transgenic plants exhibited enhanced drought tolerance and increased recovery after exposed to dehydration treatment. The stomatal density on leaves of the transgenic plants increased significantly due to the smaller cell size. However, the stomatal aperture on the leaves of the transgenic plants reduced significantly compared with wild type plants. More epidermal wax deposition on transgenic leaves was observed. Furthermore, several genes involved in wax synthesis and regulation, including CER1, CER2, KCS1 and KCS2, were upregulated in the transgenic plants. Our results indicate a potential to utilize BnLAS in the improvement of drought tolerance in plants.

  2. Decreased striatal dopamine release underlies increased expression of long-term synaptic potentiation at corticostriatal synapses 24 hours after 3-nitropropionic acid induced chemical hypoxia

    PubMed Central

    Akopian, Garnik; Crawford, Cynthia; Beal, M. Flint; Cappelletti, Maurand; Jakowec, Michael W.; Petzinger, Giselle M.; Zheng, Ling; Gheorghe, Stacey L.; Reichel, Carmela M.; Chow, Robert; Walsh, John P

    2008-01-01

    The striatum is particularly sensitive to the irreversible inhibitor of succinate dehyrdrogenase 3-nitropropionic acid (3-NP). In the present study we examined early changes in behavior and dopamine and glutamate synaptic physiology created by a single systemic injection of 3-NP in Fischer 344 rats. Hind limb dystonia was seen 2 hours after 3-NP injections and rats performed poorly on balance beam and rota-rod motor tests 24 hours later. Systemic 3-NP increased NMDA receptor-dependent long-term potentiation (LTP) at corticostriatal synapses over the same time period. The 3-NP induced corticostriatal LTP was not due to increased NMDA receptor number or function, since 3-NP did not change MK-801 binding or NMDA/AMPA receptor current ratios. The LTP seen 24 hours after 3-NP was D1 receptor-dependent and reversed by exogenous addition of dopamine or a D2 receptor agonist to brain slices. High performance liquid chromatography and fast scan cyclic voltammetry revealed a decrease in dopamine content and release in rats injected 24 hours earlier with 3-NP, and much like the enhanced LTP, dopamine changes were reversed by 48 hours. Tyrosine hydroxylase expression was not changed and there was no evidence of striatal cell loss at 24–48 hours after 3-NP exposure. Sprague-Dawley rats showed similar physiological responses to systemic 3-NP, albeit with reduced sensitivity. Thus, 3-NP causes significant changes in motor behavior marked by parallel changes in striatal dopamine release and corticostriatal synaptic plasticity. PMID:18799690

  3. Modeling bipolar disorder in mice by increasing acetylcholine or dopamine: Chronic lithium treats most, but not all features

    PubMed Central

    van Enkhuizen, Jordy; Milienne-Petiot, Morgane; Geyer, Mark A.; Young, Jared W.

    2015-01-01

    Rationale Bipolar disorder (BD) is a disabling and life-threatening disease characterized by states of depression and mania. New and efficacious treatments have not been forthcoming partly due to a lack of well-validated models representing both facets of BD. Objectives We hypothesized that cholinergic- and dopaminergic-pharmacological manipulations would model depression and mania respectively, each attenuated by lithium treatment. Methods C57BL/6J mice received the acetylcholinesterase inhibitor physostigmine or saline before testing for ‘behavioral despair’ (immobility) in the tail-suspension test (TST) and forced-swim test (FST). Physostigmine effects on exploration and sensorimotor gating were assessed using the cross-species behavioral pattern monitor (BPM) and prepulse inhibition (PPI) paradigms. Other C57BL/6J mice received chronic lithium drinking water (300, 600, or 1200 mg/l) before assessing their effects alone in the BPM or with physostigmine on FST performance. Another group was tested with acute GBR12909 (dopamine transporter inhibitor) and chronic lithium (1000 mg/l) in the BPM. Results Physostigmine (0.03 mg/kg) increased immobility in the TST and FST without affecting activity, exploration, or PPI. Lithium (600 mg/l) resulted in low therapeutic serum concentrations and normalized the physostigmine-increased immobility in the FST. GBR12909 induced mania-like behavior in the BPM of which hyper-exploration was attenuated, though not reversed, after chronic lithium (1000 mg/ml). Conclusions Increased cholinergic levels induced depression-like behavior and hyperdopaminergia induced mania-like behavior in mice, while chronic lithium treated some, but not all, facets of these effects. These data support a cholinergic-monoaminergic mechanism for modeling BD aspects and provide a way to assess novel therapeutics. PMID:26141192

  4. Claudin-3 Overexpression Increases the Malignant Potential of Colorectal Cancer Cells: Roles of ERK1/2 and PI3K-Akt as Modulators of EGFR signaling

    PubMed Central

    de Souza, Waldemir F.; Fortunato-Miranda, Natalia; Robbs, Bruno K.; de Araujo, Wallace M.; de-Freitas-Junior, Julio C.; Bastos, Lilian G.; Viola, João P. B.; Morgado-Díaz, José A.

    2013-01-01

    The altered expressions of claudin proteins have been reported during the tumorigenesis of colorectal cancer. However, the molecular mechanisms that regulate these events in this cancer type are poorly understood. Here, we report that epidermal growth factor (EGF) increases the expression of claudin-3 in human colorectal adenocarcinoma HT-29 cells. This increase was related to increased cell migration and the formation of anchorage-dependent and anchorage-independent colonies. We further showed that the ERK1/2 and PI3K-Akt pathways were involved in the regulation of these effects because specific pharmacological inhibition blocked these events. Genetic manipulation of claudin-1 and claudin-3 in HT-29 cells showed that the overexpression of claudin-1 resulted in decreased cell migration; however, migration was not altered in cells that overexpressed claudin-3. Furthermore, the overexpression of claudin-3, but not that of claudin-1, increased the tight junction-related paracellular flux of macromolecules. Additionally, an increased formation of anchorage-dependent and anchorage-independent colonies were observed in cells that overexpressed claudin-3, while no such changes were observed when claudin-1 was overexpressed. Finally, claudin-3 silencing alone despite induce increase proliferation, and the formation of anchoragedependent and -independent colonies, it was able to prevent the EGF-induced increased malignant potential. In conclusion, our results show a novel role for claudin-3 overexpression in promoting the malignant potential of colorectal cancer cells, which is potentially regulated by the EGF-activated ERK1/2 and PI3K-Akt pathways. PMID:24069372

  5. Overexpression of the alpha-2,6-sialyltransferase in MDCK cells increases influenza virus sensitivity to neuraminidase inhibitors.

    PubMed

    Matrosovich, Mikhail; Matrosovich, Tatyana; Carr, Jackie; Roberts, Noel A; Klenk, Hans-Dieter

    2003-08-01

    No reliable cell culture assay is currently available for monitoring human influenza virus sensitivity to neuraminidase inhibitors (NAI). This can be explained by the observation that because of a low concentration of sialyl-alpha2,6-galactose (Sia[alpha2,6]Gal)-containing virus receptors in conventional cell lines, replication of human virus isolates shows little dependency on viral neuraminidase. To test whether overexpression of Sia(alpha2,6)Gal moieties in cultured cells could make them suitable for testing human influenza virus sensitivity to NAI, we stably transfected MDCK cells with cDNA of human 2,6-sialyltransferase (SIAT1). Transfected cells expressed twofold-higher amounts of 6-linked sialic acids and twofold-lower amounts of 3-linked sialic acids than parent MDCK cells as judged by staining with Sambucus nigra agglutinin and Maackia amurensis agglutinin, respectively. After transfection, binding of a clinical human influenza virus isolate was increased, whereas binding of its egg-adapted variant which preferentially bound 3-linked receptors was decreased. The sensitivity of human influenza A and B viruses to the neuraminidase inhibitor oseltamivir carboxylate was substantially improved in the SIAT1-transfected cell line and was consistent with their sensitivity in neuraminidase enzyme assay and with the hemagglutinin (HA) receptor-binding phenotype. MDCK cells stably transfected with SIAT1 may therefore be a suitable system for testing influenza virus sensitivity to NAI. PMID:12857911

  6. Striatal dopamine D2/3 receptor availability increases after long-term bariatric surgery-induced weight loss.

    PubMed

    van der Zwaal, Esther M; de Weijer, Barbara A; van de Giessen, Elsmarieke M; Janssen, Ignace; Berends, Frits J; van de Laar, Arnold; Ackermans, Mariette T; Fliers, Eric; la Fleur, Susanne E; Booij, Jan; Serlie, Mireille J

    2016-07-01

    In several studies reduced striatal dopamine D2/3 receptor (D2/3R) availability was reported in obese subjects compared to lean controls. Whether this is a reversible phenomenon remained uncertain. We previously determined the short-term effect of Roux-en-Y gastric bypass surgery (RYGB) on striatal D2/3R availability (using [(123)I]IBZM SPECT) in 20 morbidly obese women. Striatal D2/3R availability was lower compared to controls at baseline, and remained unaltered after 6 weeks, despite significant weight loss. To determine whether long-term bariatric surgery-induced weight loss normalizes striatal D2/3R binding, we repeated striatal D2/3R binding measurements at least 2 years after RYGB in 14 subjects of the original cohort. In addition, we assessed long-term changes in body composition, eating behavior and fasting plasma levels of leptin, ghrelin, insulin and glucose. Mean body mass index declined from 46±7kg/m(2) to 32±6kg/m(2), which was accompanied by a significant increase in striatal D2/3R availability (p=0.031). Striatal D2/3R availability remained significantly reduced compared to the age-matched controls (BMI 22±2kg/m(2); p=0.01). Changes in striatal D2/3R availability did not correlate with changes in body weight/fat, insulin sensitivity, ghrelin or leptin levels. Scores on eating behavior questionnaires improved and changes in the General Food Craving Questionnaire-State showed a borderline significant correlation with changes in striatal D2/3R availability. These findings show that striatal D2/3R availability increases after long-term bariatric-surgery induced weight loss, suggesting that reduced D2/3R availability in obesity is a reversible phenomenon. PMID:27184782

  7. Intranasal dopamine application increases dopaminergic activity in the neostriatum and nucleus accumbens and enhances motor activity in the open field.

    PubMed

    de Souza Silva, M A; Topic, B; Huston, J P; Mattern, C

    2008-03-01

    Dopamine (DA) plays an important role in a number of behavioral processes and neurological disorders. The intranasal administration of DA provides improved brain penetrability in comparison to systemic administration. We investigated the effects of intranasal administration of DA on the activity of dopaminergic neurons of the mesostriatal and mesolimbic systems and on motor activity. Rats previously implanted with guide-cannulae in the neostriatum (NS) and nucleus accumbens (NAc) were submitted to microdialysis procedure under urethane anesthesia. Vehicle or DA (0.03, 0.3, or 3.0 mg/kg) was administered bilaterally into the nostrils. In a separate study, animals received an intraperitoneal (i.p.) injection of vehicle or DA (0.03, 0.3, 3.0, or 30.0 mg/kg). Samples were collected every 10 min and analyzed for the content of DA and metabolites using high-performance liquid chromatography. For the open field study, rats were given intranasal vehicle or DA (0.03, 0.3, or 3.0 mg/kg) and placed into the field for 30 min. Motor activity (locomotion and rearing) and grooming were analyzed in blocks of 10 min using Ethovision. Intranasal DA (3.0 mg/kg) significantly increased DA levels in the NS and NAc immediately after administration. A comparable effect was obtained only after i.p. administration of 30 mg/kg DA. In the open field, the 3.0 mg/kg dose significantly decreased grooming behavior in the second 10 min interval and significantly increased locomotor activity in the third 10 min interval. The data indicate that intranasal administration of DA can influence dopaminergic functions and motor activity, and has a potential application in the therapy of diseases affecting the dopaminergic system.

  8. Overexpression of Heat Shock Factor Gene HsfA3 Increases Galactinol Levels and Oxidative Stress Tolerance in Arabidopsis.

    PubMed

    Song, Chieun; Chung, Woo Sik; Lim, Chae Oh

    2016-06-30

    Heat shock factors (Hsfs) are central regulators of abiotic stress responses, especially heat stress responses, in plants. In the current study, we characterized the activity of the Hsf gene HsfA3 in Arabidopsis under oxidative stress conditions. HsfA3 transcription in seedlings was induced by reactive oxygen species (ROS), exogenous hydrogen peroxide (H2O2), and an endogenous H2O2 propagator, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB). HsfA3-overexpressing transgenic plants exhibited increased oxidative stress tolerance compared to untransformed wild-type plants (WT), as revealed by changes in fresh weight, chlorophyll fluorescence, and ion leakage under light conditions. The expression of several genes encoding galactinol synthase (GolS), a key enzyme in the biosynthesis of raffinose family oligosaccharides (RFOs), which function as antioxidants in plant cells, was induced in HsfA3 overexpressors. In addition, galactinol levels were higher in HsfA3 overexpressors than in WT under unstressed conditions. In transient transactivation assays using Arabidopsis leaf protoplasts, HsfA3 activated the transcription of a reporter gene driven by the GolS1 or GolS2 promoter. Electrophoretic mobility shift assays showed that GolS1 and GolS2 are directly regulated by HsfA3. Taken together, these findings provide evidence that GolS1 and GolS2 are directly regulated by HsfA3 and that GolS enzymes play an important role in improving oxidative stress tolerance by increasing galactinol biosynthesis in Arabidopsis. PMID:27109422

  9. Overexpression of Heat Shock Factor Gene HsfA3 Increases Galactinol Levels and Oxidative Stress Tolerance in Arabidopsis

    PubMed Central

    Song, Chieun; Chung, Woo Sik; Lim, Chae Oh

    2016-01-01

    Heat shock factors (Hsfs) are central regulators of abiotic stress responses, especially heat stress responses, in plants. In the current study, we characterized the activity of the Hsf gene HsfA3 in Arabidopsis under oxidative stress conditions. HsfA3 transcription in seedlings was induced by reactive oxygen species (ROS), exogenous hydrogen peroxide (H2O2), and an endogenous H2O2 propagator, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB). HsfA3-overexpressing transgenic plants exhibited increased oxidative stress tolerance compared to untransformed wild-type plants (WT), as revealed by changes in fresh weight, chlorophyll fluorescence, and ion leakage under light conditions. The expression of several genes encoding galactinol synthase (GolS), a key enzyme in the biosynthesis of raffinose family oligosaccharides (RFOs), which function as antioxidants in plant cells, was induced in HsfA3 overexpressors. In addition, galactinol levels were higher in HsfA3 overexpressors than in WT under unstressed conditions. In transient transactivation assays using Arabidopsis leaf protoplasts, HsfA3 activated the transcription of a reporter gene driven by the GolS1 or GolS2 promoter. Electrophoretic mobility shift assays showed that GolS1 and GolS2 are directly regulated by HsfA3. Taken together, these findings provide evidence that GolS1 and GolS2 are directly regulated by HsfA3 and that GolS enzymes play an important role in improving oxidative stress tolerance by increasing galactinol biosynthesis in Arabidopsis. PMID:27109422

  10. C-kit overexpression correlates with KIT gene copy numbers increases in phyllodes tumors of the breast.

    PubMed

    Liu, Junjun; Liu, Xiaozhen; Feng, Xiaolong; Liu, Jian; Lv, Shuhua; Zhang, Wei; Niu, Yun

    2015-01-01

    We determined c-kit expression in the stroma and epithelia of benign, borderline, and malignant phyllodes tumors (PTs), respectively, as well as the relationship between c-kit expression in stromal elements and KIT gene copy number variations (CNVs). To assess c-kit expression and KIT CNVs, 348 PT cases were studied: 120 (34.4 %) benign cases, 115 (33.1 %) borderline cases, and 113 (32.5 %) malignant cases. All of these cases were evaluated for c-kit (CD117) expression using immunohistochemistry. Forty-two cases (29 c-kit-positive in the stromal cells cases and 13 negative cases) were investigated for KIT gene CNVs via genomic polymerase chain reaction (PCR). The overall rate of c-kit positivity in the stroma was 46.8 %, as well as 24.2, 53.1, and 64.6 %, respectively, in PTs of three different grades. However, in the majority of cases, the epithelia were c-kit positive (98.2 %), and the positivity was 100, 99.1, and 95 % in PTs of three different grades, respectively. There was a significant change in the expression of c-kit in the stroma and epithelia according to grade (P < 0.001, P = 0.014). From the genomic PCR results, we can confirm that c-kit positivity in the stroma is directly correlated with KIT gene copy numbers increases (P = 0.003, P = 0.041). We demonstrated that c-kit expression in the stroma of PTs is positively associated with malignancy. c-Kit epithelial positivity was inversely correlated with PTs malignancy. c-Kit overexpression in the stroma was related to KIT gene copy numbers increases. PMID:25534827

  11. A murine dopamine neuron-specific cDNA library and microarray: increased COX1 expression during methamphetamine neurotoxicity.

    PubMed

    Barrett, T; Xie, T; Piao, Y; Dillon-Carter, O; Kargul, G J; Lim, M K; Chrest, F J; Wersto, R; Rowley, D L; Juhaszova, M; Zhou, L; Vawter, M P; Becker, K G; Cheadle, C; Wood, W H; McCann, U D; Freed, W J; Ko, M S; Ricaurte, G A; Donovan, D M

    2001-10-01

    Due to brain tissue heterogeneity, the molecular genetic profile of any neurotransmitter-specific neuronal subtype is unknown. The purpose of this study was to purify a population of dopamine neurons, construct a cDNA library, and generate an initial gene expression profile and a microarray representative of dopamine neuron transcripts. Ventral mesencephalic dopamine neurons were purified by fluorescent-activated cell sorting from embryonic day 13.5 transgenic mice harboring a 4.5-kb rat tyrosine hydroxylase promoter-lacZ fusion. Nine-hundred sixty dopamine neuron cDNA clones were sequenced and arrayed for use in studies of gene expression changes during methamphetamine neurotoxicity. A neurotoxic dose of methamphetamine produced a greater than twofold up-regulation of the mitochondrial cytochrome c oxidase polypeptide I transcript from adult mouse substantia nigra at 12 h posttreatment. This is the first work to describe a gene expression profile for a neuronal subtype and to identify gene expression changes during methamphetamine neurotoxicity. PMID:11592851

  12. Further increased production of free fatty acids by overexpressing a predicted transketolase gene of the pentose phosphate pathway in Aspergillus oryzae faaA disruptant.

    PubMed

    Tamano, Koichi; Miura, Ai

    2016-09-01

    Free fatty acids are useful as source materials for the production of biodiesel fuel and various chemicals such as pharmaceuticals and dietary supplements. Previously, we attained a 9.2-fold increase in free fatty acid productivity by disrupting a predicted acyl-CoA synthetase gene (faaA, AO090011000642) in Aspergillus oryzae. In this study, we achieved further increase in the productivity by overexpressing a predicted transketolase gene of the pentose phosphate pathway in the faaA disruptant. The A. oryzae genome is predicted to have three transketolase genes and overexpression of AO090023000345, one of the three genes, resulted in phenotypic change and further increase (corresponding to an increased production of 0.38 mmol/g dry cell weight) in free fatty acids at 1.4-fold compared to the faaA disruptant. Additionally, the biomass of hyphae increased at 1.2-fold by the overexpression. As a result, free fatty acid production yield per liter of liquid culture increased at 1.7-fold by the overexpression.

  13. Hoxb4 Overexpression in CD4 Memory Phenotype T Cells Increases the Central Memory Population upon Homeostatic Proliferation

    PubMed Central

    Fournier, Marilaine; Labrecque, Nathalie; Bijl, Janet J.

    2013-01-01

    Memory T cell populations allow a rapid immune response to pathogens that have been previously encountered and thus form the basis of success in vaccinations. However, the molecular pathways underlying the development and maintenance of these cells are only starting to be unveiled. Memory T cells have the capacity to self renew as do hematopoietic stem cells, and overlapping gene expression profiles suggested that these cells might use the same self-renewal pathways. The transcription factor Hoxb4 has been shown to promote self-renewal divisions of hematopoietic stem cells resulting in an expansion of these cells. In this study we investigated whether overexpression of Hoxb4 could provide an advantage to CD4 memory phenotype T cells in engrafting the niche of T cell deficient mice following adoptive transfer. Competitive transplantation experiments demonstrated that CD4 memory phenotype T cells derived from mice transgenic for Hoxb4 contributed overall less to the repopulation of the lymphoid organs than wild type CD4 memory phenotype T cells after two months. These proportions were relatively maintained following serial transplantation in secondary and tertiary mice. Interestingly, a significantly higher percentage of the Hoxb4 CD4 memory phenotype T cell population expressed the CD62L and Ly6C surface markers, characteristic for central memory T cells, after homeostatic proliferation. Thus Hoxb4 favours the maintenance and increase of the CD4 central memory phenotype T cell population. These cells are more stem cell like and might eventually lead to an advantage of Hoxb4 T cells after subjecting the cells to additional rounds of proliferation. PMID:24324706

  14. Overexpression of SAMDC1 gene in Arabidopsis thaliana increases expression of defense-related genes as well as resistance to Pseudomonas syringae and Hyaloperonospora arabidopsidis

    PubMed Central

    Marco, Francisco; Busó, Enrique; Carrasco, Pedro

    2014-01-01

    It has been previously described that elevation of endogenous spermine levels in Arabidopsis could be achieved by transgenic overexpression of S-Adenosylmethionine decarboxylase (SAMDC) or Spermine synthase (SPMS). In both cases, spermine accumulation had an impact on the plant transcriptome, with up-regulation of a set of genes enriched in functional categories involved in defense-related processes against both biotic and abiotic stresses. In this work, the response of SAMDC1-overexpressing plants against bacterial and oomycete pathogens has been tested. The expression of several pathogen defense-related genes was induced in these plants as well as in wild type plants exposed to an exogenous supply of spermine. SAMDC1-overexpressing plants showed an increased tolerance to infection by Pseudomonas syringae and by Hyaloperonospora arabidopsidis. Both results add more evidence to the hypothesis that spermine plays a key role in plant resistance to biotic stress. PMID:24734036

  15. Over-expression of MAGED4B increases cell migration and growth in oral squamous cell carcinoma and is associated with poor disease outcome.

    PubMed

    Chong, Chan Eng; Lim, Kue Peng; Gan, Chai Phei; Marsh, Christina A; Zain, Rosnah Binti; Abraham, Mannil Thomas; Prime, Stephen S; Teo, Soo-Hwang; Silvio Gutkind, J; Patel, Vyomesh; Cheong, Sok Ching

    2012-08-01

    MAGE proteins have been shown to be good targets for cancer immunotherapy. We demonstrate that MAGED4B is over-expressed in more than 50% of Oral Squamous Cell Carcinoma (OSCC) tissues and the expression of MAGED4B is associated with lymph node metastasis and poor disease specific survival. OSCC cell lines that over-express MAGED4B promote migration in vitro, exhibit an increase in cell growth both in vitro and in vivo, and are more resistant to apoptosis compared to control cells. Our data suggest that MAGED4B over-expression is a driver in oral carcinogenesis and argues strongly that this protein may represent a potential therapeutic target in OSCC. PMID:22459352

  16. Amphetamine paradoxically augments exocytotic dopamine release and phasic dopamine signals.

    PubMed

    Daberkow, D P; Brown, H D; Bunner, K D; Kraniotis, S A; Doellman, M A; Ragozzino, M E; Garris, P A; Roitman, M F

    2013-01-01

    Drugs of abuse hijack brain-reward circuitry during the addiction process by augmenting action potential-dependent phasic dopamine release events associated with learning and goal-directed behavior. One prominent exception to this notion would appear to be amphetamine (AMPH) and related analogs, which are proposed instead to disrupt normal patterns of dopamine neurotransmission by depleting vesicular stores and promoting nonexocytotic dopamine efflux via reverse transport. This mechanism of AMPH action, though, is inconsistent with its therapeutic effects and addictive properties, which are thought to be reliant on phasic dopamine signaling. Here we used fast-scan cyclic voltammetry in freely moving rats to interrogate principal neurochemical responses to AMPH in the striatum and relate these changes to behavior. First, we showed that AMPH dose-dependently enhanced evoked dopamine responses to phasic-like current pulse trains for up to 2 h. Modeling the data revealed that AMPH inhibited dopamine uptake but also unexpectedly potentiated vesicular dopamine release. Second, we found that AMPH increased the amplitude, duration, and frequency of spontaneous dopamine transients, the naturally occurring, nonelectrically evoked, phasic increases in extracellular dopamine. Finally, using an operant sugar reward paradigm, we showed that low-dose AMPH augmented dopamine transients elicited by sugar-predictive cues. However, operant behavior failed at high-dose AMPH, which was due to phasic dopamine hyperactivity and the decoupling of dopamine transients from the reward predictive cue. These findings identify upregulation of exocytotic dopamine release as a key AMPH action in behaving animals and support a unified mechanism of abused drugs to activate phasic dopamine signaling. PMID:23303926

  17. Over-expression of OsPTR6 in rice increased plant growth at different nitrogen supplies but decreased nitrogen use efficiency at high ammonium supply.

    PubMed

    Fan, Xiaorong; Xie, Dan; Chen, Jingguang; Lu, Haiyan; Xu, Yanling; Ma, Cui; Xu, Guohua

    2014-10-01

    Nitrogen (N) plays a critical role in plant growth and productivity and PTR/NRT1 transporters are critical for rice growth. In this study, OsPTR6, a PTR/NRT1 transporter, was over-expressed in the Nipponbare rice cultivar by Agrobacterium tumefaciens transformation using the ubiquitin (Ubi) promoter. Three single-copy T2 generation transgenic lines, named OE1, OE5 and OE6, were produced and subjected to hydroponic growth experiments in different nitrogen treatments. The results showed the plant height and biomass of the over-expression lines were increased, and plant N accumulation and glutamine synthetase (GS) activities were enhanced at 5.0mmol/L NH4(+) and 2.5mmol/L NH4NO3. The expression of OsATM1 genes in over-expression lines showed that the OsPTR6 over expression increased OsAMT1.1, OsATM1.2 and OsAMT1.3 expression at 0.2 and 5.0mmol/L NH4(+) and 2.5mmol/L NH4NO3. However, nitrogen utilisation efficiency (NUE) was decreased at 5.0mmol/LNH4(+). These data suggest that over-expression of the OsPTR6 gene could increase rice growth through increasing ammonium transporter expression and glutamine synthetase activity (GSA), but decreases nitrogen use efficiency under conditions of high ammonium supply.

  18. Overexpression of alpha-synuclein at non-toxic levels increases dopaminergic cell death induced by copper exposure via modulation of protein degradation pathways.

    PubMed

    Anandhan, Annadurai; Rodriguez-Rocha, Humberto; Bohovych, Iryna; Griggs, Amy M; Zavala-Flores, Laura; Reyes-Reyes, Elsa M; Seravalli, Javier; Stanciu, Lia A; Lee, Jaekwon; Rochet, Jean-Christophe; Khalimonchuk, Oleh; Franco, Rodrigo

    2015-09-01

    Gene multiplications or point mutations in alpha (α)-synuclein are associated with familial and sporadic Parkinson's disease (PD). An increase in copper (Cu) levels has been reported in the cerebrospinal fluid and blood of PD patients, while occupational exposure to Cu has been suggested to augment the risk to develop PD. We aimed to elucidate the mechanisms by which α-synuclein and Cu regulate dopaminergic cell death. Short-term overexpression of wild type (WT) or mutant A53T α-synuclein had no toxic effect in human dopaminergic cells and primary midbrain cultures, but it exerted a synergistic effect on Cu-induced cell death. Cell death induced by Cu was potentiated by overexpression of the Cu transporter protein 1 (Ctr1) and depletion of intracellular glutathione (GSH) indicating that the toxic effects of Cu are linked to alterations in its intracellular homeostasis. Using the redox sensor roGFP, we demonstrated that Cu-induced oxidative stress was primarily localized in the cytosol and not in the mitochondria. However, α-synuclein overexpression had no effect on Cu-induced oxidative stress. WT or A53T α-synuclein overexpression exacerbated Cu toxicity in dopaminergic and yeast cells in the absence of α-synuclein aggregation. Cu increased autophagic flux and protein ubiquitination. Impairment of autophagy by overexpression of a dominant negative Atg5 form or inhibition of the ubiquitin/proteasome system (UPS) with MG132 enhanced Cu-induced cell death. However, only inhibition of the UPS stimulated the synergistic toxic effects of Cu and α-synuclein overexpression. Our results demonstrate that α-synuclein stimulates Cu toxicity in dopaminergic cells independent from its aggregation via modulation of protein degradation pathways.

  19. Overexpression of alpha-synuclein at non-toxic levels increases dopaminergic cell death induced by copper exposure via modulation of protein degradation pathways

    PubMed Central

    Anandhan, Annadurai; Rodriguez-Rocha, Humberto; Bohovych, Iryna; Griggs, Amy M.; Zavala-Flores, Laura; Reyes-Reyes, Elsa M.; Seravalli, Javier; Stanciu, Lia A.; Lee, Jaekwon; Rochet, Jean-Christophe; Khalimonchuk, Oleh; Franco, Rodrigo

    2014-01-01

    Gene multiplications or point mutations in alpha (α)-synuclein are associated with familial and sporadic Parkinson’s disease (PD). An increase in copper (Cu) levels has been reported in the cerebrospinal fluid and blood of PD patients, while occupational exposure to Cu has been suggested to augment the risk to develop PD. We aimed to elucidate the mechanisms by which α-synuclein and Cu regulate dopaminergic cell death. Short-term overexpression of WT or A53T α-synuclein had no toxic effect in human dopaminergic cells and primary midbrain cultures, but it exerted a synergistic effect on Cu-induced cell death. Cell death induced by Cu was potentiated by overexpression of the Cu transporter protein 1 (Ctr1) and depletion of intracellular glutathione (GSH) indicating that the toxic effects of Cu are linked to alterations in its intracellular homeostasis. Using the redox sensor roGFP, we demonstrated that Cu-induced oxidative stress was primarily localized in the cytosol and not in the mitochondria. However, α-synuclein overexpression had no effect on Cu-induced oxidative stress. WT or A53T α-synuclein overexpression exacerbated Cu toxicity in dopaminergic cells and yeast in the absence of α-synuclein aggregation. Cu increased autophagic flux and protein ubiquitination. Impairment of autophagy by overexpression of a dominant negative Atg5 form or inhibition of the ubiquitin/proteasome system (UPS) with MG132 enhanced Cu-induced cell death. However, only inhibition of the UPS stimulated the synergistic toxic effects of Cu and α-synuclein overexpression. Our results demonstrate that α-synuclein stimulates Cu toxicity in dopaminergic cells independent from its aggregation via modulation of protein degradation pathways. PMID:25497688

  20. Overexpression of acyl-CoA synthetase-1 increases lipid deposition in hepatic (HepG2) cells and rodent liver in vivo.

    PubMed

    Parkes, Heidi A; Preston, Elaine; Wilks, Donna; Ballesteros, Mercedes; Carpenter, Lee; Wood, Leonie; Kraegen, Edward W; Furler, Stuart M; Cooney, Gregory J

    2006-10-01

    Accumulation of intracellular lipid in obesity is associated with metabolic disease in many tissues including liver. Storage of fatty acid as triglyceride (TG) requires the activation of fatty acids to long-chain acyl-CoAs (LC-CoA) by the enzyme acyl-CoA synthetase (ACSL). There are five known isoforms of ACSL (ACSL1, -3, -4, -5, -6), which vary in their tissue specificity and affinity for fatty acid substrates. To investigate the role of ACSL1 in the regulation of lipid metabolism, we used adenoviral-mediated gene transfer to overexpress ACSL1 in the human hepatoma cell-line HepG2 and in liver of rodents. Infection of HepG2 cells with the adenoviral construct AdACSL1 increased ACSL activity >10-fold compared with controls after 24 h. HepG2 cells overexpressing ACSL1 had a 40% higher triglyceride (TG) content (93 +/- 3 vs. 67 +/- 2 nmol/mg protein in controls, P < 0.05) after 24-h exposure to 1 mM oleate. Furthermore, ACSL1 overexpression produced a 60% increase in cellular LCA-CoA content (160 +/- 6 vs. 100 +/- 6 nmol/g protein in controls, P < 0.05) and increased [(14)C]oleate incorporation into TG without significantly altering fatty acid oxidation. In mice, AdACSL1 administration increased ACSL1 mRNA and protein more than fivefold over controls at 4 days postinfection. ACSL1 overexpression caused a twofold increase in TG content in mouse liver (39 +/- 4 vs. 20 +/- 2 mumol/g wet wt in controls, P < 0.05), and overexpression in rat liver increased [1-(14)C]palmitate clearance into liver TG. These in vitro and in vivo results suggest a pivotal role for ACSL1 in regulating TG synthesis in liver. PMID:16705061

  1. Prolonged survival in hepatocarcinoma patients with increased regucalcin gene expression: HepG2 cell proliferation is suppressed by overexpression of regucalcin in vitro.

    PubMed

    Yamaguchi, Masayoshi; Osuka, Satoru; Weitzmann, M Neale; El-Rayes, Bassel F; Shoji, Mamoru; Murata, Tomiyasu

    2016-10-01

    Hepatocellular carcinoma (HCC) is one of the most common malignant cancers worldwide and ranks third in overall global cancer-related mortality rates. Importantly, in this study gene expression data demonstrate that prolonged survival in HCC patients is associated with increased regucalcin gene expression. Regucalcin has been shown to play a pivotal role as a transcription repressor and diminished expression or activity of regucalcin may play a key role in the development of human carcinogenesis. Indeed, overexpression of regucalcin suppressed the proliferation, cell death, and migration of human HCC HepG2 cells in vitro. Mechanistically, regucalcin induced G1 and G2/M phase cell cycle arrest of HepG2 cells through suppression of multiple signaling pathways including Ras, Akt, MAP kinase and SAPK/JNK and by increasing the tumor suppressors p53 and Rb. Furthermore, the oncogenes c-fos and c-myc were suppressed by overexpression of regucalcin, and overexpression of regucalcin caused an increase in p21 and a decrease in NF-κB p65 and β-catenin. These findings suggest that regucalcin may play a potential role as a suppressor of human HCC, and that diminished expression of regucalcin may predispose patients to development of HCC. Overexpression of regucalcin may constitute a novel therapeutic approach to treating HCC. PMID:27633001

  2. D1-like dopamine receptors downregulate Na+-K+-ATPase activity and increase cAMP production in the posterior gills of the blue crab Callinectes sapidus.

    PubMed

    Arnaldo, Francis B; Villar, Van Anthony M; Konkalmatt, Prasad R; Owens, Shaun A; Asico, Laureano D; Jones, John E; Yang, Jian; Lovett, Donald L; Armando, Ines; Jose, Pedro A; Concepcion, Gisela P

    2014-09-15

    Dopamine-mediated regulation of Na(+)-K(+)-ATPase activity in the posterior gills of some crustaceans has been reported to be involved in osmoregulation. The dopamine receptors of invertebrates are classified into three groups based on their structure and pharmacology: D1- and D2-like receptors and a distinct invertebrate receptor subtype (INDR). We tested the hypothesis that a D1-like receptor is expressed in the blue crab Callinectes sapidus and regulates Na(+)-K(+)-ATPase activity. RT-PCR, using degenerate primers, showed the presence of D1βR mRNA in the posterior gill. The blue crab posterior gills showed positive immunostaining for a dopamine D5 receptor (D5R or D1βR) antibody in the basolateral membrane and cytoplasm. Confocal microscopy showed colocalization of Na(+)-K(+)-ATPase and D1βR in the basolateral membrane. To determine the effect of D1-like receptor stimulation on Na(+)-K(+)-ATPase activity, intact crabs acclimated to low salinity for 6 days were given an intracardiac infusion of the D1-like receptor agonist fenoldopam, with or without the D1-like receptor antagonist SCH23390. Fenoldopam increased cAMP production twofold and decreased Na(+)-K(+)-ATPase activity by 50% in the posterior gills. This effect was blocked by coinfusion with SCH23390, which had no effect on Na(+)-K(+)-ATPase activity by itself. Fenoldopam minimally decreased D1βR protein expression (10%) but did not affect Na(+)-K(+)-ATPase α-subunit protein expression. This study shows the presence of functional D1βR in the posterior gills of euryhaline crabs chronically exposed to low salinity and highlights the evolutionarily conserved function of the dopamine receptors on sodium homeostasis.

  3. D1-like dopamine receptors downregulate Na+-K+-ATPase activity and increase cAMP production in the posterior gills of the blue crab Callinectes sapidus

    PubMed Central

    Arnaldo, Francis B.; Villar, Van Anthony M.; Konkalmatt, Prasad R.; Owens, Shaun A.; Asico, Laureano D.; Jones, John E.; Yang, Jian; Lovett, Donald L.; Armando, Ines; Concepcion, Gisela P.

    2014-01-01

    Dopamine-mediated regulation of Na+-K+-ATPase activity in the posterior gills of some crustaceans has been reported to be involved in osmoregulation. The dopamine receptors of invertebrates are classified into three groups based on their structure and pharmacology: D1- and D2-like receptors and a distinct invertebrate receptor subtype (INDR). We tested the hypothesis that a D1-like receptor is expressed in the blue crab Callinectes sapidus and regulates Na+-K+-ATPase activity. RT-PCR, using degenerate primers, showed the presence of D1βR mRNA in the posterior gill. The blue crab posterior gills showed positive immunostaining for a dopamine D5 receptor (D5R or D1βR) antibody in the basolateral membrane and cytoplasm. Confocal microscopy showed colocalization of Na+-K+-ATPase and D1βR in the basolateral membrane. To determine the effect of D1-like receptor stimulation on Na+-K+-ATPase activity, intact crabs acclimated to low salinity for 6 days were given an intracardiac infusion of the D1-like receptor agonist fenoldopam, with or without the D1-like receptor antagonist SCH23390. Fenoldopam increased cAMP production twofold and decreased Na+-K+-ATPase activity by 50% in the posterior gills. This effect was blocked by coinfusion with SCH23390, which had no effect on Na+-K+-ATPase activity by itself. Fenoldopam minimally decreased D1βR protein expression (10%) but did not affect Na+-K+-ATPase α-subunit protein expression. This study shows the presence of functional D1βR in the posterior gills of euryhaline crabs chronically exposed to low salinity and highlights the evolutionarily conserved function of the dopamine receptors on sodium homeostasis. PMID:25080496

  4. Increased PKCα activity by Rack1 overexpression is responsible for chemotherapy resistance in T-cell acute lymphoblastic leukemia-derived cell line.

    PubMed

    Lei, Jie; Li, Qi; Gao, Ying; Zhao, Lei; Liu, Yanbo

    2016-01-01

    Chemoresistant mechanisms in T-cell acute lymphoblastic leukemia (T-ALL) patients are not clarified. The apoptotic signaling mediated by receptor of activated C kinase 1 (Rack1), protein kinase C (PKC) and FEM1 homolog b (FEM1b) was investigated in two T-ALL-derived cell lines (Jurkat and CCRF-CEM) following treatment with chemotherapy drugs vincristine and prednisone. Serum starvation or chemotherapeutic drugs significantly reduced Rack1 level and PKC activation, while promoted cellular apoptosis in both cell lines. Rack1 overexpression protected T-ALL cell against starvation or chemotherapeutic drug-induced apoptosis. Moreover, Rack1 overexpression reduced the level of cytochrome c and active caspase 3 as well as FEM1b and apoptotic protease activating factor-1 (Apaf-1), and inhibited induction of cellular apoptosis in chemotherapeutic drug-treated Jurkat cell. Interaction of Rack1 and PKCα, not PKCβ, was detected in both cell lines. Of note, Rack1 overexpression abrogated reduction of PKC kinase activity in chemotherapeutic drug-treated T-ALL cell. PKC kinase inhibitor Go6976 or siPKCα inhibited downregulation of FEM1b and/or Apaf-1, and thus increased cellular apoptosis in Rack1-overexpressed T-ALL cell receiving chemotherapeutic drugs. Accordingly, our data provided evidence that increased Rack1-mediated upregulation of PKC kinase activity may be responsible for the development of chemoresistance in T-ALL-derived cell line potentially by reducing FEM1b and Apaf-1 level. PMID:27644318

  5. Overexpressing Ferredoxins in Chlamydomonas reinhardtii Increase Starch and Oil Yields and Enhance Electric Power Production in a Photo Microbial Fuel Cell.

    PubMed

    Huang, Li-Fen; Lin, Ji-Yu; Pan, Kui-You; Huang, Chun-Kai; Chu, Ying-Kai

    2015-08-14

    Ferredoxins (FDX) are final electron carrier proteins in the plant photosynthetic pathway, and function as major electron donors in diverse redox-driven metabolic pathways. We previously showed that overexpression of a major constitutively expressed ferredoxin gene PETF in Chlamydomonas decreased the reactive oxygen species (ROS) level and enhanced tolerance to heat stress. In addition to PETF, an endogenous anaerobic induced FDX5 was overexpressed in transgenic Chlamydomonas lines here to address the possible functions of FDX5. All the independent FDX transgenic lines showed decreased cellular ROS levels and enhanced tolerance to heat and salt stresses. The transgenic Chlamydomonas lines accumulated more starch than the wild-type line and this effect increased almost three-fold in conditions of nitrogen depletion. Furthermore, the lipid content was higher in the transgenic lines than in the wild-type line, both with and without nitrogen depletion. Two FDX-overexpressing Chlamydomonas lines were assessed in a photo microbial fuel cell (PMFC); power density production by the transgenic lines was higher than that of the wild-type cells. These findings suggest that overexpression of either PETF or FDX5 can confer tolerance against heat and salt stresses, increase starch and oil production, and raise electric power density in a PMFC.

  6. Increased PKCα activity by Rack1 overexpression is responsible for chemotherapy resistance in T-cell acute lymphoblastic leukemia-derived cell line

    PubMed Central

    Lei, Jie; Li, Qi; Gao, Ying; Zhao, Lei; Liu, Yanbo

    2016-01-01

    Chemoresistant mechanisms in T-cell acute lymphoblastic leukemia (T-ALL) patients are not clarified. The apoptotic signaling mediated by receptor of activated C kinase 1 (Rack1), protein kinase C (PKC) and FEM1 homolog b (FEM1b) was investigated in two T-ALL-derived cell lines (Jurkat and CCRF-CEM) following treatment with chemotherapy drugs vincristine and prednisone. Serum starvation or chemotherapeutic drugs significantly reduced Rack1 level and PKC activation, while promoted cellular apoptosis in both cell lines. Rack1 overexpression protected T-ALL cell against starvation or chemotherapeutic drug-induced apoptosis. Moreover, Rack1 overexpression reduced the level of cytochrome c and active caspase 3 as well as FEM1b and apoptotic protease activating factor-1 (Apaf-1), and inhibited induction of cellular apoptosis in chemotherapeutic drug-treated Jurkat cell. Interaction of Rack1 and PKCα, not PKCβ, was detected in both cell lines. Of note, Rack1 overexpression abrogated reduction of PKC kinase activity in chemotherapeutic drug-treated T-ALL cell. PKC kinase inhibitor Go6976 or siPKCα inhibited downregulation of FEM1b and/or Apaf-1, and thus increased cellular apoptosis in Rack1-overexpressed T-ALL cell receiving chemotherapeutic drugs. Accordingly, our data provided evidence that increased Rack1-mediated upregulation of PKC kinase activity may be responsible for the development of chemoresistance in T-ALL-derived cell line potentially by reducing FEM1b and Apaf-1 level. PMID:27644318

  7. Overexpressing Ferredoxins in Chlamydomonas reinhardtii Increase Starch and Oil Yields and Enhance Electric Power Production in a Photo Microbial Fuel Cell.

    PubMed

    Huang, Li-Fen; Lin, Ji-Yu; Pan, Kui-You; Huang, Chun-Kai; Chu, Ying-Kai

    2015-01-01

    Ferredoxins (FDX) are final electron carrier proteins in the plant photosynthetic pathway, and function as major electron donors in diverse redox-driven metabolic pathways. We previously showed that overexpression of a major constitutively expressed ferredoxin gene PETF in Chlamydomonas decreased the reactive oxygen species (ROS) level and enhanced tolerance to heat stress. In addition to PETF, an endogenous anaerobic induced FDX5 was overexpressed in transgenic Chlamydomonas lines here to address the possible functions of FDX5. All the independent FDX transgenic lines showed decreased cellular ROS levels and enhanced tolerance to heat and salt stresses. The transgenic Chlamydomonas lines accumulated more starch than the wild-type line and this effect increased almost three-fold in conditions of nitrogen depletion. Furthermore, the lipid content was higher in the transgenic lines than in the wild-type line, both with and without nitrogen depletion. Two FDX-overexpressing Chlamydomonas lines were assessed in a photo microbial fuel cell (PMFC); power density production by the transgenic lines was higher than that of the wild-type cells. These findings suggest that overexpression of either PETF or FDX5 can confer tolerance against heat and salt stresses, increase starch and oil production, and raise electric power density in a PMFC. PMID:26287179

  8. Overexpressing Ferredoxins in Chlamydomonas reinhardtii Increase Starch and Oil Yields and Enhance Electric Power Production in a Photo Microbial Fuel Cell

    PubMed Central

    Huang, Li-Fen; Lin, Ji-Yu; Pan, Kui-You; Huang, Chun-Kai; Chu, Ying-Kai

    2015-01-01

    Ferredoxins (FDX) are final electron carrier proteins in the plant photosynthetic pathway, and function as major electron donors in diverse redox-driven metabolic pathways. We previously showed that overexpression of a major constitutively expressed ferredoxin gene PETF in Chlamydomonas decreased the reactive oxygen species (ROS) level and enhanced tolerance to heat stress. In addition to PETF, an endogenous anaerobic induced FDX5 was overexpressed in transgenic Chlamydomonas lines here to address the possible functions of FDX5. All the independent FDX transgenic lines showed decreased cellular ROS levels and enhanced tolerance to heat and salt stresses. The transgenic Chlamydomonas lines accumulated more starch than the wild-type line and this effect increased almost three-fold in conditions of nitrogen depletion. Furthermore, the lipid content was higher in the transgenic lines than in the wild-type line, both with and without nitrogen depletion. Two FDX-overexpressing Chlamydomonas lines were assessed in a photo microbial fuel cell (PMFC); power density production by the transgenic lines was higher than that of the wild-type cells. These findings suggest that overexpression of either PETF or FDX5 can confer tolerance against heat and salt stresses, increase starch and oil production, and raise electric power density in a PMFC. PMID:26287179

  9. Dopamine D2 Receptor-Mediated Regulation of Pancreatic β Cell Mass.

    PubMed

    Sakano, Daisuke; Choi, Sungik; Kataoka, Masateru; Shiraki, Nobuaki; Uesugi, Motonari; Kume, Kazuhiko; Kume, Shoen

    2016-07-12

    Understanding the molecular mechanisms that regulate β cell mass and proliferation is important for the treatment of diabetes. Here, we identified domperidone (DPD), a dopamine D2 receptor (DRD2) antagonist that enhances β cell mass. Over time, islet β cell loss occurs in dissociation cultures, and this was inhibited by DPD. DPD increased proliferation and decreased apoptosis of β cells through increasing intracellular cAMP. DPD prevented β cell dedifferentiation, which together highly contributed to the increased β cell mass. DRD2 knockdown phenocopied the effects of domperidone and increased the number of β cells. Drd2 overexpression sensitized the dopamine responsiveness of β cells and increased apoptosis. Further analysis revealed that the adenosine agonist 5'-N-ethylcarboxamidoadenosine, a previously identified promoter of β cell proliferation, acted with DPD to increase the number of β cells. In humans, dopamine also modulates β cell mass through DRD2 and exerts an inhibitory effect on adenosine signaling. PMID:27373926

  10. Docosahexaenoic Acid Modulates a HER2-Associated Lipogenic Phenotype, Induces Apoptosis, and Increases Trastuzumab Action in HER2-Overexpressing Breast Carcinoma Cells

    PubMed Central

    Ravacci, Graziela Rosa; Brentani, Maria Mitzi; Tortelli, Tharcisio Citrângulo; Torrinhas, Raquel Suzana M. M.; Santos, Jéssica Reis; Logullo, Angela Flávia; Waitzberg, Dan Linetzky

    2015-01-01

    In breast cancer, lipid metabolic alterations have been recognized as potential oncogenic stimuli that may promote malignancy. To investigate whether the oncogenic nature of lipogenesis closely depends on the overexpression of HER2 protooncogene, the normal breast cell line, HB4a, was transfected with HER2 cDNA to obtain HER2-overexpressing HB4aC5.2 cells. Both cell lines were treated with trastuzumab and docosahexaenoic acid. HER2 overexpression was accompanied by an increase in the expression of lipogenic genes involved in uptake (CD36), transport (FABP4), and storage (DGAT) of exogenous fatty acids (FA), as well as increased activation of “de novo” FA synthesis (FASN). We further investigate whether this lipogenesis reprogramming might be regulated by mTOR/PPARγ pathway. Inhibition of the mTORC1 pathway markers, p70S6 K1, SREBP1, and LIPIN1, as well as an increase in DEPTOR expression (the main inhibitor of the mTOR) was detected in HB4aC5.2. Based on these results, a PPARγ selective antagonist, GW9662, was used to treat both cells lines, and the lipogenic genes remained overexpressed in the HB4aC5.2 but not HB4a cells. DHA treatment inhibited all lipogenic genes (except for FABP4) in both cell lines yet only induced death in the HB4aC5.2 cells, mainly when associated with trastuzumab. Neither trastuzumab nor GW9662 alone was able to induce cell death. In conclusion, oncogenic transformation of breast cells by HER2 overexpression may require a reprogramming of lipogenic genetic that is independent of mTORC1 pathway and PPARγ activity. This reprogramming was inhibited by DHA. PMID:26640797

  11. Translation initiation of ornithine decarboxylase and nucleocytoplasmic transport of cyclin D1 mRNA are increased in cells overexpressing eukaryotic initiation factor 4E.

    PubMed Central

    Rousseau, D; Kaspar, R; Rosenwald, I; Gehrke, L; Sonenberg, N

    1996-01-01

    The structure of m7GpppN (where N is any nucleotide), termed cap, is present at the 5' end of all eukaryotic cellular mRNAs (except organellar). The eukaryotic initiation factor 4E (eIF-4E) binds to the cap and facilitates the formation of translation initiation complexes. eIF-4E is implicated in control of cell growth, as its overexpression causes malignant transformation of rodent cells and deregulates HeLa cell growth. It was suggested that overexpression of eIF-4E results in the enhanced translation of poorly translated mRNAs that encode growth-promoting proteins. Indeed, enhanced expression of several proteins, including cyclin D1 and ornithine decarboxylase (ODC), was documented in eIF-4E-overexpressing NTH 3T3 cells. However, the mechanism underlying this increase has not been elucidated. Here, we studied the mode by which eIF-4E increases the expression of cyclin D1 and ODC. We show that the increase in the amount of cyclin D1 and ODC is directly proportional to the degree of eIF-4E overexpression. Two mechanisms, which are not mutually exclusive, are responsible for the increase. In eIF-4E-overexpressing cells the rate of translation initiation of ODC mRNA was increased inasmuch as the mRNA sedimented with heavier polysomes. For cyclin D1 mRNA, translation initiation was not increased, but rather its amount in the cytoplasm increased, without a significant increase in total mRNA. Whereas, in the parental NIH 3T3 cell line, a large proportion of the cyclin D1 mRNA was confined to the nucleus, in eIF-4E-overexpressing cells the vast majority of the mRNA was present in the cytoplasm. These results indicate that eIF-4E affects directly or indirectly mRNA nucleocytoplasmic transport, in addition to its role in translation initiation. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8577715

  12. Erianthus arundinaceus HSP70 (EaHSP70) overexpression increases drought and salinity tolerance in sugarcane (Saccharum spp. hybrid).

    PubMed

    Augustine, Sruthy Maria; Narayan, J Ashwin; Syamaladevi, Divya P; Appunu, C; Chakravarthi, M; Ravichandran, V; Subramonian, N

    2015-03-01

    Heat shock proteins (HSPs) have a major role in stress tolerance mechanisms in plants. Our studies have shown that the expression of HSP70 is enhanced under water stress in Erianthus arundinaceus. In this paper, we evaluate the effects of overexpression of EaHSP70 driven by Port Ubi 2.3 promoter in sugarcane. The transgenic events exhibit significantly higher gene expression, cell membrane thermostability, relative water content, gas exchange parameters, chlorophyll content and photosynthetic efficiency. The overexpression of EaHSP70 transgenic sugarcane led to the upregulation of stress-related genes. The transformed sugarcane plants had better chlorophyll retention and higher germination ability than control plants under salinity stress. Our results suggest that EaHSP70 plays an important role in sugarcane acclimation to drought and salinity stresses and its potential for genetic engineering of sugarcane for drought and salt tolerance.

  13. SOD1 Overexpression Preserves Baroreflex Control of Heart Rate with an Increase of Aortic Depressor Nerve Function

    PubMed Central

    Hatcher, Jeffrey; Gu, He; Cheng, Zixi (Jack)

    2016-01-01

    Overproduction of reactive oxygen species (ROS), such as the superoxide radical (O2∙−), is associated with diseases which compromise cardiac autonomic function. Overexpression of SOD1 may offer protection against ROS damage to the cardiac autonomic nervous system, but reductions of O2∙− may interfere with normal cellular functions. We have selected the C57B6SJL-Tg (SOD1)2 Gur/J mouse as a model to determine whether SOD1 overexpression alters cardiac autonomic function, as measured by baroreflex sensitivity (BRS) and aortic depressor nerve (ADN) recordings, as well as evaluation of baseline heart rate (HR) and mean arterial pressure (MAP). Under isoflurane anesthesia, C57 wild-type and SOD1 mice were catheterized with an arterial pressure transducer and measurements of HR and MAP were taken. After establishing a baseline, hypotension and hypertension were induced by injection of sodium nitroprusside (SNP) and phenylephrine (PE), respectively, and ΔHR versus ΔMAP were recorded as a measure of baroreflex sensitivity (BRS). SNP and PE treatment were administered sequentially after a recovery period to measure arterial baroreceptor activation by recording aortic depressor nerve activity. Our findings show that overexpression of SOD1 in C57B6SJL-Tg (SOD1)2 Gur/J mouse preserved the normal HR, MAP, and BRS but enhanced aortic depressor nerve function. PMID:26823951

  14. PTEN Overexpression Cooperates With Lithium to Reduce the Malignancy and to Increase Cell Death by Apoptosis via PI3K/Akt Suppression in Colorectal Cancer Cells.

    PubMed

    de Araujo, Wallace Martins; Robbs, Bruno Kaufmann; Bastos, Lilian G; de Souza, Waldemir F; Vidal, Flávia C B; Viola, João P B; Morgado-Diaz, Jose A

    2016-02-01

    Lithium is a well-established non-competitive inhibitor of glycogen synthase kinase-3β (GSK-3β), a kinase that is involved in several cellular processes related to cancer progression. GSK-3β is regulated upstream by PI3K/Akt, which is negatively modulated by PTEN. The role that lithium plays in cancer is controversial because lithium can activate or inhibit survival signaling pathways depending on the cell type. In this study, we analyzed the mechanisms by which lithium can modulate events related to colorectal cancer (CRC) progression and evaluated the role that survival signaling pathways such as PI3K/Akt and PTEN play in this context. We show that the administration of lithium decreased the proliferative potential of CRC cells in a GSK-3β-independent manner but induced the accumulation of cells in G2/M phase. Furthermore, high doses of lithium increased apoptosis, which was accompanied by decreased proteins levels of Akt and PTEN. Then, cells that were induced to overexpress PTEN were treated with lithium; we observed that low doses of lithium strongly increased apoptosis. Additionally, PTEN overexpression reduced proliferation, but this effect was minor compared with that in cells treated with lithium alone. Furthermore, we demonstrated that PTEN overexpression and lithium treatment separately reduced cell migration, colony formation, and invasion, and these effects were enhanced when lithium treatment and PTEN overexpression were combined. In conclusion, our findings indicate that PTEN overexpression and lithium treatment cooperate to reduce the malignancy of CRC cells and highlight lithium and PTEN as potential candidates for studies to identify new therapeutic approaches for CRC treatment.

  15. Overexpression of angiopoietin 2 promotes the formation of oral squamous cell carcinoma by increasing epithelial–mesenchymal transition-induced angiogenesis

    PubMed Central

    Li, C; Li, Q; Cai, Y; He, Y; Lan, X; Wang, W; Liu, J; Wang, S; Zhu, G; Fan, J; Zhou, Y; Sun, R

    2016-01-01

    Oral squamous cell carcinoma (OSCC) is the most common cancer of the head and neck and is associated with a high rate of lymph node metastasis. The initial step in the metastasis and transition of tumors is epithelial–mesenchymal transition (EMT)-induced angiogenesis, which can be mediated by angiopoietin 2 (ANG2), a key regulatory factor in angiogenesis. In the present study, immunohistochemistry and real-time quantitative reverse transcriptase (qRT-PCR) were used to measure the expression of ANG2 in OSCC tissues. Plasmids encoding ANG2 mRNA were used for increased ANG2 expression in the OSCC cell line TCA8113. The short interfering RNA (siRNA)-targeting ANG2 mRNA sequences were used to inhibit ANG2 expression in TCA8113 cells. Subsequently, transwell assays were performed to examine the effects of ANG2 on TCA8113 cell migration and invasion. Furthermore, in vivo assays were performed to assess the effect of ANG2 on tumor growth. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assays and immunohistochemistry were used to examine cell apoptosis and angiogenesis in tumor tissues, respectively. Finally, western blot analysis was performed to evaluate tumor formation-related proteins in OSCC tissues. We found that protein expression of ANG2 was remarkably upregulated in OSCC tissues. Overexpression of ANG2 increased the migration and invasion of TCA8113 cells by regulating EMT. Further investigations showed that overexpression of ANG2 increased tumor growth in nude mice, and angiogenesis of OSCC tissues increased in the presence of ANG2 overexpression. Overexpression of ANG2 also reduced cell apoptosis in tumor tissue cells. Finally, we found that overexpression of ANG2 resulted in changes in the expression of tumor formation-related proteins including vimentin, E-cadherin, Bim, PUMA, Bcl-2, Bax, Cyclin D1, PCNA and CD31. Our findings show that ANG2 has an important role in the migration and invasion of OSCC. More importantly, further

  16. Overexpression of angiopoietin 2 promotes the formation of oral squamous cell carcinoma by increasing epithelial-mesenchymal transition-induced angiogenesis.

    PubMed

    Li, C; Li, Q; Cai, Y; He, Y; Lan, X; Wang, W; Liu, J; Wang, S; Zhu, G; Fan, J; Zhou, Y; Sun, R

    2016-09-01

    Oral squamous cell carcinoma (OSCC) is the most common cancer of the head and neck and is associated with a high rate of lymph node metastasis. The initial step in the metastasis and transition of tumors is epithelial-mesenchymal transition (EMT)-induced angiogenesis, which can be mediated by angiopoietin 2 (ANG2), a key regulatory factor in angiogenesis. In the present study, immunohistochemistry and real-time quantitative reverse transcriptase (qRT-PCR) were used to measure the expression of ANG2 in OSCC tissues. Plasmids encoding ANG2 mRNA were used for increased ANG2 expression in the OSCC cell line TCA8113. The short interfering RNA (siRNA)-targeting ANG2 mRNA sequences were used to inhibit ANG2 expression in TCA8113 cells. Subsequently, transwell assays were performed to examine the effects of ANG2 on TCA8113 cell migration and invasion. Furthermore, in vivo assays were performed to assess the effect of ANG2 on tumor growth. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assays and immunohistochemistry were used to examine cell apoptosis and angiogenesis in tumor tissues, respectively. Finally, western blot analysis was performed to evaluate tumor formation-related proteins in OSCC tissues. We found that protein expression of ANG2 was remarkably upregulated in OSCC tissues. Overexpression of ANG2 increased the migration and invasion of TCA8113 cells by regulating EMT. Further investigations showed that overexpression of ANG2 increased tumor growth in nude mice, and angiogenesis of OSCC tissues increased in the presence of ANG2 overexpression. Overexpression of ANG2 also reduced cell apoptosis in tumor tissue cells. Finally, we found that overexpression of ANG2 resulted in changes in the expression of tumor formation-related proteins including vimentin, E-cadherin, Bim, PUMA, Bcl-2, Bax, Cyclin D1, PCNA and CD31. Our findings show that ANG2 has an important role in the migration and invasion of OSCC. More importantly, further

  17. Increased abscisic acid levels in transgenic maize overexpressing AtLOS5 mediated root ion fluxes and leaf water status under salt stress.

    PubMed

    Zhang, Juan; Yu, Haiyue; Zhang, Yushi; Wang, Yubing; Li, Maoying; Zhang, Jiachang; Duan, Liusheng; Zhang, Mingcai; Li, Zhaohu

    2016-03-01

    Abscisic acid (ABA) is a vital cellular signal in plants, and effective ABA signalling is pivotal for stress tolerance. AtLOS5 encoding molybdenum cofactor sulphurase is a key regulator of ABA biosynthesis. Here, transgenic AtLOS5 plants were generated to explore the role of AtLOS5 in salt tolerance in maize. AtLOS5 overexpression significantly up-regulated the expression of ZmVp14-2, ZmAO, and ZmMOCO, and increased aldehyde oxidase activities, which enhanced ABA accumulation in transgenic plants under salt stress. Concurrently, AtLOS5 overexpression induced the expression of ZmNHX1, ZmCBL4, and ZmCIPK16, and enhanced the root net Na(+) efflux and H(+) influx, but decreased net K(+) efflux, which maintained a high cytosolic K(+)/Na(+) ratio in transgenic plants under salt stress. However, amiloride or sodium orthovanadate could significantly elevate K(+) effluxes and decrease Na(+) efflux and H(+) influx in salt-treated transgenic roots, but the K(+) effluxes were inhibited by TEA, suggesting that ion fluxes regulated by AtLOS5 overexpression were possibly due to activation of Na(+)/H(+) antiport and K(+) channels across the plasma membrane. Moreover, AtLOS5 overexpression could up-regulate the transcripts of ZmPIP1:1, ZmPIP1:5, and ZmPIP2:4, and enhance root hydraulic conductivity. Thus transgenic plants had higher leaf water potential and turgor, which was correlated with greater biomass accumulation under salt stress. Thus AtLOS5 overexpression induced the expression of ABA biosynthetic genes to promote ABA accumulation, which activated ion transporter and PIP aquaporin gene expression to regulate root ion fluxes and water uptake, thus maintaining high cytosolic K(+) and Na(+) homeostasis and better water status in maize exposed to salt stress. PMID:26743432

  18. Increased abscisic acid levels in transgenic maize overexpressing AtLOS5 mediated root ion fluxes and leaf water status under salt stress

    PubMed Central

    Zhang, Juan; Yu, Haiyue; Zhang, Yushi; Wang, Yubing; Li, Maoying; Zhang, Jiachang; Duan, Liusheng; Zhang, Mingcai; Li, Zhaohu

    2016-01-01

    Abscisic acid (ABA) is a vital cellular signal in plants, and effective ABA signalling is pivotal for stress tolerance. AtLOS5 encoding molybdenum cofactor sulphurase is a key regulator of ABA biosynthesis. Here, transgenic AtLOS5 plants were generated to explore the role of AtLOS5 in salt tolerance in maize. AtLOS5 overexpression significantly up-regulated the expression of ZmVp14-2, ZmAO, and ZmMOCO, and increased aldehyde oxidase activities, which enhanced ABA accumulation in transgenic plants under salt stress. Concurrently, AtLOS5 overexpression induced the expression of ZmNHX1, ZmCBL4, and ZmCIPK16, and enhanced the root net Na+ efflux and H+ influx, but decreased net K+ efflux, which maintained a high cytosolic K+/Na+ ratio in transgenic plants under salt stress. However, amiloride or sodium orthovanadate could significantly elevate K+ effluxes and decrease Na+ efflux and H+ influx in salt-treated transgenic roots, but the K+ effluxes were inhibited by TEA, suggesting that ion fluxes regulated by AtLOS5 overexpression were possibly due to activation of Na+/H+ antiport and K+ channels across the plasma membrane. Moreover, AtLOS5 overexpression could up-regulate the transcripts of ZmPIP1:1, ZmPIP1:5, and ZmPIP2:4, and enhance root hydraulic conductivity. Thus transgenic plants had higher leaf water potential and turgor, which was correlated with greater biomass accumulation under salt stress. Thus AtLOS5 overexpression induced the expression of ABA biosynthetic genes to promote ABA accumulation, which activated ion transporter and PIP aquaporin gene expression to regulate root ion fluxes and water uptake, thus maintaining high cytosolic K+ and Na+ homeostasis and better water status in maize exposed to salt stress. PMID:26743432

  19. COPS5 protein overexpression increases amyloid plaque burden, decreases spinophilin-immunoreactive puncta, and exacerbates learning and memory deficits in the mouse brain.

    PubMed

    Wang, Ruizhi; Wang, Hongjie; Carrera, Ivan; Xu, Shaohua; Lakshmana, Madepalli K

    2015-04-01

    Brain accumulation of neurotoxic amyloid β (Aβ) peptide because of increased processing of amyloid precursor protein (APP), resulting in loss of synapses and neurodegeneration, is central to the pathogenesis of Alzheimer disease (AD). Therefore, the identification of molecules that regulate Aβ generation and those that cause synaptic damage is crucial for future therapeutic approaches for AD. We demonstrated previously that COPS5 regulates Aβ generation in neuronal cell lines in a RanBP9-dependent manner. Consistent with the data from cell lines, even by 6 months, COPS5 overexpression in APΔE9 mice (APΔE9/COPS5-Tg) significantly increased Aβ40 levels by 32% (p < 0.01) in the cortex and by 28% (p < 0.01) in the hippocampus, whereas the increases for Aβ42 were 37% (p < 0.05) and 34% (p < 0.05), respectively. By 12 months, the increase was even more robust. Aβ40 levels increased by 63% (p < 0.001) in the cortex and by 65% (p < 0.001) in the hippocampus. Similarly, Aβ42 levels were increased by 69% (p < 0.001) in the cortex and by 71% (p < 0.011) in the hippocampus. Increased Aβ levels were translated into an increased amyloid plaque burden both in the cortex (54%, p < 0.01) and hippocampus (64%, p < 0.01). Interestingly, COPS5 overexpression increased RanBP9 levels in the brain, which, in turn, led to increased amyloidogenic processing of APP, as reflected by increased levels of sAPPβ and decreased levels of sAPPα. Furthermore, COPS5 overexpression reduced spinophilin in both the cortex (19%, p < 0.05) and the hippocampus (20%, p < 0.05), leading to significant deficits in learning and memory skills. Therefore, like RanBP9, COPS5 also plays a pivotal role in amyloid pathology in vivo.

  20. Increased copy-number and not DNA hypomethylation causes overexpression of the candidate proto-oncogene CYP24A1 in colorectal cancer.

    PubMed

    Höbaus, Julia; Hummel, Doris M; Thiem, Ursula; Fetahu, Irfete S; Aggarwal, Abhishek; Müllauer, Leonhard; Heller, Gerwin; Egger, Gerda; Mesteri, Ildiko; Baumgartner-Parzer, Sabina; Kallay, Enikö

    2013-09-15

    In colorectal cancer (CRC) the vitamin D catabolizing enzyme 1,25-dihydroxyvitamin D 24-hydroxylase (CYP24A1) is overexpressed with a potentially significant, positive impact on the catabolism of 1,25-dihydroxyvitamin D3 (1,25-D3 ). However, the underlying mechanism of CYP24A1 overexpression is poorly understood. In the present study, we investigated possible causes including hypomethylation of the CYP24A1 promoter, amplification of the CYP24A1 gene locus (20q13.2), and altered expression of CYP24A1-specific transcription factors. We quantified CYP24A1 gene copy-number, performed bisulfite sequencing of the CYP24A1 promoter to assess DNA methylation, and measured mRNA expression of CYP24A1, 25-hydroxyvitamin D 1α-hydroxylase (CYP27B1), vitamin D receptor (VDR) and retinoid X receptor (RXR). We found that 77 (60%) out of 127 colorectal tumors showed increased CYP24A1 gene copy-number and that more than 6 copies of CYP24A1 correlated positively with CYP24A1 mRNA expression suggestive of a causal relationship. No differences in CYP24A1 promoter methylation were found between tumor tissue and adjacent mucosa from the same patient or between tissues with high or low mRNA expression, thus excluding DNA hypomethylation as a possible cause of CYP24A1 overexpression in CRC. Furthermore, mRNA expression of several factors involved in replication licensing positively correlated with CYP24A1 mRNA expression, raising the possibility that CYP24A1 overexpression might favor increased proliferation in tumors by suppressing local 1,25-D3 levels. We conclude that high copy-number gain is a key determinant of CYP24A1 overexpression in CRC. Other postulated causes of CYP24A1 overexpression including promoter hypomethylation and enhanced VDR and/or RXR expression do not appear to be involved.

  1. The overexpression of the pine transcription factor PpDof5 in Arabidopsis leads to increased lignin content and affects carbon and nitrogen metabolism.

    PubMed

    Rueda-López, Marina; Cañas, Rafael A; Canales, Javier; Cánovas, Francisco M; Ávila, Concepción

    2015-12-01

    PpDof 5 is a regulator of the expression of glutamine synthetase (GS; EC 6.3.1.2) genes in photosynthetic and non-photosynthetic tissues of maritime pine. We have used Arabidopsis thaliana as a model system to study PpDof 5 function in planta, generating transgenic lines overexpressing the pine transcription factor. The overexpression of PpDof 5 resulted in a substantial increase of lignin content with a simultaneous regulation of carbon and nitrogen key genes. In addition, partitioning in carbon and nitrogen compounds was spread via various secondary metabolic pathways. These results suggest pleiotropic effects of PpDof 5 expression on various metabolic pathways of carbon and nitrogen metabolism. Plants overexpressing PpDof 5 exhibited upregulation of genes encoding enzymes for sucrose and starch biosynthesis, with a parallel increase in the content of soluble sugars. When the plants were grown under nitrate as the sole nitrogen source, they exhibited a significant regulation of the expression of genes involved mainly in signaling, but similar growth rates to wild-type plants. However, plants grown under ammonium exhibited major induction of the expression of photosynthetic genes and differential expression of ammonium and nitrate transporters. All these data suggest that in addition to controlling ammonium assimilation, PpDof 5 could be also involved in the regulation of other pathways in carbon and nitrogen metabolism in pine trees. PMID:26333592

  2. Targeted overexpression of inducible 6-phosphofructo-2-kinase in adipose tissue increases fat deposition but protects against diet-induced insulin resistance and inflammatory responses.

    PubMed

    Huo, Yuqing; Guo, Xin; Li, Honggui; Xu, Hang; Halim, Vera; Zhang, Weiyu; Wang, Huan; Fan, Yang-Yi; Ong, Kuok Teong; Woo, Shih-Lung; Chapkin, Robert S; Mashek, Douglas G; Chen, Yanming; Dong, Hui; Lu, Fuer; Wei, Lai; Wu, Chaodong

    2012-06-15

    Increasing evidence demonstrates the dissociation of fat deposition, the inflammatory response, and insulin resistance in the development of obesity-related metabolic diseases. As a regulatory enzyme of glycolysis, inducible 6-phosphofructo-2-kinase (iPFK2, encoded by PFKFB3) protects against diet-induced adipose tissue inflammatory response and systemic insulin resistance independently of adiposity. Using aP2-PFKFB3 transgenic (Tg) mice, we explored the ability of targeted adipocyte PFKFB3/iPFK2 overexpression to modulate diet-induced inflammatory responses and insulin resistance arising from fat deposition in both adipose and liver tissues. Compared with wild-type littermates (controls) on a high fat diet (HFD), Tg mice exhibited increased adiposity, decreased adipose inflammatory response, and improved insulin sensitivity. In a parallel pattern, HFD-fed Tg mice showed increased hepatic steatosis, decreased liver inflammatory response, and improved liver insulin sensitivity compared with controls. In both adipose and liver tissues, increased fat deposition was associated with lipid profile alterations characterized by an increase in palmitoleate. Additionally, plasma lipid profiles also displayed an increase in palmitoleate in HFD-Tg mice compared with controls. In cultured 3T3-L1 adipocytes, overexpression of PFKFB3/iPFK2 recapitulated metabolic and inflammatory changes observed in adipose tissue of Tg mice. Upon treatment with conditioned medium from iPFK2-overexpressing adipocytes, mouse primary hepatocytes displayed metabolic and inflammatory responses that were similar to those observed in livers of Tg mice. Together, these data demonstrate a unique role for PFKFB3/iPFK2 in adipocytes with regard to diet-induced inflammatory responses in both adipose and liver tissues.

  3. Quantitative Proteomics Analysis of Camelina sativa Seeds Overexpressing the AGG3 Gene to Identify the Proteomic Basis of Increased Yield and Stress Tolerance.

    PubMed

    Alvarez, Sophie; Roy Choudhury, Swarup; Sivagnanam, Kumaran; Hicks, Leslie M; Pandey, Sona

    2015-06-01

    Camelina sativa, a close relative of Arabidopsis, is an oilseed plant that is emerging as an important biofuel resource. The genome and transcriptome maps of Camelina have become available recently, but its proteome composition remained unexplored. A labeling LC-based quantitative proteomics approach was applied to decipher the Camelina seed proteome, which led to the identification of 1532 proteins. In addition, the effect of overexpression of the Arabidopsis G-protein γ subunit 3 (AGG3) on the Camelina seed proteome was elucidated to identify the proteomic basis of its increased seed size and improved stress tolerance. The comparative analysis showed a significantly higher expression of proteins involved in primary and secondary metabolism, nucleic acid and protein metabolism, and abscisic acid related responses, corroborating the physiological effects of AGG3 overexpression. More importantly, the proteomic data suggested involvement of the AGG3 protein in the regulation of oxidative stress and heavy metal stress tolerance. These observations were confirmed by the physiological and biochemical characterization of AGG3-overexpressing seeds, which exhibit a higher tolerance to exogenous cadmium in a glutathione-dependent manner. The activity of multiple redox-regulating enzymes is higher in seeds expressing enhanced levels of AGG3. Overall, these data provide critical evidence for the role of redox regulation by the AGG3 protein in mediating important seed-related traits.

  4. Overexpression of a Weed (Solanum americanum) Proteinase Inhibitor in Transgenic Tobacco Results in Increased Glandular Trichome Density and Enhanced Resistance to Helicoverpa armigera and Spodoptera litura

    PubMed Central

    Luo, Ming; Wang, Zhaoyu; Li, Huapeng; Xia, Kuai-Fei; Cai, Yinpeng; Xu, Zeng-Fu

    2009-01-01

    In this study we produced transgenic tobacco plants by overexpressing a serine proteinase inhibitor gene, SaPIN2a, from the American black nightshade Solanum americanum under the control of the CaMV 35S promoter using Agrobacterium tumefaciens-mediated transformation. SaPIN2a was properly transcribed and translated as indicated by Northern blot and Western blot analyses. Functional integrity of SaPIN2a in transgenic plants was confirmed by proteinase inhibitory activity assay. Bioassays for insect resistance showed that SaPIN2a-overexpressing transgenic tobacco plants were more resistant to cotton bollworm (Helicoverpa armigera) and tobacco cutworm (Spodoptera litura) larvae, two devastating pests of important crop plants, than the control plants. Interestingly, overexpression of SaPIN2a in transgenic tobacco plants resulted in a significant increase in glandular trichome density and a promotion of trichome branching, which could also provide an additional resistance mechanism in transgenic plants against insect pests. Therefore, SaPIN2a could be used as an alternative proteinase inhibitor for the production of insect-resistant transgenic plants. PMID:19468345

  5. Increased desensitization of dopamine D₂ receptor-mediated response in the ventral tegmental area in the absence of adenosine A(2A) receptors.

    PubMed

    Al-Hasani, R; Foster, J D; Metaxas, A; Ledent, C; Hourani, S M O; Kitchen, I; Chen, Y

    2011-09-01

    G-protein coupled receptors interact to provide additional regulatory mechanisms for neurotransmitter signaling. Adenosine A(2A) receptors are expressed at a high density in striatal neurons, where they closely interact with dopamine D₂ receptors and modulate effects of dopamine and responses to psychostimulants. A(2A) receptors are expressed at much lower densities in other forebrain neurons but play a more prominent yet opposing role to striatal receptors in response to psychostimulants in mice. It is, therefore, possible that A(2A) receptors expressed at low levels elsewhere in the brain may also regulate neurotransmitter systems and modulate neuronal functions. Dopamine D₂ receptors play an important role in autoinhibition of neuronal firing in dopamine neurons of the ventral tegmental area (VTA) and dopamine release in other brain areas. Here, we examined the effect of A(2A) receptor deletion on D₂ receptor-mediated inhibition of neuronal firing in dopamine neurons in the VTA. Spontaneous activity of dopamine neurons was recorded in midbrain slices, and concentration-dependent effects of the dopamine D₂ receptor agonist, quinpirole, was compared between wild-type and A(2A) knockout mice. The potency of quinpirole applied in single concentrations and the expression of D₂ receptors were not altered in the VTA of the knockout mice. However, quinpirole applied in stepwise escalating concentrations caused significantly reduced maximal inhibition in A(2A) knockout mice, indicating an enhanced agonist-induced desensitization of D₂ receptors in the absence of A(2A) receptors. The A(2A) receptor agonist, CGS21680, did not exert any effect on dopamine neuron firing or response to quinpirole, revealing a novel non-pharmacological interaction between adenosine A(2A) receptors and dopaminergic neurotransmission in midbrain dopamine neurons. Altered D₂ receptor desensitization may result in changes in dopamine neuron firing rate and pattern and dopamine

  6. FcRn overexpression in transgenic mice results in augmented APC activity and robust immune response with increased diversity of induced antibodies.

    PubMed

    Végh, Attila; Farkas, Anita; Kövesdi, Dorottya; Papp, Krisztián; Cervenak, Judit; Schneider, Zita; Bender, Balázs; Hiripi, László; László, Glória; Prechl, József; Matkó, János; Kacskovics, Imre

    2012-01-01

    Our previous studies have shown that overexpression of bovine FcRn (bFcRn) in transgenic (Tg) mice leads to an increase in the humoral immune response, characterized by larger numbers of Ag-specific B cells and other immune cells in secondary lymphoid organs and higher levels of circulating Ag-specific antibodies (Abs). To gain additional insights into the mechanisms underlying this increase in humoral immune response, we further characterized the bFcRn Tg mice. Our Western blot analysis showed strong expression of the bFcRn transgene in peritoneal macrophages and bone marrow derived dendritic cells; and a quantitative PCR analysis demonstrated that the expression ratios of the bFcRn to mFcRn were 2.6- and 10-fold in these cells, respectively. We also found that overexpression of bFcRn enhances the phagocytosis of Ag-IgG immune complexes (ICs) by both macrophages and dendritic cells and significantly improves Ag presentation by dendritic cells. Finally, we determined that immunized bFcRn mice produce a much greater diversity of Ag-specific IgM, whereas only the levels, but not the diversity, of IgG is increased by overexpression of bFcRn. We suggest that the increase in diversity of IgG in Tg mice is prevented by a selective bias towards immunodominant epitopes of ovalbumin, which was used in this study as a model antigen. These results are also in line with our previous reports describing a substantial increase in the levels of Ag-specific IgG in FcRn Tg mice immunized with Ags that are weakly immunogenic and, therefore, not affected by immunodominance. PMID:22558422

  7. Alterations in plasma membrane promote overexpression and increase of sodium influx through epithelial sodium channel in hypertensive platelets.

    PubMed

    Cerecedo, D; Martínez-Vieyra, Ivette; Sosa-Peinado, Alejandro; Cornejo-Garrido, Jorge; Ordaz-Pichardo, Cynthia; Benítez-Cardoza, Claudia

    2016-08-01

    Platelets are small, anucleated cell fragments that activate in response to a wide variety of stimuli, triggering a complex series of intracellular pathways leading to a hemostatic thrombus formation at vascular injury sites. However, in essential hypertension, platelet activation contributes to causing myocardial infarction and ischemic stroke. Reported abnormalities in platelet functions, such as platelet hyperactivity and hyperaggregability to several agonists, contribute to the pathogenesis and complications of thrombotic events associated with hypertension. Platelet membrane lipid composition and fluidity are determining for protein site accessibility, structural arrangement of platelet surface, and response to appropriate stimuli. The present study aimed to demonstrate whether structural and biochemical abnormalities in lipid membrane composition and fluidity characteristic of platelets from hypertensive patients influence the expression of the Epithelial Sodium Channel (ENaC), fundamental for sodium influx during collagen activation. Wb, cytometry and quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) assays demonstrated ENaC overexpression in platelets from hypertensive subjects and in relation to control subjects. Additionally, our results strongly suggest a key role of β-dystroglycan as a scaffold for the organization of ENaC and associated proteins. Understanding of the mechanisms of platelet alterations in hypertension should provide valuable information for the pathophysiology of hypertension. PMID:27137675

  8. Alterations in plasma membrane promote overexpression and increase of sodium influx through epithelial sodium channel in hypertensive platelets.

    PubMed

    Cerecedo, D; Martínez-Vieyra, Ivette; Sosa-Peinado, Alejandro; Cornejo-Garrido, Jorge; Ordaz-Pichardo, Cynthia; Benítez-Cardoza, Claudia

    2016-08-01

    Platelets are small, anucleated cell fragments that activate in response to a wide variety of stimuli, triggering a complex series of intracellular pathways leading to a hemostatic thrombus formation at vascular injury sites. However, in essential hypertension, platelet activation contributes to causing myocardial infarction and ischemic stroke. Reported abnormalities in platelet functions, such as platelet hyperactivity and hyperaggregability to several agonists, contribute to the pathogenesis and complications of thrombotic events associated with hypertension. Platelet membrane lipid composition and fluidity are determining for protein site accessibility, structural arrangement of platelet surface, and response to appropriate stimuli. The present study aimed to demonstrate whether structural and biochemical abnormalities in lipid membrane composition and fluidity characteristic of platelets from hypertensive patients influence the expression of the Epithelial Sodium Channel (ENaC), fundamental for sodium influx during collagen activation. Wb, cytometry and quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) assays demonstrated ENaC overexpression in platelets from hypertensive subjects and in relation to control subjects. Additionally, our results strongly suggest a key role of β-dystroglycan as a scaffold for the organization of ENaC and associated proteins. Understanding of the mechanisms of platelet alterations in hypertension should provide valuable information for the pathophysiology of hypertension.

  9. Overexpression of angiopoietin-1 increases CD133+/c-kit+ cells and reduces myocardial apoptosis in db/db mouse infarcted hearts.

    PubMed

    Zeng, Heng; Li, Lanfang; Chen, Jian-Xiong

    2012-01-01

    Hematopoietic progenitor CD133(+)/c-kit(+) cells have been shown to be involved in myocardial healing following myocardial infarction (MI). Previously we demonstrated that angiopoietin-1(Ang-1) is beneficial in the repair of diabetic infarcted hearts. We now investigate whether Ang-1 affects CD133(+)/c-kit(+) cell recruitment to the infarcted myocardium thereby mediating cardiac repair in type II (db/db) diabetic mice. db/db mice were administered either adenovirus Ang-1 (Ad-Ang-1) or Ad-β-gal systemically immediately after ligation of the left anterior descending coronary artery (LAD). Overexpression of Ang-1 resulted in a significant increase in CXCR-4/SDF-1α expression and promoted CD133(+)/c-kit(+), CD133(+)/CXCR-4(+) and CD133(+)/SDF-1α(+) cell recruitment into ischemic hearts. Overexpression of Ang-1 led to significant increases in number of CD31(+) and smooth muscle-like cells and VEGF expression in bone marrow (BM). This was accompanied by significant decreases in cardiac apoptosis and fibrosis and an increase in myocardial capillary density. Ang-1 also upregulated Jagged-1, Notch3 and apelin expression followed by increases in arteriole formation in the infarcted myocardium. Furthermore, overexpression of Ang-1 resulted in a significant improvement of cardiac functional recovery after 14 days of ischemia. Our data strongly suggest that Ang-1 attenuates cardiac apoptosis and promotes cardiac repair by a mechanism involving in promoting CD133(+)/c-kit(+) cells and angiogenesis in diabetic db/db mouse infarcted hearts. PMID:22558265

  10. Parvalbumin overexpression alters immune-mediated increases in intracellular calcium, and delays disease onset in a transgenic model of familial amyotrophic lateral sclerosis

    NASA Technical Reports Server (NTRS)

    Beers, D. R.; Ho, B. K.; Siklos, L.; Alexianu, M. E.; Mosier, D. R.; Mohamed, A. H.; Otsuka, Y.; Kozovska, M. E.; McAlhany, R. E.; Smith, R. G.; Appel, S. H.

    2001-01-01

    Intracellular calcium is increased in vulnerable spinal motoneurons in immune-mediated as well as transgenic models of amyotrophic lateral sclerosis (ALS). To determine whether intracellular calcium levels are influenced by the calcium-binding protein parvalbumin, we developed transgenic mice overexpressing parvalbumin in spinal motoneurons. ALS immunoglobulins increased intracellular calcium and spontaneous transmitter release at motoneuron terminals in control animals, but not in parvalbumin overexpressing transgenic mice. Parvalbumin transgenic mice interbred with mutant SOD1 (mSOD1) transgenic mice, an animal model of familial ALS, had significantly reduced motoneuron loss, and had delayed disease onset (17%) and prolonged survival (11%) when compared with mice with only the mSOD1 transgene. These results affirm the importance of the calcium binding protein parvalbumin in altering calcium homeostasis in motoneurons. The increased motoneuron parvalbumin can significantly attenuate the immune-mediated increases in calcium and to a lesser extent compensate for the mSOD1-mediated 'toxic-gain-of-function' in transgenic mice.

  11. Psychostimulants affect dopamine transmission through both dopamine transporter-dependent and independent mechanisms

    PubMed Central

    dela Peña, Ike; Gevorkiana, Ruzanna; Shi, Wei-Xing

    2015-01-01

    The precise mechanisms by which cocaine and amphetamine-like psychostimulants exert their reinforcing effects are not yet fully defined. It is widely believed, however, that these drugs produce their effects by enhancing dopamine neurotransmission in the brain, especially in limbic areas such as the nucleus accumbens, by inducing dopamine transporter-mediated reverse transport and/or blocking dopamine reuptake though the dopamine transporter. Here, we present the evidence that aside from dopamine transporter, non-dopamine transporter-mediated mechanisms also participate in psychostimulant-induced dopamine release and contribute to the behavioral effects of these drugs, such as locomotor activation and reward. Accordingly, psychostimulants could increase norepinephrine release in the prefrontal cortex, the latter then alters the firing pattern of dopamine neurons resulting in changes in action potential-dependent dopamine release. These alterations would further affect the temporal pattern of dopamine release in the nucleus accumbens, thereby modifying information processing in that area. Hence, a synaptic input to a nucleus accumbens neuron may be enhanced or inhibited by dopamine depending on its temporal relationship to dopamine release. Specific temporal patterns of dopamine release may also be required for certain forms of synaptic plasticity in the nucleus accumbens. Together, these effects induced by psychostimulants, mediated through a non-dopamine transporter-mediated mechanism involving norepinephrine and the prefrontal cortex, may also contribute importantly to the reinforcing properties of these drugs. PMID:26209364

  12. Psychostimulants affect dopamine transmission through both dopamine transporter-dependent and independent mechanisms.

    PubMed

    dela Peña, Ike; Gevorkiana, Ruzanna; Shi, Wei-Xing

    2015-10-01

    The precise mechanisms by which cocaine and amphetamine-like psychostimulants exert their reinforcing effects are not yet fully defined. It is widely believed, however, that these drugs produce their effects by enhancing dopamine neurotransmission in the brain, especially in limbic areas such as the nucleus accumbens, by inducing dopamine transporter-mediated reverse transport and/or blocking dopamine reuptake though the dopamine transporter. Here, we present the evidence that aside from dopamine transporter, non-dopamine transporter-mediated mechanisms also participate in psychostimulant-induced dopamine release and contribute to the behavioral effects of these drugs, such as locomotor activation and reward. Accordingly, psychostimulants could increase norepinephrine release in the prefrontal cortex, the latter then alters the firing pattern of dopamine neurons resulting in changes in action potential-dependent dopamine release. These alterations would further affect the temporal pattern of dopamine release in the nucleus accumbens, thereby modifying information processing in that area. Hence, a synaptic input to a nucleus accumbens neuron may be enhanced or inhibited by dopamine depending on its temporal relationship to dopamine release. Specific temporal patterns of dopamine release may also be required for certain forms of synaptic plasticity in the nucleus accumbens. Together, these effects induced by psychostimulants, mediated through a non-dopamine transporter-mediated mechanism involving norepinephrine and the prefrontal cortex, may also contribute importantly to the reinforcing properties of these drugs. PMID:26209364

  13. TFEB Overexpression in the P301S Model of Tauopathy Mitigates Increased PHF1 Levels and Lipofuscin Puncta and Rescues Memory Deficits12

    PubMed Central

    Wang, Hongjie; Wang, Ruizhi; Carrera, Ivan; Xu, Shaohua

    2016-01-01

    Abstract Transcription factor EB (TFEB) was recently shown to be a master regulator of autophagy lysosome pathway. Here, we successfully generated and characterized transgenic mice overexpressing flag-TFEB. Enhanced autophagy in the flag-TFEB transgenic mice was confirmed by an increase in the cellular autophagy markers, as determined by both immunoblots and transmission electron microscopy. Surprisingly, in the flag-TFEB mice we observed increased activity of senescence-associated β-galactosidase by ∼66% of neurons in the cortex (p < 0.001) and 73% of neurons in the hippocampus (p < 0.001). More importantly, flag-TFEB expression remarkably reduced the levels of paired-helical filament (PHF)-tau from 372% in the P301S model of tauopathy to 171% (p < 0.001) in the cortex, and from 436% to 212% (p < 0.001) in the hippocampus. Significantly, reduced levels of NeuN in the cortex (38%, p < 0.001) and hippocampus (25%, p < 0.05) of P301S mice were almost completely restored to WT levels in the P301S/flag-TFEB double-transgenic mice. Also, levels of spinophilin in both the cortex (p < 0.001) and hippocampus (p < 0.001) were restored almost to WT levels. Most importantly, the age-associated lipofuscin granules, which are generally presumed to be nondegradable, were reduced by 57% (p < 0.001) in the cortex and by 55% (p < 0.001) in the hippocampus in the double-transgenic mice. Finally, TFEB overexpression in the P301S mice markedly reversed learning deficits in terms of spatial memory (Barnes maze), as well as working and reference memories (T maze). These data suggest that the overexpression of TFEB can reliably enhance autophagy in vivo, reduce levels of PHF-tau, and thereby reverse the deposition of lipofuscin granules and memory deficits. PMID:27257626

  14. Genetic Modification of Mesenchymal Stem Cells Overexpressing CCR1 Increases Cell Viability, Migration, Engraftment and Capillary Density in the Injured Myocardium

    PubMed Central

    Huang, Jing; Zhang, Zhiping; Guo, Jian; Ni, Aiguo; Deb, Arjun; Zhang, Lunan; Mirotsou, Maria; Pratt, Richard E; Dzau, Victor J

    2010-01-01

    Rationale Although mesenchymal stem cell (MSC) transplantation has been shown to promote cardiac repair in acute myocardial injury in vivo, its overall restorative capacity appears to be restricted mainly due to poor cell viability and low engraftment in the ischemic myocardium. Specific chemokines are upregulated in the infarcted myocardium. However the expression levels of the corresponding chemokine receptors (e.g. CCR1, CXCR2) in MSCs are very low. We hypothesized that this discordance may account for the poor MSC engraftment and survival. Objective To determine whether overexpression of CCR1 or CXCR2 chemokine receptors in MSCs augments their cell survival, migration and engraftment after injection in the infarcted myocardium. Methods and Results Overexpression of CCR1, but not CXCR2, dramatically increased chemokine-induced murine MSC migration and protected MSC from apoptosis in vitro. Moreover, when MSCs were injected intramyocardially one hour after coronary artery ligation, CCR1-MSCs accumulated in the infarcted myocardium at significantly higher levels than control-MSCs or CXCR2-MSCs three days post-myocardial infarction (MI). CCR1-MSC injected hearts exhibited a significant reduction in infarct size, reduced cardiomyocytes apoptosis and increased capillary density in injured myocardium three days post-MI. Furthermore, intramyocardial injection of CCR1-MSCs prevented cardiac remodeling and restored cardiac function 4 weeks post-MI. Conclusions Our results demonstrate the in vitro and in vivo salutary effects of genetic modification of stem cells. Specifically, overexpression of chemokine receptor enhances the migration, survival and engraftment of MSCs, and may provide a new therapeutic strategy for the injured myocardium. PMID:20378860

  15. FTO Is Increased in Muscle During Type 2 Diabetes, and Its Overexpression in Myotubes Alters Insulin Signaling, Enhances Lipogenesis and ROS Production, and Induces Mitochondrial Dysfunction

    PubMed Central

    Bravard, Amélie; Lefai, Etienne; Meugnier, Emmanuelle; Pesenti, Sandra; Disse, Emmanuel; Vouillarmet, Julien; Peretti, Nöel; Rabasa-Lhoret, Rémi; Laville, Martine; Vidal, Hubert; Rieusset, Jennifer

    2011-01-01

    OBJECTIVE A strong association between genetic variants and obesity was found for the fat mass and obesity-associated gene (FTO). However, few details are known concerning the expression and function of FTO in skeletal muscle of patients with metabolic diseases. RESEARCH DESIGN AND METHODS We investigated basal FTO expression in skeletal muscle from obese nondiabetic subjects and type 1 and type 2 diabetic patients, compared with age-matched control subjects, and its regulation in vivo by insulin, glucose, or rosiglitazone. The function of FTO was further studied in myotubes by overexpression experiments. RESULTS We found a significant increase of FTO mRNA and protein levels in muscle from type 2 diabetic patients, whereas its expression was unchanged in obese or type 1 diabetic patients. Moreover, insulin or glucose infusion during specific clamps did not regulate FTO expression in skeletal muscle from control or type 2 diabetic patients. Interestingly, rosiglitazone treatment improved insulin sensitivity and reduced FTO expression in muscle from type 2 diabetic patients. In myotubes, adenoviral FTO overexpression increased basal protein kinase B phosphorylation, enhanced lipogenesis and oxidative stress, and reduced mitochondrial oxidative function, a cluster of metabolic defects associated with type 2 diabetes. CONCLUSIONS This study demonstrates increased FTO expression in skeletal muscle from type 2 diabetic patients, which can be normalized by thiazolidinedione treatment. Furthermore, in vitro data support a potential implication of FTO in oxidative metabolism, lipogenesis and oxidative stress in muscle, suggesting that it could be involved in the muscle defects that characterize type 2 diabetes. PMID:20943749

  16. Initiation of calorie restriction in middle-aged male rats attenuates aging-related motoric decline and bradykinesia without increased striatal dopamine.

    PubMed

    Salvatore, Michael F; Terrebonne, Jennifer; Fields, Victoria; Nodurft, Danielle; Runfalo, Cori; Latimer, Brian; Ingram, Donald K

    2016-01-01

    Aging-related bradykinesia affects ∼ 15% of those reaching age 65 and 50% of those reaching their 80s. Given this high risk and lack of pharmacologic therapeutics, noninvasive lifestyle strategies should be identified to diminish its risk and identify the neurobiological targets to reduce aging-related bradykinesia. Early-life, long-term calorie restriction (CR) attenuates aging-related bradykinesia in rodents. Here, we addressed whether CR initiation at middle age could attenuate aging-related bradykinesia and motoric decline measured as rotarod performance. A 30% CR regimen was implemented for 6 months duration in 12-month-old male Brown-Norway Fischer 344 F1 hybrid rats after establishing individual baseline locomotor activities. Locomotor capacity was assessed every 6 weeks thereafter. The ad libitum group exhibited predictably decreased locomotor activity, except movement speed, out to 18 months of age. In contrast, in the CR group, movement number and horizontal activity did not decrease during the 6-month trial, and aging-related decline in rotarod performance was attenuated. The response to CR was influenced by baseline locomotor activity. The lower the locomotor activity level at baseline, the greater the response to CR. Rats in the lower 50th percentile surpassed their baseline level of activity, whereas rats in the top 50th percentile decreased at 6 weeks and then returned to baseline by 12 weeks of CR. We hypothesized that nigrostriatal dopamine tissue content would be greater in the CR group and observed a modest increase only in substantia nigra with no group differences in striatum, nucleus accumbens, or ventral tegmental area. These results indicate that initiation of CR at middle age may reduce aging-related bradykinesia, and, furthermore, subjects with below average locomotor activity may increase baseline activity. Sustaining nigral dopamine neurotransmission may be one component of preserving locomotor capabilities during aging.

  17. Orexigenic effects of endomorphin-2 (EM-2) related to decreased CRH gene expression and increased dopamine and norepinephrine activity in the hypothalamus.

    PubMed

    Brunetti, Luigi; Ferrante, Claudio; Orlando, Giustino; Recinella, Lucia; Leone, Sheila; Chiavaroli, Annalisa; Di Nisio, Chiara; Shohreh, Rugia; Manippa, Fabio; Ricciuti, Adriana; Mollica, Adriano; Vacca, Michele

    2013-10-01

    Endomorphin-1 (EM-1) and endomorphin-2 (EM-2) are opioid peptides which are selective partial agonists of μ-opioid receptor. We studied the effects of EM-2 injected into the arcuate nucleus (ARC) of the hypothalamus on feeding behavior and gene expression of orexigenic [agouti-related peptide (AgRP), neuropeptide Y (NPY) and orexin-A] and anorexigenic [cocaine and amphetamine-regulated transcript (CART), corticotrophin releasing hormone (CRH) and proopiomelanocortin (POMC)] peptides in male Wistar rats fed a standard laboratory diet. Furthermore, we evaluated the effects of EM-2 on dopamine (DA), norepinephrine (NE) and serotonin (5-hydroxytryptamine, 5-HT) steady state concentrations, in the hypothalamus. 64 rats (16 for each group of treatment) were injected into the ARC, at 9.00 am, with either vehicle or EM-2 (0.50-0.75 μmol/kg) or EM-2 (0.50 μmol/kg) plus β-funaltrexamine (0.20 μmol/kg). Food intake was recorded through 24h following injection, and hypothalamic DA, NE, 5-HT levels and neuropeptide gene expression were evaluated 24h after EM-2 administration. Compared to vehicle, EM-2 significantly increased food intake, throughout 24h post-injection. Furthermore, EM-2 treatment led to a significant increase of DA and NE concentrations and a decrease of CRH mRNA levels. On the other hand, β-funaltrexamine administration reverted both feeding stimulatory and neuromodulatory effects induced by EM-2. We can conclude that the orexigenic effect of μ-opioid receptor activation by EM-2 could be related to both inhibition of CRH and stimulation of dopamine and norepinephrine levels, in the hypothalamus.

  18. Up-regulation of TRPV1 in mononuclear cells of end-stage kidney disease patients increases susceptibility to N-arachidonoyl-dopamine (NADA)-induced cell death.

    PubMed

    Saunders, Cassandra I; Fassett, Robert G; Geraghty, Dominic P

    2009-10-01

    Transient receptor potential vanilloid (TRPV) 1 channels function as sensors for a variety of noxious and inflammatory signals, including capsaicin, heat and protons, and are up-regulated under inflammatory conditions. As end-stage kidney disease (ESKD) is associated with chronic inflammation, impaired immunity and depressed lymphocyte numbers, we sought to determine whether altered TRPV1 (and related TRPV2) expression in immune cells might be a contributing factor. TRPV1 and TRPV2 mRNA expression in peripheral blood mononuclear cells (PBMC) was similar in controls and ESKD patients by quantitative real-time RT-PCR. However, using immunocytochemistry, TRPV1-immunoreactivity was significantly higher and TRPV2-immunoreactivity was significantly lower in PBMC from ESKD patients compared to controls. The plant-derived TRPV1 agonists, capsaicin and resiniferatoxin (RTX) and the putative endovanilloid/endocannabinoids, N-arachidonoyl-dopamine (NADA) and N-oleoyl-dopamine (OLDA), induced concentration-dependent death of PBMC from healthy donors with a rank order of potency of RTX>NADA>OLDA>capsaicin. TRPV1 (5'-iodoresiniferatoxin) and cannabinoid (CB2; AM630) receptor antagonists blocked the cytotoxic effect of NADA. In subsequent experiments, PBMC from ESKD patients exhibited significantly increased susceptibility to NADA-induced death compared to PBMC from controls. The apparent up-regulation of TRPV1 may be a response to the inflammatory milieu in which PBMC exist in ESKD and may be responsible for the increased susceptibility of these cells to NADA-induced death, providing a possible explanation as to why ESKD patients have reduced lymphocyte counts and impaired immune function. Thus, TRPV1 (and possibly CB2) antagonists may have potential for the treatment of immune dysfunction in ESKD.

  19. Overexpressing GSH1 and AsPCS1 simultaneously increases the tolerance and accumulation of cadmium and arsenic in Arabidopsis thaliana.

    PubMed

    Guo, Jiangbo; Dai, Xiaojing; Xu, Wenzhong; Ma, Mi

    2008-07-01

    The goal of this study was to develop transgenic plants with increased tolerance for and accumulation of heavy metals and metalloids from soil by simultaneous overexpression of AsPCS1 and GSH1 (derived from garlic and baker's yeast) in Arabidopsis thaliana. Phytochelatins (PCs) and glutathione (GSH) are the main binding peptides involved in chelating heavy metal ions in plants and other living organisms. Single-gene transgenic lines had higher tolerance to and accumulated more Cd and As than wild-type. Compared to single-gene transgenic lines, dual-gene transformants exhibited significantly higher tolerance to and accumulated more Cd and As. One of the dual-gene transgenic lines, PG1, accumulated twice the amount of Cd as single-gene transgenic lines. Simultaneous overexpression of AsPCS1 and GSH1 led to elevated total PC production in transgenic Arabidopsis. These results indicate that such a stacking of modified genes is capable of increasing Cd and As tolerance and accumulation in transgenic lines, and represents a highly promising new tool for use in phytoremediation efforts.

  20. Overexpression of Prolyl-4-Hydroxylase-α1 Stabilizes but Increases Shear Stress-Induced Atherosclerotic Plaque in Apolipoprotein E-Deficient Mice

    PubMed Central

    Liu, Xin-xin; Li, Meng-meng; Zhang, Yu; Chen, Liang; Wang, Lin; Di, Ming-xue

    2016-01-01

    The rupture and erosion of atherosclerotic plaque can induce coronary thrombosis. Prolyl-4-hydroxylase (P4H) plays a central role in the synthesis of all known types of collagens, which are the most abundant constituent of the extracellular matrix in atherosclerotic plaque. The pathogenesis of atherosclerosis is thought to be in part caused by shear stress. In this study, we aimed to investigate a relationship between P4Hα1 and shear stress-induced atherosclerotic plaque. Carotid arteries of ApoE−/− mice were exposed to low and oscillatory shear stress conditions by the placement of a shear stress cast for 2 weeks; we divided 60 male ApoE−/− mice into three groups for treatments with saline (mock) (n = 20), empty lentivirus (lenti-EGFP) (n = 20), and lentivirus-P4Hα1 (lenti-P4Hα1) (n = 20). Our results reveal that after 2 weeks of lenti-P4Hα1 treatment both low and oscillatory shear stress-induced plaques increased collagen and the thickness of fibrous cap and decreased macrophage accumulation but no change in lipid accumulation. We also observed that overexpression of P4Ha1 increased plaque size. Our study suggests that P4Hα1 overexpression might be a potential therapeutic target in stabilizing vulnerable plaques.

  1. Increased oxidative stress and toxicity in ADH and CYP2E1 overexpressing human hepatoma VL-17A cells exposed to high glucose.

    PubMed

    Chandrasekaran, Karthikeyan; Swaminathan, Kavitha; Kumar, S Mathan; Clemens, Dahn L; Dey, Aparajita

    2012-05-01

    High glucose mediated oxidative stress and cell death is a well documented phenomenon. Using VL-17A cells which are HepG2 cells over-expressing alcohol dehydrogenase (ADH) and cytochrome P450 2E1 (CYP2E1) and control HepG2 cells, the association of ADH and CYP2E1 with high glucose mediated oxidative stress and toxicity in liver cells was investigated. Cell viability was measured and apoptosis or necrosis was determined through caspase-3 activity, Annexin V-propidium iodide staining and detecting decreases in mitochondrial membrane potential. Reactive oxygen species, lipid peroxidation and the formation of advanced glycated-end products were assessed. The levels of several antioxidants which included glutathione, glutathione peroxidase, catalase and superoxide dismutase were altered in high glucose treated VL-17A cells. Greater toxicity was observed in VL-17A cells exposed to high glucose when compared to HepG2 cells. Oxidative stress parameters were greatly increased in high glucose exposed VL-17A cells and apoptotic cell death was observed. Inhibition of CYP2E1 or caspase 3 or addition of the antioxidant trolox led to significant decreases in high glucose mediated oxidative stress and toxicity. Thus, the over-expression of ADH and CYP2E1 in liver cells is associated with increased high glucose mediated oxidative stress and toxicity.

  2. Ectopic overexpression of SlHsfA3, a heat stress transcription factor from tomato, confers increased thermotolerance and salt hypersensitivity in germination in transgenic Arabidopsis.

    PubMed

    Li, Zhenjun; Zhang, Lili; Wang, Aoxue; Xu, Xiangyang; Li, Jingfu

    2013-01-01

    Plant heat stress transcription factors (Hsfs) are the critical components involved in mediating responses to various environmental stressors. However, the detailed roles of many plant Hsfs are far from fully understood. In this study, an Hsf (SlHsfA3) was isolated from the cultivated tomato (Solanum lycopersicum, Sl) and functionally characterized at the genetic and developmental levels. The nucleus-localized SlHsfA3 was basally and ubiquitously expressed in different plant organs. The expression of SlHsfA3 was induced dramatically by heat stress, moderately by high salinity, and slightly by drought, but was not induced by abscisic acid (ABA). The ectopic overexpression of SlHsfA3 conferred increased thermotolerance and late flowering phenotype to transgenic Arabidopsis plants. Moreover, SlHsfA3 played a negative role in controlling seed germination under salt stress. RNA-sequencing data demonstrated that a number of heat shock proteins (Hsps) and stress-associated genes were induced in Arabidopsis plants overexpressing SlHsfA3. A gel shift experiment and transient expression assays in Nicotiana benthamiana leaves demonstrated that SlHsfA3 directly activates the expression of SlHsp26.1-P and SlHsp21.5-ER. Taken together, our results suggest that SlHsfA3 behaves as a typical Hsf to contribute to plant thermotolerance. The late flowering and seed germination phenotypes and the RNA-seq data derived from SlHsfA3 overexpression lines lend more credence to the hypothesis that plant Hsfs participate in diverse physiological and biochemical processes related to adverse conditions. PMID:23349984

  3. Overexpression of the sweet potato IbOr gene results in the increased accumulation of carotenoid and confers tolerance to environmental stresses in transgenic potato.

    PubMed

    Goo, Young-Min; Han, Eun-Heui; Jeong, Jae Cheol; Kwak, Sang-Soo; Yu, Jaeju; Kim, Yun-Hee; Ahn, Mi-Jeong; Lee, Shin-Woo

    2015-01-01

    In a previous study, we have evidenced that the overexpression of the IbOr gene isolated from sweet potato conferred a tolerance activity against salinity and methyl viologen (MV) treatment in transgenic sweet potato calli along with an enhanced carotenoid content. In this study, to further examine the function of the IbOr gene in heterologous organism, we transformed the IbOr gene into potato under the direction of SWPA2 promoter, a strong inducible promoter upon treatment with various environmental stresses. Consistently with our previous study of sweet potato calli, the level of total carotenoid was elevated up to 2.7-fold (38.1 μg g(-1)DW) compared to the non-transgenic control, Atlantic cultivar. However, the composition of carotenoid was not influenced by the overexpression of the IbOr gene since only pre-existing carotenoids in the non-transgenic control including violaxanthin, lutien and β-carotene were elevated at a similar level of total carotenoids. In general, the transcript levels for most of carotenogenesis-related genes were elevated in transgenic tuber, whereas they remained at similar levels in transgenic leaf tissues compared to those of non-transgenic controls. The increased levels of carotenoid content in the leaf or tuber tissue of transgenic lines were correlated with the enhanced tolerance activity against salt- or MV-mediated oxidative stresses and DPPH radical-scavenging activity. Our preliminary results suggest that further investigation is required for the development of a crop tolerant to salinity and other environmental stresses through the overexpression of the IbOr gene.

  4. Ectopic Overexpression of SlHsfA3, a Heat Stress Transcription Factor from Tomato, Confers Increased Thermotolerance and Salt Hypersensitivity in Germination in Transgenic Arabidopsis

    PubMed Central

    Li, Zhenjun; Zhang, Lili; Wang, Aoxue; Xu, Xiangyang; Li, Jingfu

    2013-01-01

    Plant heat stress transcription factors (Hsfs) are the critical components involved in mediating responses to various environmental stressors. However, the detailed roles of many plant Hsfs are far from fully understood. In this study, an Hsf (SlHsfA3) was isolated from the cultivated tomato (Solanum lycopersicum, Sl) and functionally characterized at the genetic and developmental levels. The nucleus-localized SlHsfA3 was basally and ubiquitously expressed in different plant organs. The expression of SlHsfA3 was induced dramatically by heat stress, moderately by high salinity, and slightly by drought, but was not induced by abscisic acid (ABA). The ectopic overexpression of SlHsfA3 conferred increased thermotolerance and late flowering phenotype to transgenic Arabidopsis plants. Moreover, SlHsfA3 played a negative role in controlling seed germination under salt stress. RNA-sequencing data demonstrated that a number of heat shock proteins (Hsps) and stress-associated genes were induced in Arabidopsis plants overexpressing SlHsfA3. A gel shift experiment and transient expression assays in Nicotiana benthamiana leaves demonstrated that SlHsfA3 directly activates the expression of SlHsp26.1-P and SlHsp21.5-ER. Taken together, our results suggest that SlHsfA3 behaves as a typical Hsf to contribute to plant thermotolerance. The late flowering and seed germination phenotypes and the RNA-seq data derived from SlHsfA3 overexpression lines lend more credence to the hypothesis that plant Hsfs participate in diverse physiological and biochemical processes related to adverse conditions. PMID:23349984

  5. Overexpression of the phosphatidylinositol synthase gene (ZmPIS) conferring drought stress tolerance by altering membrane lipid composition and increasing ABA synthesis in maize.

    PubMed

    Liu, Xiuxia; Zhai, Shumei; Zhao, Yajie; Sun, Baocheng; Liu, Cheng; Yang, Aifang; Zhang, Juren

    2013-05-01

    Phosphatidylinositol (PtdIns) synthase is a key enzyme in the phospholipid pathway and catalyses the formation of PtdIns. PtdIns is not only a structural component of cell membranes, but also the precursor of the phospholipid signal molecules that regulate plant response to environment stresses. Here, we obtained transgenic maize constitutively overexpressing or underexpressing PIS from maize (ZmPIS) under the control of a maize ubiquitin promoter. Transgenic plants were confirmed by PCR, Southern blotting analysis and real-time RT-PCR assay. The electrospray ionization tandem mass spectrometry (ESI-MS/MS)-based lipid profiling analysis showed that, under drought stress conditions, the overexpression of ZmPIS in maize resulted in significantly elevated levels of most phospholipids and galactolipids in leaves compared with those in wild type (WT). At the same time, the expression of some genes involved in the phospholipid metabolism pathway and the abscisic acid (ABA) biosynthesis pathway including ZmPLC, ZmPLD, ZmDGK1, ZmDGK3, ZmPIP5K9, ZmABA1, ZmNCED, ZmAAO1, ZmAAO2 and ZmSCA1 was markedly up-regulated in the overexpression lines after drought stress. Consistent with these results, the drought stress tolerance of the ZmPIS sense transgenic plants was enhanced significantly at the pre-flowering stages compared with WT maize plants. These results imply that ZmPIS regulates the plant response to drought stress through altering membrane lipid composition and increasing ABA synthesis in maize.

  6. Microinjections of acetaldehyde or salsolinol into the posterior ventral tegmental area increase dopamine release in the nucleus accumbens shell

    PubMed Central

    Deehan, Gerald A.; Engleman, Eric A.; Ding, Zheng-Ming; McBride, William J.; Rodd, Zachary A.

    2014-01-01

    Background Published findings indicate that acetaldehyde (ACD; the first metabolite of EtOH) and salsolinol (SAL; formed through the non-enzymatic condensation of ACD and dopamine) can be formed following ethanol (EtOH) consumption. Both ACD and SAL exhibit reinforcing properties within the posterior ventral tegmental area (pVTA) and both exhibit an inverted “U-shaped” dose-response curve. The current study was undertaken to examine the dose-response effects of microinjections of ACD or SAL into the pVTA on DA efflux in the nucleus accumbens shell (AcbSh). Methods For the first experiment, separate groups of male Wistar rats received pulse microinjections of aCSF or 12, 23 or 90 µM ACD into the pVTA while extracellular DA levels were concurrently measured in the AcbSh. The second experiment was similarly conducted, except rats were given microinjections of aCSF or 0.03, 0.3, 1.0 or 3.0 µM SAL, while extracellular levels of DA were measured in the AcbSh. Results Both ACD and SAL produced a dose-dependent inverted “U-shaped” response on DA release in the AcbSh, with 23 µM ACD (200% baseline) and 0.3 µM SAL (300% baseline) producing maximal peak responses with higher concentrations of ACD (90 µM) and SAL (3.0 µM) producing significantly lower DA efflux. Conclusions The findings from the current study indicate that local application of intermediate concentrations of ACD and SAL stimulated DA neurons in the pVTA, whereas higher concentrations may be having secondary effects within the pVTA that inhibit DA neuronal activity. The present results parallel studies on the reinforcing effects of ACD and SAL in the pVTA and support the idea that the reinforcing effects of ACD and SAL within the pVTA are mediated by activating DA neurons. PMID:23278868

  7. Selection Based on FOXA2 Expression Is Not Sufficient to Enrich for Dopamine Neurons From Human Pluripotent Stem Cells

    PubMed Central

    Aguila, Julio Cesar; Blak, Alexandra; van Arensbergen, Joris; Sousa, Amaia; Vázquez, Nerea; Aduriz, Ariane; Gayosso, Mayela; Lopez Mato, Maria Paz; Lopez de Maturana, Rakel; Hedlund, Eva; Sonntag, Kai-Christian

    2014-01-01

    Human embryonic and induced pluripotent stem cells are potential cell sources for regenerative approaches in Parkinson disease. Inductive differentiation protocols can generate midbrain dopamine neurons but result in heterogeneous cell mixtures. Therefore, selection strategies are necessary to obtain uniform dopamine cell populations. Here, we developed a selection approach using lentivirus vectors to express green fluorescent protein under the promoter region of FOXA2, a transcription factor that is expressed in the floor plate domain that gives rise to dopamine neurons during embryogenesis. We first validated the specificity of the vectors in human cell lines against a promoterless construct. We then selected FOXA2-positive neural progenitors from several human pluripotent stem cell lines, which demonstrated a gene expression profile typical for the ventral domain of the midbrain and floor plate, but failed to enrich for dopamine neurons. To investigate whether this was due to the selection approach, we overexpressed FOXA2 in neural progenitors derived from human pluripotent stem cell lines. FOXA2 forced expression resulted in an increased expression of floor plate but not mature neuronal markers. Furthermore, selection of the FOXA2 overexpressing fraction also failed to enrich for dopamine neurons. Collectively, our results suggest that FOXA2 is not sufficient to induce a dopaminergic fate in this system. On the other hand, our study demonstrates that a combined approach of promoter activation and lentivirus vector technology can be used as a versatile tool for the selection of a defined cell population from a variety of human pluripotent stem cell lines. PMID:25024431

  8. Overexpression of ACC gene from oleaginous yeast Lipomyces starkeyi enhanced the lipid accumulation in Saccharomyces cerevisiae with increased levels of glycerol 3-phosphate substrates.

    PubMed

    Wang, Jiancai; Xu, Ronghua; Wang, Ruling; Haque, Mohammad Enamul; Liu, Aizhong

    2016-06-01

    The conversion of acetyl-CoA to malonyl-CoA by acetyl-CoA carboxylase (ACC) is the rate-limiting step in fatty acid biosynthesis. In this study, a gene coding for ACC was isolated and characterized from an oleaginous yeast, Lipomyces starkeyi. Real-time quantitative PCR (qPCR) analysis of L. starkeyi acetyl-CoA carboxylase gene (LsACC1) showed that the expression levels were upregulated with the fast accumulation of lipids. The LsACC1 was co-overexpressed with the glycerol 3-phosphate dehydrogenase gene (GPD1), which regulates lipids biosynthesis by supplying another substrates glycerol 3-phosphate for storage lipid assembly, in the non-oleaginous yeast Saccharomyces cerevisiae. Further, the S. cerevisiae acetyl-CoA carboxylase (ScACC1) was transferred with GPD1 and its function was analyzed in comparison with LsACC1. The results showed that overexpressed LsACC1 and GPD1 resulted in a 63% increase in S. cerevisiae. This study gives new data in understanding of the molecular mechanisms underlying the regulation of fatty acids and lipid biosynthesis in yeasts.

  9. Constitutive overexpression of the sucrose transporter SoSUT1 in potato plants increases arbuscular mycorrhiza fungal root colonization under high, but not under low, soil phosphorus availability.

    PubMed

    Gabriel-Neumann, Elke; Neumann, Günter; Leggewie, Georg; George, Eckhard

    2011-06-15

    The sucrose transporter SUT1 functions in phloem loading of photoassimilates in solanaceous plant species. In the present study, wildtype and transgenic potato plants with either constitutive overexpression or antisense inhibition of SUT1 were grown under high or low phosphorus (P) fertilization levels in the presence or absence of the arbuscular mycorrhizal (AM) fungus Glomus intraradices. At a low soil P fertilization level, the extent of AM fungal root colonization was not different among the genotypes. In all plants, the AM symbiosis contributed significantly to P uptake under these conditions. In response to a high soil P fertilization level, all genotypes showed a decrease in AM fungal root colonization, indicating that the expression level of SUT1 does not constitute a major mechanism of control over AM development in response to the soil P availability. However, plants with overexpression of SUT1 showed a higher extent of AM fungal root colonization compared with the other genotypes when the soil P availability was high. Whether an increased symbiotic C supply, alterations in the phytohormonal balance, or a decreased synthesis of antimicrobial compounds was the major cause for this effect requires further investigation. In plants with impaired phloem loading, a low C status of plant sink tissues did apparently not negatively affect plant C supply to the AM symbiosis. It is possible that, at least during vegetative and early generative growth, source rather than sink tissues exert control over amounts of C supplied to AM fungi.

  10. Overexpression of ErbB2 renders breast cancer cells susceptible to 3-BrPA through the increased dissociation of hexokinase II from mitochondrial outer membrane

    PubMed Central

    GAO, SUJIE; CHEN, XUEBO; JIN, HONGYONG; REN, SHENGNAN; LIU, ZHUO; FANG, XUEDONG; ZHANG, GUIZHEN

    2016-01-01

    ErbB2 is known to upregulate glycolysis in breast cancer, however, the precise mechanisms remain unclear. In the present study, ErbB2 upregulated Hexokinase II (HK II) activity by increasing the binding of HK II to the mitochondrial outer membrane. Dysregulated glucose metabolism in high ErbB2-expressing breast cancer cells induces susceptibility to glucose starvation and glycolysis inhibition. Additionally, HK II has a tendency to dissociate from the mitochondria outer membrane in ErbB2-overexpressing cells following treatment with the HK II inhibitor, 3-BrPA. Furthermore, 3-BrPA treatment results in decreased mitochondria membrane potential and release of cytochrome c into cytoplasm in ErbB2-overexpressing cells, leading to activation of the mitochondrial apoptotic signaling pathway. In summary, the results demonstrate a novel mechanism for ErbB2-activated glycolysis and reveal that 3-BrPA is effective in reducing ErbB2-positive breast cancer cell viability by targeting HK II in vitro and in vivo. PMID:26893781

  11. Constitutive overexpression of the sucrose transporter SoSUT1 in potato plants increases arbuscular mycorrhiza fungal root colonization under high, but not under low, soil phosphorus availability.

    PubMed

    Gabriel-Neumann, Elke; Neumann, Günter; Leggewie, Georg; George, Eckhard

    2011-06-15

    The sucrose transporter SUT1 functions in phloem loading of photoassimilates in solanaceous plant species. In the present study, wildtype and transgenic potato plants with either constitutive overexpression or antisense inhibition of SUT1 were grown under high or low phosphorus (P) fertilization levels in the presence or absence of the arbuscular mycorrhizal (AM) fungus Glomus intraradices. At a low soil P fertilization level, the extent of AM fungal root colonization was not different among the genotypes. In all plants, the AM symbiosis contributed significantly to P uptake under these conditions. In response to a high soil P fertilization level, all genotypes showed a decrease in AM fungal root colonization, indicating that the expression level of SUT1 does not constitute a major mechanism of control over AM development in response to the soil P availability. However, plants with overexpression of SUT1 showed a higher extent of AM fungal root colonization compared with the other genotypes when the soil P availability was high. Whether an increased symbiotic C supply, alterations in the phytohormonal balance, or a decreased synthesis of antimicrobial compounds was the major cause for this effect requires further investigation. In plants with impaired phloem loading, a low C status of plant sink tissues did apparently not negatively affect plant C supply to the AM symbiosis. It is possible that, at least during vegetative and early generative growth, source rather than sink tissues exert control over amounts of C supplied to AM fungi. PMID:21382646

  12. Over-Expression of a Tobacco Nitrate Reductase Gene in Wheat (Triticum aestivum L.) Increases Seed Protein Content and Weight without Augmenting Nitrogen Supplying

    PubMed Central

    Zhao, Xiao-Qiang; Nie, Xuan-Li; Xiao, Xing-Guo

    2013-01-01

    Heavy nitrogen (N) application to gain higher yield of wheat (Triticum aestivum L.) resulted in increased production cost and environment pollution. How to diminish the N supply without losing yield and/or quality remains a challenge. To meet the challenge, we integrated and expressed a tobacco nitrate reductase gene (NR) in transgenic wheat. The 35S-NR gene was transferred into two winter cultivars, “Nongda146” and “Jimai6358”, by Agrobacterium-mediation. Over-expression of the transgene remarkably enhanced T1 foliar NR activity and significantly augmented T2 seed protein content and 1000-grain weight in 63.8% and 68.1% of T1 offspring (total 67 individuals analyzed), respectively. Our results suggest that constitutive expression of foreign nitrate reductase gene(s) in wheat might improve nitrogen use efficiency and thus make it possible to increase seed protein content and weight without augmenting N supplying. PMID:24040315

  13. Over-expression of NADH-dependent oxidoreductase (fucO) for increasing furfural or 5-hydroxymethylfurfural tolerance

    DOEpatents

    Miller, Elliot N.; Zhang, Xueli; Yomano, Lorraine P.; Wang, Xuan; Shanmugam, Keelnatham T.; Ingram, Lonnie O'Neal

    2015-10-13

    The subject invention pertains to the discovery that the NADH-dependent propanediol oxidoreductase (FucO) can reduce furfural. This allows for a new approach to improve furfural tolerance in bacterial and/or yeast cells used to produce desired products. Thus, novel biocatalysts (bacterial, fungal or yeast cells) exhibiting increased tolerance to furfural and 5-hydroxymethylfurfural (5-HMF) are provided as are methods of making and using such biocatalysts for the production of a desired product.

  14. Overexpression of Glutamate Decarboxylase in Mesenchymal Stem Cells Enhances Their Immunosuppressive Properties and Increases GABA and Nitric Oxide Levels

    PubMed Central

    González, Marisol; Vilches, Rodrigo; Rojas, Pablo; Vásquez, Manuel; Kurte, Mónica; Vega-Letter, Ana María; Carrión, Flavio; Figueroa, Fernando; Rojas, Patricio; Irarrázabal, Carlos

    2016-01-01

    The neurotransmitter GABA has been recently identified as a potent immunosuppressive agent that targets both innate and adaptive immune systems and prevents disease progression of several autoimmunity models. Mesenchymal stem cells (MSCs) are self-renewing progenitor cells that differentiate into various cell types under specific conditions, including neurons. In addition, MSC possess strong immunosuppressive capabilities. Upon cytokine priming, undifferentiated MSC suppress T-cell proliferation via cell-to-cell contact mechanisms and the secretion of soluble factors like nitric oxide, prostaglandin E2 and IDO. Although MSC and MSC-derived neuron-like cells express some GABAergic markers in vitro, the role for GABAergic signaling in MSC-mediated immunosuppression remains completely unexplored. Here, we demonstrate that pro-inflammatory cytokines selectively regulate GAD-67 expression in murine bone marrow-MSC. However, expression of GAD-65 is required for maximal GABA release by MSC. Gain of function experiments using GAD-67 and GAD-65 co-expression demonstrates that GAD increases immunosuppressive function in the absence of pro-inflammatory licensing. Moreover, GAD expression in MSC evokes an increase in both GABA and NO levels in the supernatants of co-cultured MSC with activated splenocytes. Notably, the increase in NO levels by GAD expression was not observed in cultures of isolated MSC expressing GAD, suggesting crosstalk between these two pathways in the setting of immunosuppression. These results indicate that GAD expression increases MSC-mediated immunosuppression via secretion of immunosuppressive agents. Our findings may help reconsider GABAergic activation in MSC for immunological disorders. PMID:27662193

  15. Stimulation of glutamate receptors in the ventral tegmental area is necessary for serotonin-2 receptor-induced increases in mesocortical dopamine release.

    PubMed

    Pehek, E A; Hernan, A E

    2015-04-01

    Modulation of dopamine (DA) released by serotonin-2 (5-HT2) receptors has been implicated in the mechanism of action of antipsychotic drugs. The mesocortical DA system has been implicated particularly in the cognitive deficits observed in schizophrenia. Agonism at 5-HT2A receptors in the prefrontal cortex (PFC) is associated with increases in cortical DA release. Evidence indicates that 5-HT2A receptors in the cortex regulate mesocortical DA release through stimulation of a "long-loop" feedback system from the PFC to the ventral tegmental area (VTA) and back. However, a causal role for VTA glutamate in the 5-HT2-induced increases in PFC DA has not been established. The present study does so by measuring 5-HT2 agonist-induced DA release in the cortex after infusions of glutamate antagonists into the VTA of the rat. Infusions of a combination of a N-methyl-d-aspartic acid (NMDA) (AP-5: 2-amino-5-phosphopentanoic acid) and an AMPA/kainate (CNQX: 6-cyano-7-nitroquinoxaline-2,3-dione) receptor antagonist into the VTA blocked the increases in cortical DA produced by administration of the 5-HT2 agonist DOI [(±)-2,5-dimethoxy-4-iodoamphetamine] (2.5mg/kg s.c.). These results demonstrate that stimulation of glutamate receptors in the VTA is necessary for 5-HT2 agonist-induced increases in cortical DA.

  16. Stimulation of glutamate receptors in the ventral tegmental area is necessary for serotonin-2 receptor-induced increases in mesocortical dopamine release.

    PubMed

    Pehek, E A; Hernan, A E

    2015-04-01

    Modulation of dopamine (DA) released by serotonin-2 (5-HT2) receptors has been implicated in the mechanism of action of antipsychotic drugs. The mesocortical DA system has been implicated particularly in the cognitive deficits observed in schizophrenia. Agonism at 5-HT2A receptors in the prefrontal cortex (PFC) is associated with increases in cortical DA release. Evidence indicates that 5-HT2A receptors in the cortex regulate mesocortical DA release through stimulation of a "long-loop" feedback system from the PFC to the ventral tegmental area (VTA) and back. However, a causal role for VTA glutamate in the 5-HT2-induced increases in PFC DA has not been established. The present study does so by measuring 5-HT2 agonist-induced DA release in the cortex after infusions of glutamate antagonists into the VTA of the rat. Infusions of a combination of a N-methyl-d-aspartic acid (NMDA) (AP-5: 2-amino-5-phosphopentanoic acid) and an AMPA/kainate (CNQX: 6-cyano-7-nitroquinoxaline-2,3-dione) receptor antagonist into the VTA blocked the increases in cortical DA produced by administration of the 5-HT2 agonist DOI [(±)-2,5-dimethoxy-4-iodoamphetamine] (2.5mg/kg s.c.). These results demonstrate that stimulation of glutamate receptors in the VTA is necessary for 5-HT2 agonist-induced increases in cortical DA. PMID:25637799

  17. Del-1 overexpression in endothelial cells increases vascular density in tissue-engineered implants containing endothelial cells and adipose-derived mesenchymal stromal cells.

    PubMed

    Ciucurel, Ema C; Sefton, Michael V

    2014-04-01

    We used a combination of strategies to stimulate the vascularization of tissue-engineered constructs in vivo including a modular approach to build larger tissues from individual building blocks ("modules") mixed together. Each building block included vascular cells by design; modules were submillimeter-sized collagen gels with an outer layer of endothelial cells (ECs), and with embedded adipose-derived mesenchymal stromal cells (adMSCs) to support EC survival and blood vessel maturation in vivo. We transduced the ECs that coat the modules with a lentiviral construct to overexpress the angiogenic extracellular matrix (ECM) protein Developmental endothelial locus-1 (Del-1). Upon injection of modules in a subcutaneous SCID/Bg mouse model, there was an increase in the number of blood vessels for implants with ECs transduced to overexpress Del-1 compared with control implants (with enhanced green fluorescent protein [eGFP]-transduced ECs) over the 21-day duration of the study. The greatest difference between Del-1 and eGFP implants and the highest number of blood vessels were observed 7 days after transplantation. The day-7 Del-1 implants also had increased SMA+ staining compared with control, suggesting increased blood vessel maturation through recruitment of SMA+ smooth muscle cells or pericytes to stabilize the newly formed blood vessels. Perfusion studies (microcomputed tomography, ultrasound imaging, and systemic injection of fluorescent UEA-1 or dextran) showed that some of the newly formed blood vessels (both donor derived and host derived, in both Del-1 and eGFP implants) were perfused and connected to the host vasculature as early as 7 days after transplantation, and at later time points as well. Nevertheless, perfusion of the implants was limited in some cases, suggesting that further improvements are necessary to normalize the vasculature at the implant site. PMID:24151812

  18. Increased sensitivity to cocaine self-administration in HIV-1 transgenic rats is associated with changes in striatal dopamine transporter binding

    PubMed Central

    McIntosh, Scot; Sexton, Tammy; Pattison, Lindsey P.; Childers, Steven R.; Hemby, Scott E.

    2015-01-01

    Cocaine abuse in HIV patients accelerates the progression and severity of neuropathology, motor impairment and cognitive dysfunction compared to non-drug using HIV patients. Cocaine and HIV interact with the dopamine transporter (DAT); however, the effect of their interaction on DAT binding remains understudied. The present study compared the dose-response functions for intravenous self-administration of cocaine and heroin between male HIV-1 transgenic (HIV-1 Tg) and Fischer 344 rats. The cocaine and heroin dose-response functions exhibit an inverted U-shape for both HIV-1 Tg and F344 rats. For cocaine, the number of infusions for each dose on the ascending limb was greater for HIV-1 Tg versus F344 rats. No significant changes in the heroin dose-response function were observed in HIV-1 Tg animals. Following the conclusion of self-administration experiments, DAT binding was assessed in striatal membranes. Saturation binding of the cocaine analog [125I] 3β-(4-iodophenyl)tropan-2β-carboxylic acid methyl ester ([125I]RTI-55) in rat striatal membranes resulted in binding curves that were best fit to a two-site binding model, allowing for calculation of dissociation constant (Kd) and binding density (Bmax) values that correspond to high- and low-affinity DAT binding sites. Control HIV-1 Tg rats exhibited a significantly greater affinity (i.e., decrease in Kd value) in the low-affinity DAT binding site compared to control F344 rats. Furthermore, cocaine self-administration in HIV-1 Tg rats increased low-affinity Kd (i.e., decreased affinity) compared to levels observed in control F344 rats. Cocaine also increased low-affinity Bmax in HIV-1 Tg rats as compared to controls, indicating an increase in the number of low-affinity DAT binding sites. F344 rats did not exhibit any change in high- or low-affinity Kd or Bmax values following cocaine or heroin self-administration. The increase in DAT affinity in cocaine HIV-1 Tg rats is consistent with the leftward shift of the

  19. Increased Sensitivity to Cocaine Self-Administration in HIV-1 Transgenic Rats is Associated with Changes in Striatal Dopamine Transporter Binding.

    PubMed

    McIntosh, Scot; Sexton, Tammy; Pattison, Lindsey P; Childers, Steven R; Hemby, Scott E

    2015-09-01

    Cocaine abuse in HIV patients accelerates the progression and severity of neuropathology, motor impairment and cognitive dysfunction compared to non-drug using HIV patients. Cocaine and HIV interact with the dopamine transporter (DAT); however, the effect of their interaction on DAT binding remains understudied. The present study compared the dose-response functions for intravenous self-administration of cocaine and heroin between male HIV-1 transgenic (HIV-1 Tg) and Fischer 344 rats. The cocaine and heroin dose-response functions exhibit an inverted U-shape for both HIV-1 Tg and F344 rats. For cocaine, the number of infusions for each dose on the ascending limb was greater for HIV-1 Tg versus F344 rats. No significant changes in the heroin dose-response function were observed in HIV-1 Tg animals. Following the conclusion of self-administration experiments, DAT binding was assessed in striatal membranes. Saturation binding of the cocaine analog [(125)I] 3β-(4-iodophenyl)tropan-2β-carboxylic acid methyl ester ([(125)I]RTI-55) in rat striatal membranes resulted in binding curves that were best fit to a two-site binding model, allowing for calculation of dissociation constant (Kd) and binding density (Bmax) values that correspond to high- and low-affinity DAT binding sites. Control HIV-1 Tg rats exhibited a significantly greater affinity (i.e., decrease in Kd value) in the low-affinity DAT binding site compared to control F344 rats. Furthermore, cocaine self-administration in HIV-1 Tg rats increased low-affinity Kd (i.e., decreased affinity) compared to levels observed in control F344 rats. Cocaine also increased low-affinity Bmax in HIV-1 Tg rats as compared to controls, indicating an increase in the number of low-affinity DAT binding sites. F344 rats did not exhibit any change in high- or low-affinity Kd or Bmax values following cocaine or heroin self-administration. The increase in DAT affinity in cocaine HIV-1 Tg rats is consistent with the leftward shift of the

  20. Immunoliposome encapsulation increases cytotoxic activity and selectivity of curcumin and resveratrol against HER2 overexpressing human breast cancer cells.

    PubMed

    Catania, Angela; Barrajón-Catalán, Enrique; Nicolosi, Silvia; Cicirata, Federico; Micol, Vicente

    2013-08-01

    Natural compounds have been studied as a source of countless bioactive compounds with diverse activities. Among them, many dietary phytochemicals have been thoroughly studied for their cytotoxic or apoptotic effects in several cellular models in order to explain their anticancer capacity. Curcumin and resveratrol are two natural compounds with a large body of evidence showing their cytotoxic activity against a wide variety of cancer cells; however, their poor absorption, bioavailability, and low selectivity have limited their clinical use. With the aim of improving bioavailability and selectivity, the antiproliferative effects of free-, liposomed-, and immunoliposomed-curcumin and/or resveratrol formulations have been compared in two human breast cancer cell lines with different HER2 expression levels. The results demonstrate that when HER2-targeted immunoliposomes are coupled to trastuzumab there is a dramatic increase in the antiproliferative effects of curcumin and resveratrol in HER2 positive human breast cancer cells in comparison to regular liposomed or free forms, indicating an increase of its therapeutic effect. The enhancement of the cytotoxic effects was also correlated to the uptake of curcumin at intracellular level, as shown by using ImageStream technique. The striking efficacy of the immunoliposomed formulation containing both resveratrol and curcumin suggests a multitargeted mechanism of action that deserves further study. These findings show the potential of HER2-targeted nanovesicles to develop new drug delivery systems for cancer therapy based on these compounds and justify further preclinical trials.

  1. The Over-expression of the Plastidial Transglutaminase from Maize in Arabidopsis Increases the Activation Threshold of Photoprotection

    PubMed Central

    Ioannidis, Nikolaos E.; Malliarakis, Dimitris; Torné, Josep M.; Santos, Mireya; Kotzabasis, Kiriakos

    2016-01-01

    Plastidial transglutaminase is one of the most promising enzymes in chloroplast bioenergetics due to its link with polyamine pathways and the cross talk with signals such as Ca2+ and GTP. Here, we show the effect of the increase of transglutaminase activity in Arabidopsis by using genetic transformation techniques. These lines fulfill their biological cycle normally (normal growth in soil, production of viable seeds) and show a relatively mild increase in transglutaminase activity (127%). These overexpressors of transglutaminase (OE TGase) have an extended stroma thylakoid network (71% higher number of PSIIβ centers), similar chlorophyll content (-4%), higher linear electron flow (+13%), and higher threshold of photoprotection activation (∼100%). On the other hand OE TGase showed a reduced maximum photochemistry of PSII (-6.5%), a smaller antenna per photosystem II (-25%), a lower photoprotective “energization” quenching or qE (-77% at 490 μmol photons m-2 s-1) due to a higher threshold of qE activation and slightly lower light induced proton motive force (-17%). The role of the polyamines and of the transglutaminase in the regulation of chemiosmosis and photoprotection in chloroplasts is discussed. PMID:27242838

  2. The Over-expression of the Plastidial Transglutaminase from Maize in Arabidopsis Increases the Activation Threshold of Photoprotection.

    PubMed

    Ioannidis, Nikolaos E; Malliarakis, Dimitris; Torné, Josep M; Santos, Mireya; Kotzabasis, Kiriakos

    2016-01-01

    Plastidial transglutaminase is one of the most promising enzymes in chloroplast bioenergetics due to its link with polyamine pathways and the cross talk with signals such as Ca(2+) and GTP. Here, we show the effect of the increase of transglutaminase activity in Arabidopsis by using genetic transformation techniques. These lines fulfill their biological cycle normally (normal growth in soil, production of viable seeds) and show a relatively mild increase in transglutaminase activity (127%). These overexpressors of transglutaminase (OE TGase) have an extended stroma thylakoid network (71% higher number of PSIIβ centers), similar chlorophyll content (-4%), higher linear electron flow (+13%), and higher threshold of photoprotection activation (∼100%). On the other hand OE TGase showed a reduced maximum photochemistry of PSII (-6.5%), a smaller antenna per photosystem II (-25%), a lower photoprotective "energization" quenching or qE (-77% at 490 μmol photons m(-2) s(-1)) due to a higher threshold of qE activation and slightly lower light induced proton motive force (-17%). The role of the polyamines and of the transglutaminase in the regulation of chemiosmosis and photoprotection in chloroplasts is discussed. PMID:27242838

  3. Cell homeostasis in a Leishmania major mutant overexpressing the spliced leader RNA is maintained by an increased proteolytic activity.

    PubMed

    Toledo, Juliano S; Ferreira, Tiago R; Defina, Tânia P A; Dossin, Fernando de M; Beattie, Kenneth A; Lamont, Douglas J; Cloutier, Serge; Papadopoulou, Barbara; Schenkman, Sergio; Cruz, Angela K

    2010-10-01

    Although several stage-specific genes have been identified in Leishmania, the molecular mechanisms governing developmental gene regulation in this organism are still not well understood. We have previously reported an attenuation of virulence in Leishmania major and L. braziliensis carrying extra-copies of the spliced leader RNA gene. Here, we surveyed the major differences in proteome and transcript expression profiles between the spliced leader RNA overexpressor and control lines using two-dimensional gel electrophoresis and differential display reverse transcription PCR, respectively. Thirty-nine genes related to stress response, cytoskeleton, proteolysis, cell cycle control and proliferation, energy generation, gene transcription, RNA processing and post-transcriptional regulation have abnormal patterns of expression in the spliced leader RNA overexpressor line. The evaluation of proteolytic pathways in the mutant revealed a selective increase of cysteine protease activity and an exacerbated ubiquitin-labeled protein population. Polysome profile analysis and measurement of cellular protein aggregates showed that protein translation in the spliced leader RNA overexpressor line is increased when compared to the control line. We found that L. major promastigotes maintain homeostasis in culture when challenged with a metabolic imbalance generated by spliced leader RNA surplus through modulation of intracellular proteolysis. However, this might interfere with a fine-tuned gene expression control necessary for the amastigote multiplication in the mammalian host.

  4. Overexpression of the Gene Encoding the Multidrug Resistance-Associated Protein Results in Increased ATP-Dependent Glutathione S-Conjugate Transport

    NASA Astrophysics Data System (ADS)

    Muller, Michael; Meijer, Coby; Zaman, Guido J. R.; Borst, Piet; Scheper, Rik J.; Mulder, Nanno H.; de Vries, Elisabeth G. E.; Jansen, Peter L. M.

    1994-12-01

    The multidrug resistance-associated protein (MRP) is a 180- to 195-kDa glycoprotein associated with multidrug resistance of human tumor cells. MRP is mainly located in the plasma membrane and it confers resistance by exporting natural product drugs out of the cell. Here we demonstrate that overexpression of the MRP gene in human cancer cells increases the ATP-dependent glutathione S-conjugate carrier activity in plasma membrane vesicles isolated from these cells. The glutathione S-conjugate export carrier is known to mediate excretion of bivalent anionic conjugates from mammalian cells and is thought to play a role in the elimination of conjugated xenobiotics. Our results suggest that MRP can cause multidrug resistance by promoting the export of drug modification products from cells and they shed light on the reported link between drug resistance and cellular glutathione and glutathione S-transferase levels.

  5. Overexpression of the Lactobacillus plantarum peptidoglycan biosynthesis murA2 gene increases the tolerance of Escherichia coli to alcohols and enhances ethanol production.

    PubMed

    Yuan, Yongbo; Bi, Changhao; Nicolaou, Sergios A; Zingaro, Kyle A; Ralston, Matthew; Papoutsakis, Eleftherios T

    2014-10-01

    A major challenge in producing chemicals and biofuels is to increase the tolerance of the host organism to toxic products or byproducts. An Escherichia coli strain with superior ethanol and more generally alcohol tolerance was identified by screening a library constructed by randomly integrating Lactobacillus plantarum genomic DNA fragments into the E. coli chromosome via Cre-lox recombination. Sequencing identified the inserted DNA fragment as the murA2 gene and its upstream intergenic 973-bp sequence, both coded on the negative genomic DNA strand. Overexpression of this murA2 gene and its upstream 973-bp sequence significantly enhanced ethanol tolerance in both E. coli EC100 and wild type E. coli MG1655 strains by 4.1-fold and 2.0-fold compared to control strains, respectively. Tolerance to n-butanol and i-butanol in E. coli MG1655 was increased by 1.85-fold and 1.91-fold, respectively. We show that the intergenic 973-bp sequence contains a native promoter for the murA2 gene along with a long 5' UTR (286 nt) on the negative strand, while a noncoding, small RNA, named MurA2S, is expressed off the positive strand. MurA2S is expressed in E. coli and may interact with murA2, but it does not affect murA2's ability to enhance alcohol tolerance in E. coli. Overexpression of murA2 with its upstream region in the ethanologenic E. coli KO11 strain significantly improved ethanol production in cultures that simulate the industrial Melle-Boinot fermentation process.

  6. Overexpression of soybean miR172c confers tolerance to water deficit and salt stress, but increases ABA sensitivity in transgenic Arabidopsis thaliana.

    PubMed

    Li, Wenbin; Wang, Tao; Zhang, Yuhang; Li, Yongguang

    2016-01-01

    MiRNAs play crucial roles in many aspects of plant development and the response to the environment. The miR172 family has been shown to participate in the control of flowering time and the response to abiotic stress. This family regulates the expression of APETALA2 (AP2)-like transcription factors in Arabidopsis. In the present study, soybean (Glycine max L. Merr.) miR172c, a member of the miR172 family, and its target gene were investigated for abiotic stress responses in transgenic Arabidopsis. gma-miR172c was induced by abscisic acid (ABA) treatments and abiotic stresses, including salt and water deficit. 5'-RACE (5'-rapid amplification of cDNA ends) assays indicated that miR172c directed Glyma01g39520 mRNA cleavage in soybeans. Overexpression of gma-miR172c in Arabidopsis resulted in reduced leaf water loss and increased survival rate under stress conditions. Meanwhile, the root length, germination rate, and cotyledon greening of transgenic plants were improved during both high salt and water deficit conditions. In addition, transgenic plants exhibited hypersensitivity to ABA during both the seed germination and post-germination seedling growth stages. Stress-related physiological indicators and the expression of stress/ABA-responsive genes were affected by abiotic treatments. The overexpression of gma-miR172c in Arabidopsis promoted earlier flowering compared with the wild type through modulation of the expression of flowering genes, such as FT and LFY during long days, especially under drought conditions. Glyma01g39520 weakened ABA sensitivity and reduced the tolerance to drought stress in the snz mutant of Arabidopsis by reducing the expression of ABI3 and ABI5. Overall, the present results demonstrate that gma-miR172c confers water deficit and salt tolerance but increased ABA sensitivity by regulating Glyma01g39520, which also accelerates flowering under abiotic stresses.

  7. Overexpression of Reg3alpha increases cell growth and the levels of cyclin D1 and CDK4 in insulinoma cells.

    PubMed

    Cui, Wei; De Jesus, Kristine; Zhao, Hong; Takasawa, Shin; Shi, Bingyin; Srikant, Coimbatore B; Liu, Jun-Li

    2009-06-01

    Regenerating gene (Reg) family protein Reg3alpha is normally expressed in pancreatic acinar and endocrine cells. In order to explore its effect on islet beta-cell replication, insulinoma MIN6 cells were stably transfected with murine Reg3alpha cDNA. Determined using real-time PCR and Western blots, the levels of Reg3alpha mRNA and protein in Reg3alpha-transfected clones were increased 10- and 6-fold, respectively. Western blots also revealed that the protein was released into the culture medium, consistent with an endocrine effect. In MTT cell proliferation assay, Reg3alpha-overexpressing cells exhibited a 2-fold increase in the rate of cell growth. In order to investigate the intracellular mechanism, we studied cell cycle regulatory proteins. In Reg3alpha-expressing cells, we detected 2.2- and 2.5-fold increased levels of cyclin D1 and CDK4, respectively, which paralleled a 1.8-fold increase in the rate of Akt phosphorylation. It is established that beta-cell replication is associated with increased cyclin D1 and CDK4 levels; deficiency in CDK4 or cyclin D2 results in reduced beta-cell mass and diabetes. Our results suggest that Reg3alpha stimulates beta-cell replication, by activating Akt kinase and increasing the levels of cyclin D1/CDK4.

  8. Dopamine and anorexia nervosa.

    PubMed

    Södersten, P; Bergh, C; Leon, M; Zandian, M

    2016-01-01

    We have suggested that reduced food intake increases the risk for anorexia nervosa by engaging mesolimbic dopamine neurons, thereby initially rewarding dieting. Recent fMRI studies have confirmed that dopamine neurons are activated in anorexia nervosa, but it is not clear whether this response is due to the disorder or to its resulting nutritional deficit. When the body senses the shortage of nutrients, it rapidly shifts behavior toward foraging for food as a normal physiological response and the mesolimbic dopamine neurons may be involved in that process. On the other hand, the altered dopamine status of anorexics has been suggested to result from a brain abnormality that underlies their complex emotional disorder. We suggest that the outcomes of the treatments that emerge from that perspective remain poor because they target the mental symptoms that are actually the consequences of the food deprivation that accompanies anorexia. On the other hand, a method that normalizes the disordered eating behavior of anorexics results in much better physiological, behavioral, and emotional outcomes.

  9. PKCζ and PKMζ are overexpressed in TCF3-rearranged paediatric acute lymphoblastic leukaemia and are associated with increased thiopurine sensitivity.

    PubMed

    Hartsink-Segers, S A; Beaudoin, J J; Luijendijk, M W J; Exalto, C; Pieters, R; Den Boer, M L

    2015-02-01

    Both tumour suppressor and oncogenic functions have been ascribed to the atypical zeta isoform of protein kinase C (PKCζ), whereas its constitutively active form PKMζ is almost exclusively expressed in the brain where it has a role in long-term memory. Using primers unique for either isoform, we found that both PKCζ and PKMζ were expressed in a subset of paediatric acute lymphoblastic leukaemia (ALL) cases carrying a TCF3 (E2A) chromosomal rearrangement. Combined PKCζ and PKMζ (PKC/Mζ) protein as well as phosphorylation levels were elevated in ALL cases, especially TCF3-rearranged precursor B-ALL cases, compared with normal bone marrow (P<0.01). Furthermore, high PKC/Mζ expression in primary ALL cells was associated with increased sensitivity to 6-thioguanine and 6-mercaptopurine (P<0.01), thiopurines used in ALL treatment. PKCζ is believed to stabilize mismatch-repair protein MSH2, facilitating thiopurine responsiveness in T-ALL. However, PKC/Mζ knockdown in a TCF3-rearranged cell line model decreased MSH2 expression but did not induce thiopurine resistance, indicative that the link between high PKC/Mζ levels and thiopurine sensitivity in paediatric precursor B-ALL is not directly causal. Collectively, our data indicate that thiopurine treatment may be effective, especially in paediatric TCF3-rearranged ALL and other patients with a high expression of PKC/Mζ.

  10. Lead uptake increases drought tolerance of wild type and transgenic poplar (Populus tremula x P. alba) overexpressing gsh 1.

    PubMed

    Samuilov, Sladjana; Lang, Friedericke; Djukic, Matilda; Djunisijevic-Bojovic, Danijela; Rennenberg, Heinz

    2016-09-01

    Growth and development of plants largely depends on their adaptation ability in a changing climate. This is particularly true on heavy metal contaminated soils, but the interaction of heavy metal stress and climate on plant performance has not been intensively investigated. The aim of the present study was to elucidate if transgenic poplars (Populus tremula x P. alba) with enhanced glutathione content possess an enhanced tolerance to drought and lead (Pb) exposure (single and in combination) and if they are good candidates for phytoremediation of Pb contaminated soil. Lead exposure reduced growth and biomass accumulation only in above-ground tissue of wild type poplar, although most of lead accumulated in the roots. Drought caused a decline of the water content rather than reduced biomass production, while Pb counteracted this decline in the combined exposure. Apparently, metals such as Pb possess a protective function against drought, because they interact with abscisic acid dependent stomatal closure. Lead exposure decreased while drought increased glutathione content in leaves of both plant types. Lead accumulation was higher in the roots of transgenic plants, presumably as a result of chelation by glutathione. Water deprivation enhanced Pb accumulation in the roots, but Pb was subject to leakage out of the roots after re-watering. Transgenic plants showed better adaptation under mild drought plus Pb exposure partially due to improved glutathione synthesis. However, the transgenic plants cannot be considered as a good candidate for phytoremediation of Pb, due to its small translocation to the shoots and its leakage out of the roots upon re-watering. PMID:27396669

  11. Overexpression of FK506-binding protein FKBP12.6 in cardiomyocytes reduces ryanodine receptor-mediated Ca(2+) leak from the sarcoplasmic reticulum and increases contractility.

    PubMed

    Prestle, J; Janssen, P M; Janssen, A P; Zeitz, O; Lehnart, S E; Bruce, L; Smith, G L; Hasenfuss, G

    2001-02-01

    The FK506-binding protein FKBP12.6 is tightly associated with the cardiac sarcoplasmic reticulum (SR) Ca(2+)-release channel (ryanodine receptor type 2 [RyR2]), but the physiological function of FKBP12.6 is unclear. We used adenovirus (Ad)-mediated gene transfer to overexpress FKBP12.6 in adult rabbit cardiomyocytes. Western immunoblot and reverse transcriptase-polymerase chain reaction analysis revealed specific overexpression of FKBP12.6, with unchanged expression of endogenous FKBP12. FKBP12.6-transfected myocytes displayed a significantly higher (21%) fractional shortening (FS) at 48 hours after transfection compared with Ad-GFP-infected control cells (4.8+/-0.2% FS versus 4+/-0.2% FS, respectively; n=79 each; P:=0.001). SR-Ca(2+) uptake rates were monitored in beta-escin-permeabilized myocytes using Fura-2. Ad-FKBP12.6-infected cells showed a statistically significant higher rate of Ca(2+) uptake of 0.8+/-0.09 nmol/s(-)(1)/10(6) cells (n=8, P:<0.05) compared with 0.52+/-0.1 nmol/s(-)(1)/10(6) cells in sham-infected cells (n=8) at a [Ca(2+)] of 1 micromol/L. In the presence of 5 micromol/L ruthenium red to block Ca(2+) efflux via RyR2, SR-Ca(2+) uptake rates were not significantly different between groups. From these measurements, we calculate that SR-Ca(2+) leak through RyR2 is reduced by 53% in FKBP12.6-overexpressing cells. Caffeine-induced contractures were significantly larger in Ad-FKBP12.6-infected myocytes compared with Ad-GFP-infected control cells, indicating a higher SR-Ca(2+) load. Taken together, these data suggest that FKBP12.6 stabilizes the closed conformation state of RyR2. This may reduce diastolic SR-Ca(2+) leak and consequently increase SR-Ca(2+) release and myocyte shortening. PMID:11157671

  12. Overexpression of a novel Arabidopsis PP2C isoform, AtPP2CF1, enhances plant biomass production by increasing inflorescence stem growth.

    PubMed

    Sugimoto, Hiroki; Kondo, Satoshi; Tanaka, Tomoko; Imamura, Chie; Muramoto, Nobuhiko; Hattori, Etsuko; Ogawa, Ken'ichi; Mitsukawa, Norihiro; Ohto, Chikara

    2014-10-01

    In contrast to mammals, higher plants have evolved to express diverse protein phosphatase 2Cs (PP2Cs). Of all Arabidopsis thaliana PP2Cs, members of PP2C subfamily A, including ABI1, have been shown to be key negative regulators of abscisic acid (ABA) signalling pathways, which regulate plant growth and development as well as tolerance to adverse environmental conditions. However, little is known about the enzymatic and signalling roles of other PP2C subfamilies. Here, we report a novel Arabidopsis subfamily E PP2C gene, At3g05640, designated AtPP2CF1. AtPP2CF1 was dramatically expressed in response to exogenous ABA and was expressed in vascular tissues and guard cells, similar to most subfamily A PP2C genes. In vitro enzymatic activity assays showed that AtPP2CF1 possessed functional PP2C activity. However, yeast two-hybrid analysis revealed that AtPP2CF1 did not interact with PYR/PYL/RCAR receptors or three SnRK2 kinases, which are ABI1-interacting proteins. This was supported by homology-based structural modelling demonstrating that the putative active- and substrate-binding site of AtPP2CF1 differed from that of ABI1. Furthermore, while overexpression of ABI1 in plants induced an ABA-insensitive phenotype, Arabidopsis plants overexpressing AtPP2CF1 (AtPP2CF1oe) were weakly hypersensitive to ABA during seed germination and drought stress. Unexpectedly, AtPP2CF1oe plants also exhibited increased biomass yield, mainly due to accelerated growth of inflorescence stems through the activation of cell proliferation and expansion. Our results provide new insights into the physiological significance of AtPP2CF1 as a candidate gene for plant growth production and for potential application in the sustainable supply of plant biomass.

  13. Overexpression of a novel Arabidopsis PP2C isoform, AtPP2CF1, enhances plant biomass production by increasing inflorescence stem growth.

    PubMed

    Sugimoto, Hiroki; Kondo, Satoshi; Tanaka, Tomoko; Imamura, Chie; Muramoto, Nobuhiko; Hattori, Etsuko; Ogawa, Ken'ichi; Mitsukawa, Norihiro; Ohto, Chikara

    2014-10-01

    In contrast to mammals, higher plants have evolved to express diverse protein phosphatase 2Cs (PP2Cs). Of all Arabidopsis thaliana PP2Cs, members of PP2C subfamily A, including ABI1, have been shown to be key negative regulators of abscisic acid (ABA) signalling pathways, which regulate plant growth and development as well as tolerance to adverse environmental conditions. However, little is known about the enzymatic and signalling roles of other PP2C subfamilies. Here, we report a novel Arabidopsis subfamily E PP2C gene, At3g05640, designated AtPP2CF1. AtPP2CF1 was dramatically expressed in response to exogenous ABA and was expressed in vascular tissues and guard cells, similar to most subfamily A PP2C genes. In vitro enzymatic activity assays showed that AtPP2CF1 possessed functional PP2C activity. However, yeast two-hybrid analysis revealed that AtPP2CF1 did not interact with PYR/PYL/RCAR receptors or three SnRK2 kinases, which are ABI1-interacting proteins. This was supported by homology-based structural modelling demonstrating that the putative active- and substrate-binding site of AtPP2CF1 differed from that of ABI1. Furthermore, while overexpression of ABI1 in plants induced an ABA-insensitive phenotype, Arabidopsis plants overexpressing AtPP2CF1 (AtPP2CF1oe) were weakly hypersensitive to ABA during seed germination and drought stress. Unexpectedly, AtPP2CF1oe plants also exhibited increased biomass yield, mainly due to accelerated growth of inflorescence stems through the activation of cell proliferation and expansion. Our results provide new insights into the physiological significance of AtPP2CF1 as a candidate gene for plant growth production and for potential application in the sustainable supply of plant biomass. PMID:25038254

  14. Increased feeding and food hoarding following food deprivation are associated with activation of dopamine and orexin neurons in male Brandt's voles.

    PubMed

    Zhang, Xue-Ying; Yang, Hui-Di; Zhang, Qiang; Wang, Zuoxin; Wang, De-Hua

    2011-01-01

    Small mammals usually face energetic challenges, such as food shortage, in the field. They have thus evolved species-specific adaptive strategies for survival and reproductive success. In the present study, we examined male Brandt's voles (Lasiopodomys brandtii) for their physiological, behavioral, and neuronal responses to food deprivation (FD) and subsequent re-feeding. Although 48 hr FD induced a decrease in body weight and the resting metabolic rate (RMR), such decreases did not reach statistical significance when compared to the control males that did not experience FD. During the first 2 hr of re-feeding following 48 hr FD, voles showed higher levels of feeding than controls. However, when permitted to hoard food, FD voles showed an increase in food hoarding, rather than feeding, compared to the controls. Further, both feeding and food hoarding induced an increase in neuronal activation, measured by Fos-ir, in a large number of brain areas examined. Interestingly, feeding and food hoarding also induced an increase in the percentage of tyrosine hydroxylase immunoreactive (TH-ir) cells that co-expressed Fos-ir in the ventral tegmental area (VTA), whereas both FD and feeding induced an increase in the percentage of orexin-ir cells that co-expressed Fos-ir in the lateral hypothalamus (LH). Food hoarding also increased orexin-ir/Fos-ir labeling in the LH. Together, our data indicate that food-deprived male Brandt's voles display enhanced feeding or food hoarding dependent upon an environmental setting. In addition, changes in central dopamine and orexin activities in selected brain areas are associated with feeding and hoarding behaviors following FD and subsequent re-feeding.

  15. Increased sulfate uptake by E. coli overexpressing the SLC26-related SulP protein Rv1739c from Mycobacterium tuberculosis

    PubMed Central

    Zolotarev, Alexander S.; Unnikrishnan, Meera; Shmukler, Boris E.; Clark, Jeffrey S.; Vandorpe, David H.; Grigorieff, Nikolaus; Rubin, Eric J.; Alper, Seth L.

    2009-01-01

    Growth and virulence of mycobacteria requires sulfur uptake. The Mycobacterium tuberculosis genome contains, in addition to the ABC sulfate permease cysTWA, three SLC26-related SulP genes of unknown function. We report that induction of Rv1739c expression in E. coli increased bacterial uptake of sulfate, but not Cl−, formate, or oxalate. Uptake was time-dependent, maximal at pH 6.0, and exhibited a K1/2 for sulfate of 4.0 μM. Na+-independent sulfate uptake was not reduced by bicarbonate, nitrate, or phosphate, but was inhibited by sulfite, selenate, thiosulfate, N-ethylmaleimide and carbonyl cyanide 3-chloro-phenylhydrazone. Sulfate uptake was also increased by overexpression of the Rv1739c transmembrane domain, but not of the cytoplasmic C-terminal STAS domain. Mutation to serine of the three cysteine residues of Rv1739c did not affect magnitude, pH-dependence, or pharmacology of sulfate uptake. Expression of Rv1739c in a M. bovis BCG strain lacking the ABC sulfate permease subunit CysA could not complement sulfate auxotrophy. Moreover, inducible expression of Rv1739c in an E. coli strain lacking CysA did not increase sulfate uptake by intact cells. Our data show that facilitation of bacterial sulfate uptake by Rv1739c requires CysA and its associated sulfate permease activity, and suggest that Rv1739c may be a CysTWA-dependent sulfate transporter. PMID:18255326

  16. Over-expression of an inactive mutant cathepsin D increases endogenous alpha-synuclein and cathepsin B activity in SH-SY5Y cells.

    PubMed

    Crabtree, Donna; Dodson, Matthew; Ouyang, Xiaosen; Boyer-Guittaut, Michaël; Liang, Qiuli; Ballestas, Mary E; Fineberg, Naomi; Zhang, Jianhua

    2014-03-01

    Parkinson's disease is a neurodegenerative movement disorder. The histopathology of Parkinson's disease comprises proteinaceous inclusions known as Lewy bodies, which contains aggregated α-synuclein. Cathepsin D (CD) is a lysosomal protease previously demonstrated to cleave α-synuclein and decrease its toxicity in both cell lines and mouse brains in vivo. Here, we show that pharmacological inhibition of CD, or introduction of catalytically inactive mutant CD, resulted in decreased CD activity and increased cathepsin B activity, suggesting a possible compensatory response to inhibition of CD activity. However, this increased cathepsin B activity was not sufficient to maintain α-synuclein degradation, as evidenced by the accumulation of endogenous α-synuclein. Interestingly, the levels of LC3, LAMP1, and LAMP2, proteins involved in autophagy-lysosomal activities, as well as total lysosomal mass as assessed by LysoTracker flow cytometry, were unchanged. Neither autophagic flux nor proteasomal activities differs between cells over-expressing wild-type versus mutant CD. These observations point to a critical regulatory role for that endogenous CD activity in dopaminergic cells in α-synuclein homeostasis which cannot be compensated for by increased Cathepsin B. These data support the potential need to enhance CD function in order to attenuate α-synuclein accumulation as a therapeutic strategy against development of synucleinopathy.

  17. Overexpression of an alfalfa GDP-mannose 3, 5-epimerase gene enhances acid, drought and salt tolerance in transgenic Arabidopsis by increasing ascorbate accumulation.

    PubMed

    Ma, Lichao; Wang, Yanrong; Liu, Wenxian; Liu, Zhipeng

    2014-11-01

    GDP-mannose 3', 5'-epimerase (GME) catalyses the conversion of GDP-D-mannose to GDP-L-galactose, an important step in the ascorbic acid (ascorbic acid) biosynthetic pathway in higher plants. In this study, a novel cDNA fragment (MsGME) encoding a GME protein was isolated and characterised from alfalfa (Medicago sativa). An expression analysis confirmed that MsGME expression was induced by salinity, PEG and acidity stresses. MsGME overexpression in Arabidopsis enhanced tolerance of the transgenic plants to salt, drought and acid. Real-time PCR analysis revealed that the transcript levels of GDP-D-mannose pyrophosphorylase (GMP), L-galactose-phosphate 1-P phosphatase (GP) and GDP-L-galactose phosphorylase (GGP) were increased in transgenic Arabidopsis (T3 generation). Moreover, the ascorbate content was increased in transgenic Arabidopsis. Our results suggest that MsGME can effectively enhance tolerance of transgenic Arabidopsis to acid, drought and salt by increasing ascorbate accumulation.

  18. Na+/H+ exchanger regulatory factor 1 overexpression-dependent increase of cytoskeleton organization is fundamental in the rescue of F508del cystic fibrosis transmembrane conductance regulator in human airway CFBE41o- cells.

    PubMed

    Favia, Maria; Guerra, Lorenzo; Fanelli, Teresa; Cardone, Rosa Angela; Monterisi, Stefania; Di Sole, Francesca; Castellani, Stefano; Chen, Mingmin; Seidler, Ursula; Reshkin, Stephan Joel; Conese, Massimo; Casavola, Valeria

    2010-01-01

    We have demonstrated that Na(+)/H(+) exchanger regulatory factor 1 (NHERF1) overexpression in CFBE41o- cells induces a significant redistribution of F508del cystic fibrosis transmembrane conductance regulator (CFTR) from the cytoplasm to the apical membrane and rescues CFTR-dependent chloride secretion. Here, we observe that CFBE41o- monolayers displayed substantial disassembly of actin filaments and that overexpression of wild-type (wt) NHERF1 but not NHERF1-Delta Ezrin-Radixin-Moesin (ERM) increased F-actin assembly and organization. Furthermore, the dominant-negative band Four-point one, Ezrin, Radixin, Moesin homology (FERM) domain of ezrin reversed the wt NHERF1 overexpression-induced increase in both F-actin and CFTR-dependent chloride secretion. wt NHERF1 overexpression enhanced the interaction between NHERF1 and both CFTR and ezrin and between ezrin and actin and the overexpression of wt NHERF1, but not NHERF1-DeltaERM, also increased the phosphorylation of ezrin in the apical region of the cell monolayers. Furthermore, wt NHERF1 increased RhoA activity and transfection of constitutively active RhoA in CFBE41o- cells was sufficient to redistribute phospho-ezrin to the membrane fraction and rescue both the F-actin content and the CFTR-dependent chloride efflux. Rho kinase (ROCK) inhibition, in contrast, reversed the wt NHERF1 overexpression-induced increase of membrane phospho-ezrin, F-actin content, and CFTR-dependent secretion. We conclude that NHERF1 overexpression in CFBE41o- rescues CFTR-dependent chloride secretion by forming the multiprotein complex RhoA-ROCK-ezrin-actin that, via actin cytoskeleton reorganization, tethers F508del CFTR to the cytoskeleton stabilizing it on the apical membrane.

  19. Na+/H+ Exchanger Regulatory Factor 1 Overexpression-dependent Increase of Cytoskeleton Organization Is Fundamental in the Rescue of F508del Cystic Fibrosis Transmembrane Conductance Regulator in Human Airway CFBE41o- Cells

    PubMed Central

    Favia, Maria; Guerra, Lorenzo; Fanelli, Teresa; Cardone, Rosa Angela; Monterisi, Stefania; Di Sole, Francesca; Castellani, Stefano; Chen, Mingmin; Seidler, Ursula; Reshkin, Stephan Joel; Conese, Massimo

    2010-01-01

    We have demonstrated that Na+/H+ exchanger regulatory factor 1 (NHERF1) overexpression in CFBE41o- cells induces a significant redistribution of F508del cystic fibrosis transmembrane conductance regulator (CFTR) from the cytoplasm to the apical membrane and rescues CFTR-dependent chloride secretion. Here, we observe that CFBE41o- monolayers displayed substantial disassembly of actin filaments and that overexpression of wild-type (wt) NHERF1 but not NHERF1-Δ Ezrin-Radixin-Moesin (ERM) increased F-actin assembly and organization. Furthermore, the dominant-negative band Four-point one, Ezrin, Radixin, Moesin homology (FERM) domain of ezrin reversed the wt NHERF1 overexpression-induced increase in both F-actin and CFTR-dependent chloride secretion. wt NHERF1 overexpression enhanced the interaction between NHERF1 and both CFTR and ezrin and between ezrin and actin and the overexpression of wt NHERF1, but not NHERF1-ΔERM, also increased the phosphorylation of ezrin in the apical region of the cell monolayers. Furthermore, wt NHERF1 increased RhoA activity and transfection of constitutively active RhoA in CFBE41o- cells was sufficient to redistribute phospho-ezrin to the membrane fraction and rescue both the F-actin content and the CFTR-dependent chloride efflux. Rho kinase (ROCK) inhibition, in contrast, reversed the wt NHERF1 overexpression-induced increase of membrane phospho-ezrin, F-actin content, and CFTR-dependent secretion. We conclude that NHERF1 overexpression in CFBE41o- rescues CFTR-dependent chloride secretion by forming the multiprotein complex RhoA-ROCK-ezrin-actin that, via actin cytoskeleton reorganization, tethers F508del CFTR to the cytoskeleton stabilizing it on the apical membrane. PMID:19889841

  20. Dopamine reward prediction error coding.

    PubMed

    Schultz, Wolfram

    2016-03-01

    Reward prediction errors consist of the differences between received and predicted rewards. They are crucial for basic forms of learning about rewards and make us strive for more rewards-an evolutionary beneficial trait. Most dopamine neurons in the midbrain of humans, monkeys, and rodents signal a reward prediction error; they are activated by more reward than predicted (positive prediction error), remain at baseline activity for fully predicted rewards, and show depressed activity with less reward than predicted (negative prediction error). The dopamine signal increases nonlinearly with reward value and codes formal economic utility. Drugs of addiction generate, hijack, and amplify the dopamine reward signal and induce exaggerated, uncontrolled dopamine effects on neuronal plasticity. The striatum, amygdala, and frontal cortex also show reward prediction error coding, but only in subpopulations of neurons. Thus, the important concept of reward prediction errors is implemented in neuronal hardware.

  1. Dopamine reward prediction error coding

    PubMed Central

    Schultz, Wolfram

    2016-01-01

    Reward prediction errors consist of the differences between received and predicted rewards. They are crucial for basic forms of learning about rewards and make us strive for more rewards—an evolutionary beneficial trait. Most dopamine neurons in the midbrain of humans, monkeys, and rodents signal a reward prediction error; they are activated by more reward than predicted (positive prediction error), remain at baseline activity for fully predicted rewards, and show depressed activity with less reward than predicted (negative prediction error). The dopamine signal increases nonlinearly with reward value and codes formal economic utility. Drugs of addiction generate, hijack, and amplify the dopamine reward signal and induce exaggerated, uncontrolled dopamine effects on neuronal plasticity. The striatum, amygdala, and frontal cortex also show reward prediction error coding, but only in subpopulations of neurons. Thus, the important concept of reward prediction errors is implemented in neuronal hardware. PMID:27069377

  2. Copper deficiency increases levels of dopamine (DA) and norepinephrine (NE) in ventromedial hypothalamus without altering feeding patterns

    SciTech Connect

    Seidel, K.E.; Castonguay, T.W.; Failla, M.L. Univ. of Maryland, College Park )

    1991-03-11

    Cu deficiency results in altered levels of catecholamines in peripheral tissues and specific regions of the CNS in rodents. Because catecholamines can affect feeding behavior, meal patterns of control and Cu deficient rats were compared using a computerized system. Cu deficiency was induced by feeding dams a low Cu diet beginning at 17d of pregnancy and weaning pups to the same diet. Between 4.5 and 6.5 wk-of-age, rats fed {minus}Cu diet ate fewer meals during the light period than did controls. However, total food intake and meal size during light and dark periods were similar for the two groups. At 6.5 wk-of-age, Cu deficiency was confirmed by stunted growth, low tissue Cu and enlarged hearts. Cardiac CA was increased 4.3-fold in Cu deficient rats, while the NE level in heart of Cu deficient rats was 54% that of control. The concentrations of both DA and NE were increased in ventromedial hypothalamus of Cu deficient rats. These results indicate that alterations in catecholamine status of ventromedial hypothalamus associated with severe Cu deficiency fail to markedly affect feeding behavior.

  3. Overexpression of the Saccharomyces cerevisiae mannosylphosphodolichol synthase-encoding gene in Trichoderma reesei results in an increased level of protein secretion and abnormal cell ultrastructure.

    PubMed

    Kruszewska, J S; Butterweck, A H; Kurzatkowski, W; Migdalski, A; Kubicek, C P; Palamarczyk, G

    1999-06-01

    Production of extracellular proteins plays an important role in the physiology of Trichoderma reesei and has potential industrial application. To improve the efficiency of protein secretion, we overexpressed in T. reesei the DPM1 gene of Saccharomyces cerevisiae, encoding mannosylphosphodolichol (MPD) synthase, under homologous, constitutively acting expression signals. Four stable transformants, each with different copy numbers of tandemly integrated DPM1, exhibited roughly double the activity of MPD synthase in the respective endoplasmic reticulum membrane fraction. On a dry-weight basis, they secreted up to sevenfold-higher concentrations of extracellular proteins during growth on lactose, a carbon source promoting formation of cellulases. Northern blot analysis showed that the relative level of the transcript of cbh1, which encodes the major cellulase (cellobiohydrolase I [CBH I]), did not increase in the transformants. On the other hand, the amount of secreted CBH I and, in all but one of the transformants, intracellular CBH I was elevated. Our results suggest that posttranscriptional processes are responsible for the increase in CBH I production. The carbohydrate contents of the extracellular proteins were comparable in the wild type and in the transformants, and no hyperglycosylation was detected. Electron microscopy of the DPM1-amplified strains revealed amorphous structure of the cell wall and over three times as many mitochondria as in the control. Our data indicate that molecular manipulation of glycan biosynthesis in Trichoderma can result in improved protein secretion.

  4. Neuroeconomics: a formal test of dopamine's role in reinforcement learning.

    PubMed

    DeWitt, Eric E J

    2014-04-14

    Over the last two decades, dopamine and reinforcement learning have been increasingly linked. Using a novel, axiomatic approach, a recent study shows that dopamine meets the necessary and sufficient conditions required by the theory to encode a reward prediction error.

  5. HMGB1 induces the overexpression of miR-222 and miR-221 and increases growth and motility in papillary thyroid cancer cells.

    PubMed

    Mardente, Stefania; Mari, Emanuela; Consorti, Fabrizio; Di Gioia, Cira; Negri, Rodolfo; Etna, Marilena; Zicari, Alessandra; Antonaci, Alfredo

    2012-12-01

    Experimental and epidemiological studies have revealed that chronic inflammation contributes to cancer progression and even predisposes to cellular transformation. Inflammatory infiltrates in papillary thyroid cancer include lymphocytes, macrophages and cytokines. High-mobility group box 1 protein (HMGB1) is a late inflammatory cytokine that signals danger to the immune system through the receptor for advanced glycation end-products (RAGE) and Toll-like receptor. The activation of the above receptors results in the secretion of growth, chemotactic and angiogenic factors that contribute to chronic inflammation. In this study, we suggest that apart from the activation of signal transduction pathways by the activation of RAGE, the indirect inhibition of cell cycle regulators [such as phosphatase and tensin homolog (PTEN)] may also cause an increase in cell growth and motility. MicroRNAs (miRNAs) have increasingly been implicated in regulating the malignant progression of cancer. MiR-221 and miR-222 have been found to be deregulated in human papillary thyroid carcinomas. They are involved in cell proliferation through the inhibition of the cell cycle regulator, p27kip1, in human papillary carcinomas. In this study, we show that HMGB1 increases the expression of miR-221 and miR-222 in primary cultures of excised papillary lesions and in an established papillary cancer cell line (BC PAP). The overexpression of oncogenic miR-221 and miR-222 caused by HMGB1 is associated with an increase in malignancy scores, namely cell growth and motility.

  6. Increased nitrogen-use efficiency in transgenic rice plants over-expressing a nitrogen-responsive early nodulin gene identified from rice expression profiling.

    PubMed

    Bi, Yong-Mei; Kant, Surya; Clarke, Joseph; Clark, Joseph; Gidda, Satinder; Ming, Feng; Xu, Jianyao; Rochon, Amanda; Shelp, Barry J; Hao, Lixin; Zhao, Rong; Mullen, Robert T; Zhu, Tong; Rothstein, Steven J

    2009-12-01

    Development of genetic varieties with improved nitrogen-use efficiency (NUE) is essential for sustainable agriculture. In this study, we developed a growth system for rice wherein N was the growth-limiting factor, and identified N-responsive genes by a whole genome transcriptional profiling approach. Some genes were selected to test their functionality in NUE by a transgenic approach. One such example with positive effects on NUE is an early nodulin gene OsENOD93-1. This OsENOD93-1 gene responded significantly to both N induction and N reduction. Transgenic rice plants over-expressing the OsENOD93-1 gene had increased shoot dry biomass and seed yield. This OsENOD93-1 gene was expressed at high levels in roots of wild-type (WT) plants, and its protein product was localized in mitochondria. Transgenic plants accumulated higher concentrations of total amino acids and total N in roots. A higher concentration of amino acids in xylem sap was detected in transgenic plants, especially under N stress. In situ hybridization revealed that OsENOD93-1 is expressed in vascular bundles, as well as in epidermis and endodermis. This work demonstrates that transcriptional profiling, coupled with a transgenic validation approach, is an effective strategy for gene discovery. The knowledge gained from this study could be applied to other important crops.

  7. Overexpression of Thiamin Biosynthesis Genes in Rice Increases Leaf and Unpolished Grain Thiamin Content But Not Resistance to Xanthomonas oryzae pv. oryzae.

    PubMed

    Dong, Wei; Thomas, Nicholas; Ronald, Pamela C; Goyer, Aymeric

    2016-01-01

    Thiamin diphosphate (ThDP), also known as vitamin B1, serves as an enzymatic cofactor in glucose metabolism, the Krebs cycle, and branched-chain amino acid biosynthesis in all living organisms. Unlike plants and microorganisms, humans are not able to synthesize ThDP de novo and must obtain it from their diet. Staple crops such as rice are poor sources of thiamin. Hence, populations that mainly consume rice commonly suffer thiamin deficiency. In addition to thiamin's nutritional function, studies in rice have shown that some thiamin biosynthesis genes are involved in resistance to Xanthomonas oryzae, which causes a serious disease in rice fields. This study shows that overexpression of two thiamin biosynthesis genes, 4-methyl-5-β-hydroxyethylthiazole phosphate synthase and 4-amino-2-methyl-5-hydroxymethylpyrimidine phosphate synthase, involved in the first steps of the thiazole and pyrimidine synthesis branches, respectively, increased thiamin content up to fivefold in unpolished seeds that retain the bran. However, thiamin levels in polished seeds with removed bran were similar to those found in polished control seeds. Plants with higher accumulation of thiamin did not show enhanced resistance to X. oryzae. These results indicate that stacking of two traits can enhance thiamin accumulation in rice unpolished grain. We discuss potential roadblocks that prevent thiamin accumulation in the endosperm.

  8. Overexpression of Thiamin Biosynthesis Genes in Rice Increases Leaf and Unpolished Grain Thiamin Content But Not Resistance to Xanthomonas oryzae pv. oryzae

    PubMed Central

    Dong, Wei; Thomas, Nicholas; Ronald, Pamela C.; Goyer, Aymeric

    2016-01-01

    Thiamin diphosphate (ThDP), also known as vitamin B1, serves as an enzymatic cofactor in glucose metabolism, the Krebs cycle, and branched-chain amino acid biosynthesis in all living organisms. Unlike plants and microorganisms, humans are not able to synthesize ThDP de novo and must obtain it from their diet. Staple crops such as rice are poor sources of thiamin. Hence, populations that mainly consume rice commonly suffer thiamin deficiency. In addition to thiamin’s nutritional function, studies in rice have shown that some thiamin biosynthesis genes are involved in resistance to Xanthomonas oryzae, which causes a serious disease in rice fields. This study shows that overexpression of two thiamin biosynthesis genes, 4-methyl-5-β-hydroxyethylthiazole phosphate synthase and 4-amino-2-methyl-5-hydroxymethylpyrimidine phosphate synthase, involved in the first steps of the thiazole and pyrimidine synthesis branches, respectively, increased thiamin content up to fivefold in unpolished seeds that retain the bran. However, thiamin levels in polished seeds with removed bran were similar to those found in polished control seeds. Plants with higher accumulation of thiamin did not show enhanced resistance to X. oryzae. These results indicate that stacking of two traits can enhance thiamin accumulation in rice unpolished grain. We discuss potential roadblocks that prevent thiamin accumulation in the endosperm. PMID:27242822

  9. Insulin increase in MAP kinase phosphorylation is shifted to early time-points by overexpressing APS, while Akt phosphorylation is not influenced.

    PubMed

    Onnockx, Sheela; Xie, Jingwei; Degraef, Chantal; Erneux, Christophe; Pirson, Isabelle

    2009-09-10

    Upon insulin stimulation, the adaptor protein APS is recruited to the insulin receptor and tyrosine phosphorylated. APS initiates the insulin-induced TC10 cascade which participates to GLUT4 translocation to the plasma membrane. Nevertheless, the molecular mechanism that governs APS and its SH2 and PH domains action on the insulin transduction cascade is not yet fully understood. Here, we show that APS co-immunoprecipitates with the class I PI 3-kinase regulatory subunit p85, through its SH2 domain but that APS does not modulate neither PtdIns(3,4,5)P3 levels nor Akt phosphorylation provoked by insulin. We have confirmed a previously described positive effect of APS overexpression on insulin-induced MAPK phosphorylation upregulation. Consequently, we analyzed the role of SH2 and PH domains of APS in the APS increased MAPK phosphorylation observed upon insulin stimulation and correlated this with the membrane localization of the protein. The effect observed on MAPK phosphorylation requires the intact PH binding domain of APS as well as its SH2 domain.

  10. Overexpression of Thiamin Biosynthesis Genes in Rice Increases Leaf and Unpolished Grain Thiamin Content But Not Resistance to Xanthomonas oryzae pv. oryzae.

    PubMed

    Dong, Wei; Thomas, Nicholas; Ronald, Pamela C; Goyer, Aymeric

    2016-01-01

    Thiamin diphosphate (ThDP), also known as vitamin B1, serves as an enzymatic cofactor in glucose metabolism, the Krebs cycle, and branched-chain amino acid biosynthesis in all living organisms. Unlike plants and microorganisms, humans are not able to synthesize ThDP de novo and must obtain it from their diet. Staple crops such as rice are poor sources of thiamin. Hence, populations that mainly consume rice commonly suffer thiamin deficiency. In addition to thiamin's nutritional function, studies in rice have shown that some thiamin biosynthesis genes are involved in resistance to Xanthomonas oryzae, which causes a serious disease in rice fields. This study shows that overexpression of two thiamin biosynthesis genes, 4-methyl-5-β-hydroxyethylthiazole phosphate synthase and 4-amino-2-methyl-5-hydroxymethylpyrimidine phosphate synthase, involved in the first steps of the thiazole and pyrimidine synthesis branches, respectively, increased thiamin content up to fivefold in unpolished seeds that retain the bran. However, thiamin levels in polished seeds with removed bran were similar to those found in polished control seeds. Plants with higher accumulation of thiamin did not show enhanced resistance to X. oryzae. These results indicate that stacking of two traits can enhance thiamin accumulation in rice unpolished grain. We discuss potential roadblocks that prevent thiamin accumulation in the endosperm. PMID:27242822

  11. Intravenous ethanol increases dopamine release in the ventral striatum in humans: PET study using bolus-plus-infusion administration of [11C]raclopride

    PubMed Central

    Aalto, Sargo; Ingman, Kimmo; Alakurtti, Kati; Kaasinen, Valtteri; Virkkala, Jussi; Någren, Kjell; Rinne, Juha O; Scheinin, Harry

    2015-01-01

    Ethanol increases the interstitial dopamine (DA) concentration in the nucleus accumbens (NAcc) of experimental animals, but positron emission tomography (PET) studies using the single-bolus protocol of the [11C]-raclopride competition paradigm have yielded conflicting results in humans. To resolve disparate previous findings, we utilized the bolus-plus-infusion (B/I) method, allowing both baseline and intervention quantification of [11C]raclopride binding during a single 105-minute PET scan, to investigate possible ethanol-induced DA release in nine healthy male subjects. A 25-minute intravenous ethanol (7.6%) infusion, resulting in a 1.3 g/L mean blood ethanol concentration, was administered using masked timing during the PET scan. Automated region-of-interest analysis testing the difference between baseline (40–50 minutes) and intervention (60–85 minutes) revealed an average 12.6% decrease in [11C]raclopride binding in the ventral striatum (VST, P=0.003) including the NAcc. In addition, a shorter time interval from the start of ethanol infusion to the first subjective effect was associated with a greater binding potential decrease bilaterally in the VST (r=0.92, P=0.004), and the feeling of pleasure was associated with a decrease in binding potential values in both the caudate nucleus (r=−0.87, P=0.003) and putamen (r=−0.74; P=0.02). These results confirm that ethanol induces rapid DA release in the limbic striatum, which can be reliably estimated using the B/I method in one imaging session. PMID:25492110

  12. Overexpression of OsCYP19-4 increases tolerance to cold stress and enhances grain yield in rice (Oryza sativa).

    PubMed

    Yoon, Dae Hwa; Lee, Sang Sook; Park, Hyun Ji; Lyu, Jae Il; Chong, Won Seog; Liu, Jang Ryol; Kim, Beom-Gi; Ahn, Jun Cheul; Cho, Hye Sun

    2016-01-01

    AtCYP19-4 (also known as CYP5) was previously identified as interacting in vitro with GNOM, a member of a large family of ARF guanine nucleotide exchange factors that is required for proper polar localization of the auxin efflux carrier PIN1. The present study demonstrated that OsCYP19-4, a gene encoding a putative homologue of AtCYP19-4, was up-regulated by several stresses and showed over 10-fold up-regulation in response to cold. The study further demonstrated that the promoter of OsCYP19-4 was activated in response to cold stress. An OsCYP19-4-GFP fusion protein was targeted to the outside of the plasma membrane via the endoplasmic reticulum as determined using brefeldin A, a vesicle trafficking inhibitor. An in vitro assay with a synthetic substrate oligomer confirmed that OsCYP19-4 had peptidyl-prolyl cis-trans isomerase activity, as was previously reported for AtCYP19-4. Rice plants overexpressing OsCYP19-4 showed cold-resistance phenotypes with significantly increased tiller and spike numbers, and consequently enhanced grain weight, compared with wild-type plants. Based on these results, the authors suggest that OsCYP19-4 is required for developmental acclimation to environmental stresses, especially cold. Furthermore, the results point to the potential of manipulating OsCYP19-4 expression to enhance cold tolerance or to increase biomass. PMID:26453745

  13. Overexpression of a rice heme activator protein gene (OsHAP2E) confers resistance to pathogens, salinity and drought, and increases photosynthesis and tiller number.

    PubMed

    Alam, Md Mahfuz; Tanaka, Toru; Nakamura, Hidemitsu; Ichikawa, Hiroaki; Kobayashi, Kappei; Yaeno, Takashi; Yamaoka, Naoto; Shimomoto, Kota; Takayama, Kotaro; Nishina, Hiroshige; Nishiguchi, Masamichi

    2015-01-01

    Heme activator protein (HAP), also known as nuclear factor Y or CCAAT binding factor (HAP/NF-Y/CBF), has important functions in regulating plant growth, development and stress responses. The expression of rice HAP gene (OsHAP2E) was induced by probenazole (PBZ), a chemical inducer of disease resistance. To characterize the gene, the chimeric gene (OsHAP2E::GUS) engineered to carry the structural gene encoding β-glucuronidase (GUS) driven by the promoter from OsHAP2E was introduced into rice. The transgenic lines of OsHAP2Ein::GUS with the intron showed high GUS activity in the wounds and surrounding tissues. When treated by salicylic acid (SA), isonicotinic acid (INA), abscisic acid (ABA) and hydrogen peroxide (H2 O2 ), the lines showed GUS activity exclusively in vascular tissues and mesophyll cells. This activity was enhanced after inoculation with Magnaporthe oryzae or Xanthomonas oryzae pv. oryzae. The OsHAP2E expression level was also induced after inoculation of rice with M. oryzae and X. oryzae pv. oryzae and after treatment with SA, INA, ABA and H2 O2, respectively. We further produced transgenic rice overexpressing OsHAP2E. These lines conferred resistance to M. oryzae or X. oryzae pv. oryzae and to salinity and drought. Furthermore, they showed a higher photosynthetic rate and an increased number of tillers. Microarray analysis showed up-regulation of defence-related genes. These results suggest that this gene could contribute to conferring biotic and abiotic resistances and increasing photosynthesis and tiller numbers. PMID:25168932

  14. Overexpression of OsCYP19-4 increases tolerance to cold stress and enhances grain yield in rice (Oryza sativa).

    PubMed

    Yoon, Dae Hwa; Lee, Sang Sook; Park, Hyun Ji; Lyu, Jae Il; Chong, Won Seog; Liu, Jang Ryol; Kim, Beom-Gi; Ahn, Jun Cheul; Cho, Hye Sun

    2016-01-01

    AtCYP19-4 (also known as CYP5) was previously identified as interacting in vitro with GNOM, a member of a large family of ARF guanine nucleotide exchange factors that is required for proper polar localization of the auxin efflux carrier PIN1. The present study demonstrated that OsCYP19-4, a gene encoding a putative homologue of AtCYP19-4, was up-regulated by several stresses and showed over 10-fold up-regulation in response to cold. The study further demonstrated that the promoter of OsCYP19-4 was activated in response to cold stress. An OsCYP19-4-GFP fusion protein was targeted to the outside of the plasma membrane via the endoplasmic reticulum as determined using brefeldin A, a vesicle trafficking inhibitor. An in vitro assay with a synthetic substrate oligomer confirmed that OsCYP19-4 had peptidyl-prolyl cis-trans isomerase activity, as was previously reported for AtCYP19-4. Rice plants overexpressing OsCYP19-4 showed cold-resistance phenotypes with significantly increased tiller and spike numbers, and consequently enhanced grain weight, compared with wild-type plants. Based on these results, the authors suggest that OsCYP19-4 is required for developmental acclimation to environmental stresses, especially cold. Furthermore, the results point to the potential of manipulating OsCYP19-4 expression to enhance cold tolerance or to increase biomass.

  15. Dopamine synapse is a neuroligin-2-mediated contact between dopaminergic presynaptic and GABAergic postsynaptic structures.

    PubMed

    Uchigashima, Motokazu; Ohtsuka, Toshihisa; Kobayashi, Kazuto; Watanabe, Masahiko

    2016-04-12

    Midbrain dopamine neurons project densely to the striatum and form so-called dopamine synapses on medium spiny neurons (MSNs), principal neurons in the striatum. Because dopamine receptors are widely expressed away from dopamine synapses, it remains unclear how dopamine synapses are involved in dopaminergic transmission. Here we demonstrate that dopamine synapses are contacts formed between dopaminergic presynaptic and GABAergic postsynaptic structures. The presynaptic structure expressed tyrosine hydroxylase, vesicular monoamine transporter-2, and plasmalemmal dopamine transporter, which are essential for dopamine synthesis, vesicular filling, and recycling, but was below the detection threshold for molecules involving GABA synthesis and vesicular filling or for GABA itself. In contrast, the postsynaptic structure of dopamine synapses expressed GABAergic molecules, including postsynaptic adhesion molecule neuroligin-2, postsynaptic scaffolding molecule gephyrin, and GABAA receptor α1, without any specific clustering of dopamine receptors. Of these, neuroligin-2 promoted presynaptic differentiation in axons of midbrain dopamine neurons and striatal GABAergic neurons in culture. After neuroligin-2 knockdown in the striatum, a significant decrease of dopamine synapses coupled with a reciprocal increase of GABAergic synapses was observed on MSN dendrites. This finding suggests that neuroligin-2 controls striatal synapse formation by giving competitive advantage to heterologous dopamine synapses over conventional GABAergic synapses. Considering that MSN dendrites are preferential targets of dopamine synapses and express high levels of dopamine receptors, dopamine synapse formation may serve to increase the specificity and potency of dopaminergic modulation of striatal outputs by anchoring dopamine release sites to dopamine-sensing targets. PMID:27035941

  16. PRKAR1A overexpression is associated with increased ECPKA autoantibody in liver fluke-associated cholangiocarcinoma: application for assessment of the risk group.

    PubMed

    Loilome, Watcharin; Yooyuen, Sasithorn; Namwat, Nisana; Sithithaworn, Paiboon; Puapairoj, Anucha; Kano, Junko; Noguchi, Masayuki; Miwa, Masanao; Yongvanit, Puangrat

    2012-12-01

    Cholangiocarcinoma (CCA) associated with Opisthorchis viverrini (Ov) chronic infection is the most frequent primary liver cancer in Thailand, and current approaches to early diagnosis and curative treatments are largely disappointing. We hypothesize a role for protein kinase A (PKA) in Ov-induced CCA. First, we studied the PKA isozyme switching in the liver from the hamster CCA model using quantitative (q) PCR, in situ hybridization, and immunohistochemical and western blot analysis. Second, the presence of extracellular PKA (ECPKA) in CCA cell lines and their conditioned media was demonstrated by western blot and PKA activity assay. Third, we determined the association between PRKAR1A expression and serum ECPKA autoantibody in patients with CCA by ELISA. We demonstrated that an increased PRKAR1A expression is restricted to the biliary cells starting from week 1, with remarkable up-regulation when CCA has completely developed by week 24. The switching of the PKA regulatory subunit isoforms from PRKAR2B/PKAII to PRKAR1A/PKAI is significantly associated with cholangiocyte proliferation. Further, we observed that human CCA cell lines express PRKAR1A but not PRKAR2B and excrete ECPKA. Finally, ECPKA autoantibodies are detected in serum of patients with CCA, adenocarcinoma, and Ov infection with periductal fibrosis, but not from Ov-infected subjects without periductal fibrosis lesion and healthy controls. We conclude that PKA isozyme switching and the PRKAR1A/PKAI pathway might contribute to the induction of cholangiocyte transformation and proliferation in Ov-induced CCA. Overexpression of PRKAR1A leads to secretion of ECPKA which is associated with serum autoantibody that may constitute a biomarker for human CCA genesis. PMID:22922884

  17. Post-infarct treatment with [Pyr(1)]apelin-13 improves myocardial function by increasing neovascularization and overexpression of angiogenic growth factors in rats.

    PubMed

    Azizi, Yaser; Faghihi, Mahdieh; Imani, Alireza; Roghani, Mehrdad; Zekri, Ali; Mobasheri, Maryam Beigom; Rastgar, Tayebeh; Moghimian, Maryam

    2015-08-15

    Ischemic heart disease is the leading cause of mortality in the world. Angiogenesis is important for cardiac repair after myocardial infarction (MI) as restores blood supply to the ischemic myocardium and preserves cardiac function. Apelin is a peptide that has been recently shown to potentiate angiogenesis. The aim of this study was to investigate angiogenic effects of [Pyr(1)]apelin-13 in the rat model of post-MI. Male Wistar rats (n=36) were randomly divided into three groups: (1) sham (2) MI and (3) MI treated with [Pyr(1)]apelin-13 (MI+Apel). MI animals were subjected to 30min left anterior descending coronary artery (LAD) ligation and 14 days of reperfusion. Twenty-four hours after LAD ligation, [Pyr(1)]apelin-13 (10nmol/kg/day) was administered i.p. for 5 days. Hemodynamic functions by catheter introduced into the left ventricle (LV), myocardial fibrosis by Masson׳s trichrome staining, gene expression of vascular endothelial growth factor-A (VEGFA), VEGF receptor-2 (Kdr), Ang-1 (angiopoietin-1), Tie2 (tyrosine kinase with immunoglobulin and epidermal growth factor homology domains 2) and eNOS by Real-time polymerase chain reaction (Real-Time PCR) and myocardial angiogenesis by CD31 imunostaining were assessed at day 14 post-MI. Post-infarct treatment with [Pyr(1)]apelin-13 improved LV function and decreased myocardial fibrosis. [Pyr(1)]apelin-13 treatment led to a significant increase in the expression of VEGFA, Kdr, Ang-1, Tie2 and eNOS. Further, treatment with [Pyr(1)]apelin-13 promoted capillary density. [Pyr(1)]apelin-13 has angiogenic and anti-fibrotic activity via formation of new blood vessels and overexpression of VEGFA, Kdr, Ang-1, Tie2 and eNOS in the infarcted myocardium which could in turn repair myocardium and improve LV function.

  18. Constitutive overexpression of a ripening-related pepper endo-1,4-beta-glucanase in transgenic tomato fruit does not increase xyloglucan depolymerization or fruit softening.

    PubMed

    Harpster, Mark H; Dawson, Debra M; Nevins, Donald J; Dunsmuir, Pamela; Brummell, David A

    2002-10-01

    The ripening-related pepper endo-1,4-beta-D-glucanase (EGase) CaCel1 was over-expressed in transgenic tomato plants under the control of the constitutive 35S promoter to investigate the effects on plant growth and fruit softening of high levels of a potential cell wall-degrading activity. In transgenic fruit, recombinant CaCel1 protein was associated with a high-salt putative cell wall fraction, and extractable CMCase activity was increased by up to 20-fold relative to controls. However, the effects of high levels of EGase activity on fruit cell wall metabolism were relatively small. The largest consequence observed was a decrease of up to 20% in the amount of matrix glycans in a 24% KOH-soluble fraction consisting of polysaccharides tightly bound to cellulose. This decrease was confined to polysaccharides other than xyloglucan, did not affect the size distribution of remaining molecules, and was not correlated with a corresponding increase in glycans in a 4% KOH-soluble fraction loosely bound to cellulose, suggesting that the missing polymers had been degraded to fragments small enough to be lost from the extracts. The amount of matrix glycans in the 4% KOH-soluble fraction was not substantially changed, but the size distribution showed a small relative increase in the amount of polymers in a peak eluting close to a linear dextran marker of 71 kDa. This could be due either to an increase in the amount of polymers of this size, or to a loss from the extract of other polymers present in peaks of higher molecular weight. Transgenic fruit were not softer than controls but appeared the same or slightly firmer at both green and red developmental stages, and no differences in plant vegetative growth were observed. CaCel1 did not cause depolymerization of tomato fruit xyloglucan in vivo, but differences in the amount or molecular weight profile of other matrix glycans were observed. The data suggest that degradation of a proportion of matrix glycans other than xyloglucan

  19. Synapsins differentially control dopamine and serotonin release.

    PubMed

    Kile, Brian M; Guillot, Thomas S; Venton, B Jill; Wetsel, William C; Augustine, George J; Wightman, R Mark

    2010-07-21

    Synapsins are a family of synaptic vesicle proteins that are important for neurotransmitter release. Here we have used triple knock-out (TKO) mice lacking all three synapsin genes to determine the roles of synapsins in the release of two monoamine neurotransmitters, dopamine and serotonin. Serotonin release evoked by electrical stimulation was identical in substantia nigra pars reticulata slices prepared from TKO and wild-type mice. In contrast, release of dopamine in response to electrical stimulation was approximately doubled in striatum of TKO mice, both in vivo and in striatal slices, in comparison to wild-type controls. This was due to loss of synapsin III, because deletion of synapsin III alone was sufficient to increase dopamine release. Deletion of synapsins also increased the sensitivity of dopamine release to extracellular calcium ions. Although cocaine did not affect the release of serotonin from nigral tissue, this drug did enhance dopamine release. Cocaine-induced facilitation of dopamine release was a function of external calcium, an effect that was reduced in TKO mice. We conclude that synapsins play different roles in the control of release of dopamine and serotonin, with release of dopamine being negatively regulated by synapsins, specifically synapsin III, while serotonin release appears to be relatively independent of synapsins. These results provide further support for the concept that synapsin function in presynaptic terminals varies according to the neurotransmitter being released. PMID:20660258

  20. Decreased spontaneous activity in AMPK α2 muscle specific kinase dead mice is not caused by changes in brain dopamine metabolism.

    PubMed

    Møller, Lisbeth L V; Sylow, Lykke; Gøtzsche, Casper R; Serup, Annette K; Christiansen, Søren H; Weikop, Pia; Kiens, Bente; Woldbye, David P D; Richter, Erik A

    2016-10-01

    It is well known that physical activity has several health benefits, yet many people do not exercise. Dopamine levels in the striatum of the brain are thought to be important for the motivation to exercise. Conversely, we hypothesized that muscle quality can affect the motivation to exercise through alterations of the brain dopamine levels specifically in the striatal region. To test this hypothesis, transgenic mice overexpressing an inactivatable dominant negative α2 AMPK construct (AMPK α2 KD) in muscles and littermate wildtype (WT) mice were tested. AMPK α2 KD mice have impaired running capacity and display reduced voluntary wheel running activity. Striatal content of dopamine and its metabolites were measured under basal physiological conditions and after cocaine-induced dopamine efflux from the ventral striatum by in vivo microdialysis. Moreover, cocaine-induced locomotor activity was tested in an open field test. Furthermore, we investigated maximal running capacity and voluntary running over a period of 19days. AMPK α2 KD mice ran 30% less in daily distance compared to WT. Furthermore, AMPK α2 KD mice showed significantly decreased locomotor activity in the open field test compared to WT when treated with saline or cocaine, respectively, but the increase induced by cocaine was similar in AMPK α2 KD and WT mice. The efflux of dopamine in ventral striatum after cocaine treatment increased similarly by 2.5-fold in the two genotypes, and basal levels of dopamine and its metabolites DOPAC and HVA were also similar between genotypes. These findings show that decreased AMPK activity in muscle leads to decreased voluntary activity which is not due to secondary abnormalities in dopamine levels in the ventral striatum or sensitivity to cocaine. Thus, decreased voluntary activity in AMPK muscle deficient mice is most likely unrelated to regulation of brain dopamine content and metabolism. PMID:27306083

  1. Overexpression of the ethylene-responsive factor gene BrERF4 from Brassica rapa increases tolerance to salt and drought in Arabidopsis plants.

    PubMed

    Seo, Yean Joo; Park, Jong-Beum; Cho, Yeon-Jeong; Jung, Choonkyun; Seo, Hak Soo; Park, Soon-Ki; Nahm, Baek Hie; Song, Jong Tae

    2010-09-01

    Ethylene-responsive factors (ERFs), within a subgroup of the AP2/ERF transcription factor family, are involved in diverse plant reactions to biotic or abiotic stresses. Here, we report that overexpression of an ERF gene from Brassica rapa ssp. pekinensis (BrERF4) led to improved tolerance to salt and drought stresses in Arabidopsis. It also significantly affected the growth and development of transgenic plants. We detected that salt-induced expressions of a transcriptional repressor gene, AtERF4, and some Ser/Thr protein phosphatase2C genes, ABI1, ABI2 and AtPP2CA, were suppressed in BrERF4-overexpressing Arabidopsis plants. Furthermore, BrERF4 was induced by treatment with ethylene or methyljasmonate, but not by abscisic acid or NaCl in B. rapa. These results suggest that BrERF4 is activated through a network of different signaling pathways in response to salinity and drought.

  2. Overexpression of the ethylene-responsive factor gene BrERF4 from Brassica rapa increases tolerance to salt and drought in Arabidopsis plants.

    PubMed

    Seo, Yean Joo; Park, Jong-Beum; Cho, Yeon-Jeong; Jung, Choonkyun; Seo, Hak Soo; Park, Soon-Ki; Nahm, Baek Hie; Song, Jong Tae

    2010-09-01

    Ethylene-responsive factors (ERFs), within a subgroup of the AP2/ERF transcription factor family, are involved in diverse plant reactions to biotic or abiotic stresses. Here, we report that overexpression of an ERF gene from Brassica rapa ssp. pekinensis (BrERF4) led to improved tolerance to salt and drought stresses in Arabidopsis. It also significantly affected the growth and development of transgenic plants. We detected that salt-induced expressions of a transcriptional repressor gene, AtERF4, and some Ser/Thr protein phosphatase2C genes, ABI1, ABI2 and AtPP2CA, were suppressed in BrERF4-overexpressing Arabidopsis plants. Furthermore, BrERF4 was induced by treatment with ethylene or methyljasmonate, but not by abscisic acid or NaCl in B. rapa. These results suggest that BrERF4 is activated through a network of different signaling pathways in response to salinity and drought. PMID:20803085

  3. Misassembly of full-length Disrupted-in-Schizophrenia 1 protein is linked to altered dopamine homeostasis and behavioral deficits

    PubMed Central

    Trossbach, S V; Bader, V; Hecher, L; Pum, M E; Masoud, S T; Prikulis, I; Schäble, S; de Souza Silva, M A; Su, P; Boulat, B; Chwiesko, C; Poschmann, G; Stühler, K; Lohr, K M; Stout, K A; Oskamp, A; Godsave, S F; Müller-Schiffmann, A; Bilzer, T; Steiner, H; Peters, P J; Bauer, A; Sauvage, M; Ramsey, A J; Miller, G W; Liu, F; Seeman, P; Brandon, N J; Huston, J P; Korth, C

    2016-01-01

    Disrupted-in-schizophrenia 1 (DISC1) is a mental illness gene first identified in a Scottish pedigree. So far, DISC1-dependent phenotypes in animal models have been confined to expressing mutant DISC1. Here we investigated how pathology of full-length DISC1 protein could be a major mechanism in sporadic mental illness. We demonstrate that a novel transgenic rat model, modestly overexpressing the full-length DISC1 transgene, showed phenotypes consistent with a significant role of DISC1 misassembly in mental illness. The tgDISC1 rat displayed mainly perinuclear DISC1 aggregates in neurons. Furthermore, the tgDISC1 rat showed a robust signature of behavioral phenotypes that includes amphetamine supersensitivity, hyperexploratory behavior and rotarod deficits, all pointing to changes in dopamine (DA) neurotransmission. To understand the etiology of the behavioral deficits, we undertook a series of molecular studies in the dorsal striatum of tgDISC1 rats. We observed an 80% increase in high-affinity DA D2 receptors, an increased translocation of the dopamine transporter to the plasma membrane and a corresponding increase in DA inflow as observed by cyclic voltammetry. A reciprocal relationship between DISC1 protein assembly and DA homeostasis was corroborated by in vitro studies. Elevated cytosolic dopamine caused an increase in DISC1 multimerization, insolubility and complexing with the dopamine transporter, suggesting a physiological mechanism linking DISC1 assembly and dopamine homeostasis. DISC1 protein pathology and its interaction with dopamine homeostasis is a novel cellular mechanism that is relevant for behavioral control and may have a role in mental illness. PMID:26754951

  4. Chromatographic analysis of dopamine metabolism in a Parkinsonian model.

    PubMed

    Baranyi, Mária; Milusheva, Elisaveta; Vizi, E Sylvester; Sperlágh, Beáta

    2006-07-01

    The present study examined the metabolism of released dopamine from rat striatum upon chronic rotenone exposure. The sample separation was carried out by two-dimensional, reversed-phase and ion pair reversed-phase chromatography using on-line solid phase extraction enrichment. Reduced dopamine content and decreased extracellular level of [(3)H] and endogenous dopamine evoked by electrical stimulation indicated the injury of dopaminergic pathway. Sensitivity of dopaminergic neurons were increased to oxidative stress with enhanced release of dopamine and formation of oxidized metabolite dopamine quinone (DAQ). Utilizing multidimensional detection, EC at -100 mV reduction potential, the method has been applied for identification of DAQ and aminochrome (DAC).

  5. Regulation of Dopamine Uptake by Vasoactive Peptides in the Kidney

    PubMed Central

    Gironacci, M. M.

    2016-01-01

    Considering the key role of renal dopamine in tubular sodium handling, we hypothesized that c-type natriuretic peptide (CNP) and Ang-(1-7) may regulate renal dopamine availability in tubular cells, contributing to Na+, K+-ATPase inhibition. Present results show that CNP did not affect either 3H-dopamine uptake in renal tissue or Na+, K+-ATPase activity; meanwhile, Ang-(1-7) was able to increase 3H-dopamine uptake and decreased Na+, K+-ATPase activity in renal cortex. Ang-(1-7) and dopamine together decreased further Na+, K+-ATPase activity showing an additive effect on the sodium pump. In addition, hydrocortisone reversed Ang-(1-7)-dopamine overinhibition on the enzyme, suggesting that this inhibition is closely related to Ang-(1-7) stimulation on renal dopamine uptake. Both anantin and cANP (4-23-amide) did not modify CNP effects on 3H-dopamine uptake by tubular cells. The Mas receptor antagonist, A-779, blocked the increase elicited by Ang-(1-7) on 3H-dopamine uptake. The stimulatory uptake induced by Ang-(1-7) was even more pronounced in the presence of losartan, suggesting an inhibitory effect of Ang-(1-7) on AT1 receptors on 3H-dopamine uptake. By increasing dopamine bioavailability in tubular cells, Ang-(1-7) enhances Na+, K+-ATPase activity inhibition, contributing to its natriuretic and diuretic effects.

  6. Regulation of Dopamine Uptake by Vasoactive Peptides in the Kidney

    PubMed Central

    Gironacci, M. M.

    2016-01-01

    Considering the key role of renal dopamine in tubular sodium handling, we hypothesized that c-type natriuretic peptide (CNP) and Ang-(1-7) may regulate renal dopamine availability in tubular cells, contributing to Na+, K+-ATPase inhibition. Present results show that CNP did not affect either 3H-dopamine uptake in renal tissue or Na+, K+-ATPase activity; meanwhile, Ang-(1-7) was able to increase 3H-dopamine uptake and decreased Na+, K+-ATPase activity in renal cortex. Ang-(1-7) and dopamine together decreased further Na+, K+-ATPase activity showing an additive effect on the sodium pump. In addition, hydrocortisone reversed Ang-(1-7)-dopamine overinhibition on the enzyme, suggesting that this inhibition is closely related to Ang-(1-7) stimulation on renal dopamine uptake. Both anantin and cANP (4-23-amide) did not modify CNP effects on 3H-dopamine uptake by tubular cells. The Mas receptor antagonist, A-779, blocked the increase elicited by Ang-(1-7) on 3H-dopamine uptake. The stimulatory uptake induced by Ang-(1-7) was even more pronounced in the presence of losartan, suggesting an inhibitory effect of Ang-(1-7) on AT1 receptors on 3H-dopamine uptake. By increasing dopamine bioavailability in tubular cells, Ang-(1-7) enhances Na+, K+-ATPase activity inhibition, contributing to its natriuretic and diuretic effects. PMID:27635280

  7. Regulation of Dopamine Uptake by Vasoactive Peptides in the Kidney.

    PubMed

    Rukavina Mikusic, N L; Kouyoumdzian, N M; Rouvier, E; Gironacci, M M; Toblli, J E; Fernández, B E; Choi, M R

    2016-01-01

    Considering the key role of renal dopamine in tubular sodium handling, we hypothesized that c-type natriuretic peptide (CNP) and Ang-(1-7) may regulate renal dopamine availability in tubular cells, contributing to Na(+), K(+)-ATPase inhibition. Present results show that CNP did not affect either (3)H-dopamine uptake in renal tissue or Na(+), K(+)-ATPase activity; meanwhile, Ang-(1-7) was able to increase (3)H-dopamine uptake and decreased Na(+), K(+)-ATPase activity in renal cortex. Ang-(1-7) and dopamine together decreased further Na(+), K(+)-ATPase activity showing an additive effect on the sodium pump. In addition, hydrocortisone reversed Ang-(1-7)-dopamine overinhibition on the enzyme, suggesting that this inhibition is closely related to Ang-(1-7) stimulation on renal dopamine uptake. Both anantin and cANP (4-23-amide) did not modify CNP effects on (3)H-dopamine uptake by tubular cells. The Mas receptor antagonist, A-779, blocked the increase elicited by Ang-(1-7) on (3)H-dopamine uptake. The stimulatory uptake induced by Ang-(1-7) was even more pronounced in the presence of losartan, suggesting an inhibitory effect of Ang-(1-7) on AT1 receptors on (3)H-dopamine uptake. By increasing dopamine bioavailability in tubular cells, Ang-(1-7) enhances Na(+), K(+)-ATPase activity inhibition, contributing to its natriuretic and diuretic effects. PMID:27635280

  8. Functional characterization of dopamine transporter in vivo using Drosophila melanogaster behavioral assays.

    PubMed

    Ueno, Taro; Kume, Kazuhiko

    2014-01-01

    Dopamine mediates diverse functions such as motivation, reward, attention, learning/memory and sleep/arousal. Recent studies using model organisms including the fruit fly, have elucidated various physiological functions of dopamine, and identified specific neural circuits for these functions. Flies with mutations in the Drosophila dopamine transporter (dDAT) gene show enhanced dopamine signaling, and short sleep and memory impairment phenotypes. However, understanding the mechanism by which dopamine signaling causes these phenotypes requires an understanding of the dynamics of dopamine release. Here we report the effects of dDAT expression on behavioral traits. We show that dDAT expression in a subset of dopaminergic neurons is sufficient for normal sleep. dDAT expression in other cell types such as Kenyon cells and glial cells can also rescue the short sleep phenotype of dDAT mutants. dDAT mutants also show a down-regulation of the D1-like dopamine receptor dDA1, and this phenotype is rescued when dDAT is expressed in the same cell types in which it rescues sleep. On the other hand, dDAT overexpression in mushroom bodies, which are the target of memory forming dopamine neurons, abolishes olfactory aversive memory. Our data demonstrate that expression of extrasynaptic dopamine transporters can rescue some aspects of dopamine signaling in dopamine transporter mutants. These results provide novel insights into regulatory systems that modulate dopamine signaling. PMID:25232310

  9. Blockade of uptake for dopamine, but not norepinephrine or 5-HT, increases selection of high effort instrumental activity: Implications for treatment of effort-related motivational symptoms in psychopathology.

    PubMed

    Yohn, Samantha E; Errante, Emily E; Rosenbloom-Snow, Aaron; Somerville, Matthew; Rowland, Margaret; Tokarski, Kristin; Zafar, Nadia; Correa, Merce; Salamone, John D

    2016-10-01

    Deficits in behavioral activation, exertion of effort, and other psychomotor/motivational symptoms are frequently seen in people with depression and other disorders. Depressed people show a decision bias towards selection of low effort activities, and animal tests of effort-related decision making are being used as models of motivational dysfunctions seen in psychopathology. The present studies investigated the ability of drugs that block dopamine transport (DAT), norepinephrine transport (NET), and serotonin transport (SERT) to modulate work output in rats responding on a test of effort-related decision making (i.e., a progressive ratio (PROG)/chow feeding choice task). With this task, rats choose between working for a preferred food (high carbohydrate pellets) by lever pressing on a PROG schedule vs. obtaining a less preferred lab chow that is freely available in the chamber. The present studies focused on the effects of the selective DAT inhibitor GBR12909, the selective SERT inhibitor fluoxetine, and the selective NET inhibitors desipramine and atomoxetine. Acute and repeated administration of GBR12909 shifted choice behavior, increasing measures of PROG lever pressing but decreasing chow intake. In contrast, fluoxetine, desipramine and atomoxetine failed to increase lever pressing output, and actually decreased it at higher doses. In the behaviorally effective dose range, GBR12909 elevated extracellular dopamine levels in accumbens core as measured by microdialysis, but fluoxetine, desipramine and atomoxetine decreased extracellular dopamine. Thus, blockade of DAT increases selection of the high effort instrumental activity, while inhibition of SERT or NET does not. These results have implications for the use of monoamine uptake inhibitors for the treatment of effort-related psychiatric symptoms in humans. PMID:27329556

  10. Blockade of uptake for dopamine, but not norepinephrine or 5-HT, increases selection of high effort instrumental activity: Implications for treatment of effort-related motivational symptoms in psychopathology.

    PubMed

    Yohn, Samantha E; Errante, Emily E; Rosenbloom-Snow, Aaron; Somerville, Matthew; Rowland, Margaret; Tokarski, Kristin; Zafar, Nadia; Correa, Merce; Salamone, John D

    2016-10-01

    Deficits in behavioral activation, exertion of effort, and other psychomotor/motivational symptoms are frequently seen in people with depression and other disorders. Depressed people show a decision bias towards selection of low effort activities, and animal tests of effort-related decision making are being used as models of motivational dysfunctions seen in psychopathology. The present studies investigated the ability of drugs that block dopamine transport (DAT), norepinephrine transport (NET), and serotonin transport (SERT) to modulate work output in rats responding on a test of effort-related decision making (i.e., a progressive ratio (PROG)/chow feeding choice task). With this task, rats choose between working for a preferred food (high carbohydrate pellets) by lever pressing on a PROG schedule vs. obtaining a less preferred lab chow that is freely available in the chamber. The present studies focused on the effects of the selective DAT inhibitor GBR12909, the selective SERT inhibitor fluoxetine, and the selective NET inhibitors desipramine and atomoxetine. Acute and repeated administration of GBR12909 shifted choice behavior, increasing measures of PROG lever pressing but decreasing chow intake. In contrast, fluoxetine, desipramine and atomoxetine failed to increase lever pressing output, and actually decreased it at higher doses. In the behaviorally effective dose range, GBR12909 elevated extracellular dopamine levels in accumbens core as measured by microdialysis, but fluoxetine, desipramine and atomoxetine decreased extracellular dopamine. Thus, blockade of DAT increases selection of the high effort instrumental activity, while inhibition of SERT or NET does not. These results have implications for the use of monoamine uptake inhibitors for the treatment of effort-related psychiatric symptoms in humans.

  11. Higher and longer stress-induced increase in dopamine concentrations in the nucleus accumbens of animals predisposed to amphetamine self-administration. A microdialysis study.

    PubMed

    Rougé-Pont, F; Piazza, P V; Kharouby, M; Le Moal, M; Simon, H

    1993-01-29

    Individual vulnerability to the reinforcing effects of drugs appears to be a crucial factor in the development of addiction in humans. In the rat, individuals at risk for psychostimulant self-administration (SA) may be identified from their locomotor reactivity to a stress situation such as exposure to a novel environment. Animals with high locomotor responses to novelty (high responders, HR) acquire amphetamine SA, while animals with low responses (low responders, LR) do not. In this study we examined by microdialysis whether stress-induced extracellular dopamine (DA) concentrations in the nucleus accumbens differed between these two groups of animals. This neurotransmitter was studied because it is thought to be involved in the reinforcing effects of psychostimulants. Furthermore, previous studies have shown that HR animals have a higher basal DOPAC/DA ratio in the nucleus accumbens and higher extracellular concentrations of dopamine in this structure in response to cocaine. The stress procedure used in this experiment consisted of a 10 min tail-pinch. HR animals displayed a higher and longer stress-induced changes in DA concentrations than the LR group. Regression analysis showed that stress-induced changes in DA levels accounted for 75% of the variance observed in the locomotor response to a novel environment. Since higher DA activity in the nucleus accumbens has been reported in animals in which the propensity to psychostimulant SA is induced by brain lesions or life events, this biochemical modification may be one neurobiological substrate of the predisposition to acquire psychostimulant self-administration. PMID:8448654

  12. Association of increased levels of TGF-β1 and p14ARF in prostate carcinoma cell lines overexpressing Egr-1.

    PubMed

    Parra, Eduardo; Gutiérrez, Luis; Ferreira, Jorge

    2014-11-01

    The present study examined the effect of the overexpression of early growth response gene (Egr-1) on transforming growth factor β-1 (TGF-β1) and p14ARF levels, in PC-3 and LNCaP prostate carcinoma cell lines. Amplification of EGR-1, TGF-β1 and p14ARF were observed in the two cell lines treated with different stimuli and resulted in a corresponding mRNA and protein expression. The downregulation of TGF-β1 and the attenuation of p14ARF expression by siRNA against Egr-1 predominantly suggested that TGF-β1 and p14ARF may be regulated by the transcription factor EGR-1. A marginal attenuation of cell growth in PC-3 and LNCaP prostate carcinoma cell lines overexpressing p14ARF was observed. Cells transfected with Egr-1 wild-type were able to grow and avoid cell cycle arrest and apoptosis in the presence or absence of p14ARF. In addition, EGR-1 stimulated the expression of TGF β-l as well as the accumulation of the p14ARF proteins. The results suggested that TGF-β1 and p14ARF activities in the presence of EGR-1 overexpression can exist independently of the presence of cells carrying a mutant p53 (PC-3 cells) or cells carrying a wild‑type p53 (LNCaP cells). Thus, the effect of EGR-1 on the growth of prostate carcinoma cells may occur through multiple mechanisms, but be independent of p53 expression control.

  13. Pharmacological characterization of the dopamine-sensitive adenylate cyclase in cockroach brain: evidence for a distinct dopamine receptor

    SciTech Connect

    Orr, G.L.; Gole, J.W.D.; Notman, H.J.; Downer, R.G.H.

    1987-12-21

    Dopamine increases cyclic AMP production in crude membrane preparations of cockroach brain with plateaus in cyclic AMP production occurring between 1-10 ..mu..M and 10 mM. Maximal production of cyclic AMP is 2.25 fold greater than that of control values. Octopamine also increases cyclic AMP production with a Ka of 1.4 ..mu..M and maximal production 3.5 fold greater than that of control. 5-Hydroxytryptamine does not increase cyclic AMP production. The effects of octopamine and dopamine are fully additive. The vertebrate dopamine agonists ADTN and epinine stimulate the dopamine-sensitive adenylate cyclase (AC) with Ka values of 4.5 and 0.6 ..mu..M respectively and with maximal effectiveness 1.7 fold greater than that of control. The selective D/sub 2/-dopamine agonist LY-171555 stimulates cyclic AMP production to a similar extent with a Ka of 50 ..mu..M. Other dopamine agonists have no stimulatory effects. With the exception of mianserin, /sup 3/H-piflutixol is displaced from brain membranes by dopamine antagonists with an order of potency similar to that observed for the inhibition of dopamine-sensitive AC. The results indicate that the octopamine- and dopamine-sensitive AC in cockroach brain can be distinguished pharmacologically and the dopamine receptors coupled to AC have pharmacological characteristics distinct from vertebrate D/sup 1/- and D/sup 2/-dopamine receptors. 33 references, 3 figures, 2 tables.

  14. Overexpression of EsMcsu1 from the halophytic plant Eutrema salsugineum promotes abscisic acid biosynthesis and increases drought resistance in alfalfa (Medicago sativa L.).

    PubMed

    Zhou, C; Ma, Z Y; Zhu, L; Guo, J S; Zhu, J; Wang, J F

    2015-01-01

    The stress phytohormone abscisic acid (ABA) plays pivotal roles in plants' adaptive responses to adverse environments. Molybdenum cofactor sulfurases influence aldehyde oxidase activity and ABA biosynthesis. In this study, we isolated a novel EsMcsu1 gene encoding a molybdenum cofactor sulfurase from Eutrema salsugineum. EsMcus1 transcriptional patterns varied between organs, and its expression was significantly upregulated by abiotic stress or ABA treatment. Alfalfa plants that overexpressed EsMcsu1 had a higher ABA content than wild-type (WT) plants under drought stress conditions. Furthermore, levels of reactive oxygen species (ROS), ion leakage, and malondialdehyde were lower in the transgenic plants than in the WT plants after drought treatment, suggesting that the transgenic plants experienced less ROS-mediated damage. However, the expression of several stress-responsive genes, antioxidant enzyme activity, and osmolyte (proline and total soluble sugar) levels in the transgenic plants were higher than those in the WT plants after drought treatment. Therefore, EsMcsu1 overexpression improved drought tolerance in alfalfa plants by activating a series of ABA-mediated stress responses. PMID:26681214

  15. Overexpression of the pumpkin (Cucurbita maxima) 16 kDa phloem protein CmPP16 increases tolerance to water deficit.

    PubMed

    Ramírez-Ortega, Francisco Arturo; Herrera-Pola, Paul Starsky; Toscano-Morales, Roberto; Xoconostle-Cázares, Beatriz; Ruiz-Medrano, Roberto

    2014-01-01

    The phloem plays an important role in the delivery of nutrients and signals between photosynthetic to heterotrophic tissues. Proteins and RNAs in the phloem translocation stream may have an important role in maintaining the integrity of the sieve tube system, as well as in long-distance signaling. CmPP16 is a pumpkin phloem protein, which has been shown to bind RNA in a non-sequence specific manner, and move it cell-to-cell and conceivably, long-distance. The protein and RNA are found in both companion cell (CC) and sieve elements (SE). However, a more precise function for this protein is not known. In this work we report the overexpression of CmPP16 fused to GFP via transformation of pumpkin (Cucurbita maxima cv. Big Max) plants in the cotyledonary stage by direct inoculation of Agrobacterium tumefaciens and Agrobacterium rhizogenes. Plants overexpressing CmPP16 did not show an obvious phenotype. However, these plants displayed higher photosynthetic capacity during drought than wild-type (WT) pumpkin or transformed with another construct. These results suggest that CmPP16 may be involved in the response to stress through long-distance signaling.

  16. Overexpression of the pumpkin (Cucurbita maxima) 16 kDa phloem protein CmPP16 increases tolerance to water deficit

    PubMed Central

    Ramírez-Ortega, Francisco Arturo; Herrera-Pola, Paul Starsky; Toscano-Morales, Roberto; Xoconostle-Cázares, Beatriz; Ruiz-Medrano, Roberto

    2014-01-01

    The phloem plays an important role in the delivery of nutrients and signals between photosynthetic to heterotrophic tissues. Proteins and RNAs in the phloem translocation stream may have an important role in maintaining the integrity of the sieve tube system, as well as in long-distance signaling. CmPP16 is a pumpkin phloem protein, which has been shown to bind RNA in a non-sequence specific manner, and move it cell-to-cell and conceivably, long-distance. The protein and RNA are found in both companion cell (CC) and sieve elements (SE). However, a more precise function for this protein is not known. In this work we report the overexpression of CmPP16 fused to GFP via transformation of pumpkin (Cucurbita maxima cv. Big Max) plants in the cotyledonary stage by direct inoculation of Agrobacterium tumefaciens and Agrobacterium rhizogenes. Plants overexpressing CmPP16 did not show an obvious phenotype. However, these plants displayed higher photosynthetic capacity during drought than wild-type (WT) pumpkin or transformed with another construct. These results suggest that CmPP16 may be involved in the response to stress through long-distance signaling. PMID:25482781

  17. Overexpression of EsMcsu1 from the halophytic plant Eutrema salsugineum promotes abscisic acid biosynthesis and increases drought resistance in alfalfa (Medicago sativa L.).

    PubMed

    Zhou, C; Ma, Z Y; Zhu, L; Guo, J S; Zhu, J; Wang, J F

    2015-12-17

    The stress phytohormone abscisic acid (ABA) plays pivotal roles in plants' adaptive responses to adverse environments. Molybdenum cofactor sulfurases influence aldehyde oxidase activity and ABA biosynthesis. In this study, we isolated a novel EsMcsu1 gene encoding a molybdenum cofactor sulfurase from Eutrema salsugineum. EsMcus1 transcriptional patterns varied between organs, and its expression was significantly upregulated by abiotic stress or ABA treatment. Alfalfa plants that overexpressed EsMcsu1 had a higher ABA content than wild-type (WT) plants under drought stress conditions. Furthermore, levels of reactive oxygen species (ROS), ion leakage, and malondialdehyde were lower in the transgenic plants than in the WT plants after drought treatment, suggesting that the transgenic plants experienced less ROS-mediated damage. However, the expression of several stress-responsive genes, antioxidant enzyme activity, and osmolyte (proline and total soluble sugar) levels in the transgenic plants were higher than those in the WT plants after drought treatment. Therefore, EsMcsu1 overexpression improved drought tolerance in alfalfa plants by activating a series of ABA-mediated stress responses.

  18. Over-expression of StAPX in tobacco improves seed germination and increases early seedling tolerance to salinity and osmotic stresses.

    PubMed

    Sun, Wei-Hong; Duan, Ming; Shu, De-Feng; Yang, Sha; Meng, Qing-Wei

    2010-08-01

    Ascorbate peroxidase plays a key role in scavenging reactive oxygen species under environmental stresses and in protecting plant cells against toxic effects. The Solanum lycopersicum thylakoid-bound ascorbate peroxidase gene (StAPX) was introduced into tobacco under the control of the cauliflower mosaic virus 35S promoter. Transformants were selected for their ability to grow on medium containing kanamycin. RNA gel blot analysis confirmed that StAPX was transferred into the tobacco genome and StAPX was induced by salt and osmotic stresses in tomato leaves. Over-expression of StAPX in tobacco improved seed germination rate and elevated stress tolerance during post-germination development. Two transgenic lines showed higher APX activity and accumulated less hydrogen peroxide than wild-type plants after stress treatments. The photosynthetic rates, the root lengths, the fresh and dry weights of the transgenic lines were distinctly higher than those of wild-type plants under stress conditions. Results indicated that the over-expression of StAPX had enhanced tolerance to salt stress and osmotic stress in transgenic tobacco plants.

  19. Constitutive or seed-specific overexpression of Arabidopsis G-protein γ subunit 3 (AGG3) results in increased seed and oil production and improved stress tolerance in Camelina sativa.

    PubMed

    Roy Choudhury, Swarup; Riesselman, Adam J; Pandey, Sona

    2014-01-01

    Heterotrimeric G-proteins consisting of Gα, Gβ and Gγ subunits play an integral role in mediating multiple signalling pathways in plants. A novel, recently identified plant-specific Gγ protein, AGG3, has been proposed to be an important regulator of organ size and mediator of stress responses in Arabidopsis, whereas its potential homologs in rice are major quantitative trait loci for seed size and panicle branching. To evaluate the role of AGG3 towards seed and oil yield improvement, the gene was overexpressed in Camelina sativa, an oilseed crop of the Brassicaceae family. Analysis of multiple homozygous T4 transgenic Camelina lines showed that constitutive overexpression of AGG3 resulted in faster vegetative as well as reproductive growth accompanied by an increase in photosynthetic efficiency. Moreover, when expressed constitutively or specifically in seed tissue, AGG3 was found to increase seed size, seed mass and seed number per plant by 15%-40%, effectively resulting in significantly higher oil yield per plant. AGG3 overexpressing Camelina plants also exhibited improved stress tolerance. These observations draw a strong link between the roles of AGG3 in regulating two critical yield parameters, seed traits and plant stress responses, and reveal an effective biotechnological tool to dramatically increase yield in agricultural crops. PMID:24102738

  20. Overexpression of the steroidogenic enzyme cytochrome P450 side chain cleavage in the ventral tegmental area increases 3α,5α-THP and reduces long-term operant ethanol self-administration.

    PubMed

    Cook, Jason B; Werner, David F; Maldonado-Devincci, Antoniette M; Leonard, Maggie N; Fisher, Kristen R; O'Buckley, Todd K; Porcu, Patrizia; McCown, Thomas J; Besheer, Joyce; Hodge, Clyde W; Morrow, A Leslie

    2014-04-23

    Neuroactive steroids are endogenous neuromodulators capable of altering neuronal activity and behavior. In rodents, systemic administration of endogenous or synthetic neuroactive steroids reduces ethanol self-administration. We hypothesized this effect arises from actions within mesolimbic brain regions that we targeted by viral gene delivery. Cytochrome P450 side chain cleavage (P450scc) converts cholesterol to pregnenolone, the rate-limiting enzymatic reaction in neurosteroidogenesis. Therefore, we constructed a recombinant adeno-associated serotype 2 viral vector (rAAV2), which drives P450scc expression and neuroactive steroid synthesis. The P450scc-expressing vector (rAAV2-P450scc) or control GFP-expressing vector (rAAV2-GFP) were injected bilaterally into the ventral tegmental area (VTA) or nucleus accumbens (NAc) of alcohol preferring (P) rats trained to self-administer ethanol. P450scc overexpression in the VTA significantly reduced ethanol self-administration by 20% over the 3 week test period. P450scc overexpression in the NAc, however, did not alter ethanol self-administration. Locomotor activity was unaltered by vector administration to either region. P450scc overexpression produced a 36% increase in (3α,5α)-3-hydroxypregnan-20-one (3α,5α-THP, allopregnanolone)-positive cells in the VTA, but did not increase 3α,5α-THP immunoreactivity in NAc. These results suggest that P450scc overexpression and the resultant increase of 3α,5α-THP-positive cells in the VTA reduces ethanol reinforcement. 3α,5α-THP is localized to neurons in the VTA, including tyrosine hydroxylase neurons, but not astrocytes. Overall, the results demonstrate that using gene delivery to modulate neuroactive steroids shows promise for examining the neuronal mechanisms of moderate ethanol drinking, which could be extended to other behavioral paradigms and neuropsychiatric pathology.

  1. The role of dopamine receptors in the neurotoxicity of methamphetamine.

    PubMed

    Ares-Santos, S; Granado, N; Moratalla, R

    2013-05-01

    Methamphetamine is a synthetic drug consumed by millions of users despite its neurotoxic effects in the brain, leading to loss of dopaminergic fibres and cell bodies. Moreover, clinical reports suggest that methamphetamine abusers are predisposed to Parkinson's disease. Therefore, it is important to elucidate the mechanisms involved in methamphetamine-induced neurotoxicity. Dopamine receptors may be a plausible target to prevent this neurotoxicity. Genetic inactivation of dopamine D1 or D2 receptors protects against the loss of dopaminergic fibres in the striatum and loss of dopaminergic neurons in the substantia nigra. Protection by D1 receptor inactivation is due to blockade of hypothermia, reduced dopamine content and turnover and increased stored vesicular dopamine in D1R(-/-) mice. However, the neuroprotective impact of D2 receptor inactivation is partially dependent on an effect on body temperature, as well as on the blockade of dopamine reuptake by decreased dopamine transporter activity, which results in reduced intracytosolic dopamine levels in D2R(-/-) mice.

  2. Dopamine synapse is a neuroligin-2–mediated contact between dopaminergic presynaptic and GABAergic postsynaptic structures

    PubMed Central

    Uchigashima, Motokazu; Ohtsuka, Toshihisa; Kobayashi, Kazuto; Watanabe, Masahiko

    2016-01-01

    Midbrain dopamine neurons project densely to the striatum and form so-called dopamine synapses on medium spiny neurons (MSNs), principal neurons in the striatum. Because dopamine receptors are widely expressed away from dopamine synapses, it remains unclear how dopamine synapses are involved in dopaminergic transmission. Here we demonstrate that dopamine synapses are contacts formed between dopaminergic presynaptic and GABAergic postsynaptic structures. The presynaptic structure expressed tyrosine hydroxylase, vesicular monoamine transporter-2, and plasmalemmal dopamine transporter, which are essential for dopamine synthesis, vesicular filling, and recycling, but was below the detection threshold for molecules involving GABA synthesis and vesicular filling or for GABA itself. In contrast, the postsynaptic structure of dopamine synapses expressed GABAergic molecules, including postsynaptic adhesion molecule neuroligin-2, postsynaptic scaffolding molecule gephyrin, and GABAA receptor α1, without any specific clustering of dopamine receptors. Of these, neuroligin-2 promoted presynaptic differentiation in axons of midbrain dopamine neurons and striatal GABAergic neurons in culture. After neuroligin-2 knockdown in the striatum, a significant decrease of dopamine synapses coupled with a reciprocal increase of GABAergic synapses was observed on MSN dendrites. This finding suggests that neuroligin-2 controls striatal synapse formation by giving competitive advantage to heterologous dopamine synapses over conventional GABAergic synapses. Considering that MSN dendrites are preferential targets of dopamine synapses and express high levels of dopamine receptors, dopamine synapse formation may serve to increase the specificity and potency of dopaminergic modulation of striatal outputs by anchoring dopamine release sites to dopamine-sensing targets. PMID:27035941

  3. Dopamine, depression and antidepressants.

    PubMed

    Dailly, Eric; Chenu, Franck; Renard, Caroline E; Bourin, Michel

    2004-12-01

    Abstract The relationship between depression and dopamine deficiency in the mesolimbic pathway has been hypothesized for many years. The experimental studies with animal models of depression and the human studies implicate the role of the dopamine system in depression. Not only do dopaminergic receptor agonists, but also antagonists such as olanzapine exhibit antidepressant effects associated with standard antidepressants in patients with treatment-resistant depression. This paradoxical result suggests that further investigations are necessary to understand the role played by dopamine in depression.

  4. Dopamine-melanin nanofilms for biomimetic structural coloration.

    PubMed

    Wu, Tong-Fei; Hong, Jong-Dal

    2015-02-01

    This article describes the formation of dopamine-melanin thin films (50-200 nm thick) at an air/dopamine solution interface under static conditions. Beneath these films, spherical melanin granules formed in bulk liquid phase. The thickness of dopamine-melanin films at the interface relied mainly on the concentration of dopamine solution and the reaction time. A plausible mechanism underlining dopamine-melanin thin film formation was proposed based on the hydrophobicity of dopamine-melanin aggregates and the mass transport of the aggregates to the air/solution interface as a result of convective flow. The thickness of the interfacial films increased linearly with the dopamine concentration and the reaction time. The dopamine-melanin thin film and granules (formed in bulk liquid phase) with a double-layered structure were transferred onto a solid substrate to mimic the (keratin layer)/(melanin granules) structure present in bird plumage, thereby preparing full dopamine-melanin thin-film reflectors. The reflected color of the thin-film reflectors depended on the film thickness, which could be adjusted according to the dopamine concentration. The reflectance of the resulted reflectors exhibited a maximal reflectance value of 8-11%, comparable to that of bird plumage (∼11%). This study provides a useful, simple, and low-cost approach to the fabrication of biomimetic thin-film reflectors using full dopamine-melanin materials.

  5. PII Overexpression in Lotus japonicus Affects Nodule Activity in Permissive Low-Nitrogen Conditions and Increases Nodule Numbers in High Nitrogen Treated Plants.

    PubMed

    D'Apuzzo, Enrica; Valkov, Vladimir Totev; Parlati, Aurora; Omrane, Selim; Barbulova, Ani; Sainz, Maria Martha; Lentini, Marco; Esposito, Sergio; Rogato, Alessandra; Chiurazzi, Maurizio

    2015-04-01

    We report here the first characterization of a GLNB1 gene coding for the PII protein in leguminous plants. The main purpose of this work was the investigation of the possible roles played by this multifunctional protein in nodulation pathways. The Lotus japonicus LjGLB1 gene shows a significant transcriptional regulation during the light-dark cycle and different nitrogen availability, conditions that strongly affect nodule formation, development, and functioning. We also report analysis of the spatial profile of expression of LjGLB1 in root and nodule tissues and of the protein's subcellular localization. Transgenic L. japonicus lines overexpressing the PII protein were obtained and tested for the analysis of the symbiotic responses in different conditions. The uncoupling of PII from its native regulation affects nitrogenase activity and nodule polyamine content. Furthermore, our results suggest the involvement of PII in the signaling of the nitrogen nutritional status affecting the legumes' predisposition for nodule formation.

  6. Decoding dopamine signaling.

    PubMed

    Bibb, James A

    2005-07-29

    Dopamine is a key neurotransmitter that is important for many physiological functions including motor control, mood, and the reward pathway. In this issue of Cell, the laboratories of Marc Caron and Li-Huei Tsai identify two very different molecules--beta-arrestin 2 and Par-4, respectively--that unexpectedly are involved in dopamine signaling via the D2 receptor. These two new signaling pathways mediate the actions of dopamine on behavior and facilitate crosstalk between different signaling pathways that are activated by binding of dopamine to the D2 receptor.

  7. Dopamine receptors in human gastrointestinal mucosa

    SciTech Connect

    Hernandez, D.E.; Mason, G.A.; Walker, C.H.; Valenzuela, J.E.

    1987-12-21

    Dopamine is a putative enteric neurotransmitter that has been implicated in exocrine secretory and motility functions of the gastrointestinal tract of several mammalian species including man. This study was designed to determine the presence of dopamine binding sites in human gastric and duodenal mucosa and to describe certain biochemical characteristics of these enteric receptor sites. The binding assay was performed in triplicate with tissue homogenates obtained from healthy volunteers of both sexes using /sup 3/H-dopamine as a ligand. The extent of nonspecific binding was determined in the presence of a 100-fold excess of unlabeled dopamine. Scatchard analysis performed with increasing concentrations of /sup 3/H-dopamine (20-500 nM) revealed a single class of saturable dopamine binding sites in gastric and duodenal mucosa. The results of this report demonstrate the presence of specific dopamine receptors in human gastric and duodenal mucosa. These biochemical data suggest that molecular abnormalities of these receptor sites may be operative in the pathogenesis of important gastrointestinal disorders. 33 references, 2 figures.

  8. Striatal pleiotrophin overexpression provides functional and morphological neuroprotection in the 6-hydroxydopamine model.

    PubMed

    Gombash, Sara E; Lipton, Jack W; Collier, Timothy J; Madhavan, Lalitha; Steece-Collier, Kathy; Cole-Strauss, Allyson; Terpstra, Brian T; Spieles-Engemann, Anne L; Daley, Brian F; Wohlgenant, Susan L; Thompson, Valerie B; Manfredsson, Fredric P; Mandel, Ronald J; Sortwell, Caryl E

    2012-03-01

    Neurotrophic factors are integrally involved in the development of the nigrostriatal system and in combination with gene therapy, possess great therapeutic potential for Parkinson's disease (PD). Pleiotrophin (PTN) is involved in the development, maintenance, and repair of the nigrostriatal dopamine (DA) system. The present study examined the ability of striatal PTN overexpression, delivered via psueudotyped recombinant adeno-associated virus type 2/1 (rAAV2/1), to provide neuroprotection and functional restoration from 6-hydroxydopamine (6-OHDA). Striatal PTN overexpression led to significant neuroprotection of tyrosine hydroxylase immunoreactive (THir) neurons in the substantia nigra pars compacta (SNpc) and THir neurite density in the striatum, with long-term PTN overexpression producing recovery from 6-OHDA-induced deficits in contralateral forelimb use. Transduced striatal PTN levels were increased threefold compared to adult striatal PTN expression and approximated peak endogenous developmental levels (P1). rAAV2/1 vector exclusively transduced neurons within the striatum and SNpc with approximately half the total striatal volume routinely transduced using our injection parameters. Our results indicate that striatal PTN overexpression can provide neuroprotection for the 6-OHDA lesioned nigrostriatal system based upon morphological and functional measures and that striatal PTN levels similar in magnitude to those expressed in the striatum during development are sufficient to provide neuroprotection from Parkinsonian insult.

  9. Differential tonic influence of lateral habenula on prefrontal cortex and nucleus accumbens dopamine release.

    PubMed

    Lecourtier, Lucas; Defrancesco, Alicia; Moghaddam, Bita

    2008-04-01

    Conditions of increased cognitive or emotional demand activate dopamine release in a regionally selective manner. Whereas the brief millisecond response of dopamine neurons to salient stimuli suggests that dopamine's influence on behaviour may be limited to signalling certain cues, the prolonged availability of dopamine in regions such as the prefrontal cortex and nucleus accumbens is consistent with the well described role of dopamine in maintaining motivation states, associative learning and working memory. The behaviourally elicited terminal release of dopamine is generally attributed to increased excitatory drive on dopamine neurons. Our findings here, however, indicate that this increase may involve active removal of a tonic inhibitory control on dopamine neurons exerted by the lateral habenula (LHb). Inhibition of LHb in behaving animals transiently increased dopamine release in the prefrontal cortex, nucleus accumbens and dorsolateral striatum. The inhibitory influence was more pronounced in the nucleus accumbens and striatum than in the prefrontal cortex. This pattern of regional dopamine activation after LHb inhibition mimicked conditions of reward availability but not increased cognitive demand. Electrical or chemical stimulation of LHb produced minimal reduction of extracellular dopamine, suggesting that in an awake brain the inhibition associated with tonic LHb activity represents a near-maximal influence on dopamine neurotransmission. These data indicate that LHb may be critical for functional differences in dopamine neurons by preferentially modulating dopamine neurons that project to the nucleus accumbens over those neurons that primarily project to the prefrontal cortex.

  10. Chronic treatment with extended release methylphenidate does not alter dopamine systems or increase vulnerability for cocaine self-administration: a study in nonhuman primates.

    PubMed

    Gill, Kathryn E; Pierre, Peter J; Daunais, James; Bennett, Allyson J; Martelle, Susan; Gage, H Donald; Swanson, James M; Nader, Michael A; Porrino, Linda J

    2012-11-01

    Despite the widespread use of stimulant medications for the treatment of attention deficit hyperactivity disorder, few studies have addressed their long-term effects on the developing brain or susceptibility to drug use in adolescence. Here, we determined the effects of chronic methylphenidate (MPH) treatment on brain dopamine (DA) systems, developmental milestones, and later vulnerability to substance abuse in juvenile nonhuman primates. Male rhesus monkeys (approximately 30 months old) were treated daily with either a sustained release formulation of MPH or placebo (N=8 per group). Doses were titrated to achieve initial drug blood serum levels within the therapeutic range in children and adjusted throughout the study to maintain target levels. Growth, including measures of crown-rump length and weight, was assessed before and after 1 year of treatment and after 3-5 months washout. In addition, positron emission tomography scans were performed to quantify binding availability of D2/D3 receptors and dopamine transporters (DATs). Distribution volume ratios were calculated to quantify binding of [¹⁸F]fluoroclebopride (DA D2/D3) and [¹⁸F]-(+)-N-(4-fluorobenzyl)-2β-propanoyl-3β-(4-chlorophenyl)tropane (DAT). Chronic MPH did not differentially alter the course of weight gain or other measures of growth, nor did it influence DAT or D2/D3 receptor availability after 1 year of treatment. However, after washout, the D2/D3 receptor availability of MPH-treated animals did not continue to decline at the same rate as control animals. Acquisition of intravenous cocaine self-administration was examined by first substituting saline for food reinforcement and then cocaine doses (0.001-0.1 mg/kg per injection) in ascending order. Each dose was available for at least five consecutive sessions. The lowest dose of cocaine that maintained response rates significantly higher than saline-contingent rates was operationally defined as acquisition of cocaine reinforcement. There

  11. The dopamine D2/D3 receptor agonist quinpirole increases checking-like behaviour in an operant observing response task with uncertain reinforcement: A novel possible model of OCD?

    PubMed Central

    Eagle, Dawn M.; Noschang, Cristie; d’Angelo, Laure-Sophie Camilla; Noble, Christie A.; Day, Jacob O.; Dongelmans, Marie Louise; Theobald, David E.; Mar, Adam C.; Urcelay, Gonzalo P.; Morein-Zamir, Sharon; Robbins, Trevor W.

    2014-01-01

    Excessive checking is a common, debilitating symptom of obsessive-compulsive disorder (OCD). In an established rodent model of OCD checking behaviour, quinpirole (dopamine D2/3-receptor agonist) increased checking in open-field tests, indicating dopaminergic modulation of checking-like behaviours. We designed a novel operant paradigm for rats (observing response task (ORT)) to further examine cognitive processes underpinning checking behaviour and clarify how and why checking develops. We investigated i) how quinpirole increases checking, ii) dependence of these effects on D2/3 receptor function (following treatment with D2/3 receptor antagonist sulpiride) and iii) effects of reward uncertainty. In the ORT, rats pressed an ‘observing’ lever for information about the location of an ‘active’ lever that provided food reinforcement. High- and low-checkers (defined from baseline observing) received quinpirole (0.5 mg/kg, 10 treatments) or vehicle. Parametric task manipulations assessed observing/checking under increasing task demands relating to reinforcement uncertainty (variable response requirement and active-lever location switching). Treatment with sulpiride further probed the pharmacological basis of long-term behavioural changes. Quinpirole selectively increased checking, both functional observing lever presses (OLPs) and non-functional extra OLPs (EOLPs). The increase in OLPs and EOLPs was long-lasting, without further quinpirole administration. Quinpirole did not affect the immediate ability to use information from checking. Vehicle and quinpirole-treated rats (VEH and QNP respectively) were selectively sensitive to different forms of uncertainty. Sulpiride reduced non-functional EOLPs in QNP rats but had no effect on functional OLPs. These data have implications for treatment of compulsive checking in OCD, particularly for serotonin-reuptake-inhibitor treatment-refractory cases, where supplementation with dopamine receptor antagonists may be

  12. Ectopic overexpression of a novel Glycine soja stress-induced plasma membrane intrinsic protein increases sensitivity to salt and dehydration in transgenic Arabidopsis thaliana plants.

    PubMed

    Wang, Xi; Cai, Hua; Li, Yong; Zhu, Yanming; Ji, Wei; Bai, Xi; Zhu, Dan; Sun, Xiaoli

    2015-01-01

    Plasma membrane intrinsic proteins (PIPs) belong to the aquaporin family and facilitate water movement across plasma membranes. Existing data indicate that PIP genes are associated with the abilities of plants to tolerate certain stress conditions. A review of our Glycine soja expressed sequence tag (EST) dataset revealed that abiotic stress stimulated expression of a PIP, herein designated as GsPIP2;1 (GenBank_Accn: FJ825766). To understand the roles of this PIP in stress tolerance, we generated a coding sequence for GsPIP2;1 by in silico elongation and cloned the cDNA by 5'-RACE. Semiquantitative RT-PCR showed that GsPIP2;1 expression was stimulated in G. soja leaves by cold, salt, or dehydration stress, whereas the same stresses suppressed GsPIP2;1 expression in the roots. Transgenic Arabidopsis thaliana plants overexpressing GsPIP2;1 grew normally under unstressed and cold conditions, but exhibited depressed tolerance to salt and dehydration stresses. Moreover, greater changes in water potential were detected in the transgenic A. thaliana shoots, implying that GsPIP2;1 may negatively impact stress tolerance by regulating water potential. These results, deviating from those obtained in previous reports, provide new insights into the relationship between PIPs and abiotic stress tolerance in plants.

  13. Consumption of tyrosine in royal jelly increases brain levels of dopamine and tyramine and promotes transition from normal to reproductive workers in queenless honey bee colonies.

    PubMed

    Matsuyama, Syuhei; Nagao, Takashi; Sasaki, Ken

    2015-01-15

    Dopamine (DA) and tyramine (TA) have neurohormonal roles in the production of reproductive workers in queenless colonies of honey bees, but the regulation of these biogenic amines in the brain are still largely unclear. Nutrition is an important factor in promoting reproduction and might be involved in the regulation of these biogenic amines in the brain. To test this hypothesis, we examined the effect of oral treatments of tyrosine (Tyr; a common precursor of DA, TA and octopamine, and a component of royal jelly) in queenless workers and quantified the resulting production of biogenic amines. Tyrosine treatments enhanced the levels of DA, TA and their metabolites in the brain. Workers fed royal jelly had significantly larger brain levels of Tyr, DA, TA and the metabolites in the brains compared with those bees fed honey or sucrose (control). Treatment with Tyr also inhibited the behavior of workers outside of the hive and promoted ovarian development. These results suggest that there is a link between nutrition and the regulation of DA and TA in the brain to promote the production of reproductive workers in queenless honey bee colonies. PMID:25448251

  14. Consumption of tyrosine in royal jelly increases brain levels of dopamine and tyramine and promotes transition from normal to reproductive workers in queenless honey bee colonies.

    PubMed

    Matsuyama, Syuhei; Nagao, Takashi; Sasaki, Ken

    2015-01-15

    Dopamine (DA) and tyramine (TA) have neurohormonal roles in the production of reproductive workers in queenless colonies of honey bees, but the regulation of these biogenic amines in the brain are still largely unclear. Nutrition is an important factor in promoting reproduction and might be involved in the regulation of these biogenic amines in the brain. To test this hypothesis, we examined the effect of oral treatments of tyrosine (Tyr; a common precursor of DA, TA and octopamine, and a component of royal jelly) in queenless workers and quantified the resulting production of biogenic amines. Tyrosine treatments enhanced the levels of DA, TA and their metabolites in the brain. Workers fed royal jelly had significantly larger brain levels of Tyr, DA, TA and the metabolites in the brains compared with those bees fed honey or sucrose (control). Treatment with Tyr also inhibited the behavior of workers outside of the hive and promoted ovarian development. These results suggest that there is a link between nutrition and the regulation of DA and TA in the brain to promote the production of reproductive workers in queenless honey bee colonies.

  15. Overexpression of a R2R3 MYB gene MdSIMYB1 increases tolerance to multiple stresses in transgenic tobacco and apples.

    PubMed

    Wang, Rong-Kai; Cao, Zhong-Hui; Hao, Yu-Jin

    2014-01-01

    MYB transcription factors (TFs) involve in plant abiotic stress tolerance and response in various plant species. In this study, rapid amplification of cDNA ends (RACE) was conducted to isolate the R2R3-MYB TF gene MdSIMYB1 from apples (Malus × domestica). The gene transcripts were abundant in the leaves, flowers and fruits, compared to other organs, and were induced by abiotic stresses and plant hormones. We observed the subcellular localization of an MdSIMYB1-GFP fusion protein in the nucleus. Furthermore, the MdSIMYB1 gene was introduced into the tobacco genome and ectopically expressed in transgenic lines. The results indicate that MdSIMYB1 transgenic tobacco seed germination is insensitive to abscisic acid and NaCl treatment. Additionally, it was found that the ectopic expression of MdSIMYB1 enhanced the tolerance of plants to high salinity, drought and cold tolerance by upregulating the stress-responsive genes NtDREB1A, NtERD10B and NtERD10C. Meanwhile, the transgenic tobacco exhibited robust root growth because of the enhanced expression of the auxin-responsive genes NtIAA4.2, NtIAA4.1 and NtIAA2.5 under stress conditions, which is conducive to stress tolerance. Finally, transgenic apple lines were obtained and tested. Transgenic apple lines that were overexpressing MdSIMYB1 exhibited a higher tolerance to abiotic stress than the wild-type control, but suppression of MdSIMYB1 resulted in lower tolerance. Our results indicate that MdSIMYB1 may be utilized as a target gene for enhancing stress tolerance in important crops.

  16. Overexpression of Rice Glutaredoxin OsGrx_C7 and OsGrx_C2.1 Reduces Intracellular Arsenic Accumulation and Increases Tolerance in Arabidopsis thaliana.

    PubMed

    Verma, Pankaj K; Verma, Shikha; Pande, Veena; Mallick, Shekhar; Deo Tripathi, Rudra; Dhankher, Om P; Chakrabarty, Debasis

    2016-01-01

    Glutaredoxins (Grxs) are a family of small multifunctional proteins involved in various cellular functions, including redox regulation and protection under oxidative stress. Despite the high number of Grx genes in plant genomes (48 Grxs in rice), the biological functions and physiological roles of most of them remain unknown. Here, the functional characterization of the two arsenic-responsive rice Grx family proteins, OsGrx_C7 and OsGrx_C2.1 are reported. Over-expression of OsGrx_C7 and OsGrx_C2.1 in transgenic Arabidopsis thaliana conferred arsenic (As) tolerance as reflected by germination, root growth assay, and whole plant growth. Also, the transgenic expression of OsGrxs displayed significantly reduced As accumulation in A. thaliana seeds and shoot tissues compared to WT plants during both AsIII and AsV stress. Thus, OsGrx_C7 and OsGrx_C2.1 seem to be an important determinant of As-stress response in plants. OsGrx_C7 and OsGrx_C2.1 transgenic showed to maintain intracellular GSH pool and involved in lowering AsIII accumulation either by extrusion or reducing uptake by altering the transcript of A. thaliana AtNIPs. Overall, OsGrx_C7 and OsGrx_C2.1 may represent a Grx family protein involved in As stress response and may allow a better understanding of the As induced stress pathways and the design of strategies for the improvement of stress tolerance as well as decreased As content in crops.

  17. AtRAV1 and AtRAV2 overexpression in cotton increases fiber length differentially under drought stress and delays flowering.

    PubMed

    Mittal, Amandeep; Jiang, Yingwen; Ritchie, Glen L; Burke, John J; Rock, Christopher D

    2015-12-01

    There is a longstanding problem of an inverse relationship between cotton fiber qualities versus high yields. To better understand drought stress signaling and adaptation in cotton (Gossypium hirsutum) fiber development, we expressed the Arabidopsis transcription factors RELATED_TO_ABA-INSENSITIVE3/VIVIPAROUS1/(RAV1) and AtRAV2, which encode APETALA2-Basic3 domain proteins shown to repress transcription of FLOWERING_LOCUS_T (FT) and to promote stomatal opening cell-autonomously. In three years of field trials, we show that AtRAV1 and AtRAV2-overexpressing cotton had ∼5% significantly longer fibers with only marginal decreases in yields under well-watered or drought stress conditions that resulted in 40-60% yield penalties and 3-7% fiber length penalties in control plants. The longer transgenic fibers from drought-stressed transgenics could be spun into yarn which was measurably stronger and more uniform than that from well-watered control fibers. The transgenic AtRAV1 and AtRAV2 lines flowered later and retained bolls at higher nodes, which correlated with repression of endogenous GhFT-Like (FTL) transcript accumulation. Elevated expression early in development of ovules was observed for GhRAV2L, GhMYB25-Like (MYB25L) involved in fiber initiation, and GhMYB2 and GhMYB25 involved in fiber elongation. Altered expression of RAVs controlling critical nodes in developmental and environmental signaling hierarchies has the potential for phenotypic modification of crops.

  18. Overexpression of Rice Glutaredoxin OsGrx_C7 and OsGrx_C2.1 Reduces Intracellular Arsenic Accumulation and Increases Tolerance in Arabidopsis thaliana

    PubMed Central

    Verma, Pankaj K.; Verma, Shikha; Pande, Veena; Mallick, Shekhar; Deo Tripathi, Rudra; Dhankher, Om P.; Chakrabarty, Debasis

    2016-01-01

    Glutaredoxins (Grxs) are a family of small multifunctional proteins involved in various cellular functions, including redox regulation and protection under oxidative stress. Despite the high number of Grx genes in plant genomes (48 Grxs in rice), the biological functions and physiological roles of most of them remain unknown. Here, the functional characterization of the two arsenic-responsive rice Grx family proteins, OsGrx_C7 and OsGrx_C2.1 are reported. Over-expression of OsGrx_C7 and OsGrx_C2.1 in transgenic Arabidopsis thaliana conferred arsenic (As) tolerance as reflected by germination, root growth assay, and whole plant growth. Also, the transgenic expression of OsGrxs displayed significantly reduced As accumulation in A. thaliana seeds and shoot tissues compared to WT plants during both AsIII and AsV stress. Thus, OsGrx_C7 and OsGrx_C2.1 seem to be an important determinant of As-stress response in plants. OsGrx_C7 and OsGrx_C2.1 transgenic showed to maintain intracellular GSH pool and involved in lowering AsIII accumulation either by extrusion or reducing uptake by altering the transcript of A. thaliana AtNIPs. Overall, OsGrx_C7 and OsGrx_C2.1 may represent a Grx family protein involved in As stress response and may allow a better understanding of the As induced stress pathways and the design of strategies for the improvement of stress tolerance as well as decreased As content in crops. PMID:27313586

  19. Overexpression of the Flii gene increases dermal-epidermal blistering in an autoimmune ColVII mouse model of epidermolysis bullosa acquisita.

    PubMed

    Kopecki, Zlatko; Arkell, Ruth M; Strudwick, Xanthe L; Hirose, Misa; Ludwig, Ralf J; Kern, Johannes S; Bruckner-Tuderman, Leena; Zillikens, Detlef; Murrell, Dedee F; Cowin, Allison J

    2011-11-01

    Epidermolysis bullosa (EB) is a severe genetic skin fragility syndrome characterized by blister formation. The molecular basis of EB is still largely unknown and wound healing in patients suffering from EB remains a major challenge to their survival. Our previous studies have identified the actin remodelling protein Flightless I (Flii) as an important mediator of wound repair. Here we identify Flii as a novel target involved in skin blistering. Flii expression was significantly elevated in 30 patients with EB, most prominently in patients with recessive dystrophic EB (RDEB) who have defects in production of type VII collagen (ColVII). Using an autoimmune ColVII murine model of EB acquisita (EBA) and an immunocompetent-ColVII-hypomorphic genetic mouse model of RDEB together with murine Flii alleles, we investigated the contribution of Flii to EB. Overexpression of Flii produced severe blistering post-induction of EBA, while decreased Flii reduced blister severity, elevated integrin expression, and improved ColVII production. Flii(+/-) blistered skin showed reduced α-SMA, TGF-β1, and Smad 2/3 expression, suggesting that decreasing Flii may affect fibrosis. In support of this, Flii-deficient fibroblasts from EBA mice were less able to contract collagen gels in vitro; however, addition of TGF-β1 restored collagen contraction, suggesting an interplay between Flii and TGF-β1. Elevated Flii gene and protein expression was further observed in the blisters of ColVII hypomorphic mice, a murine model of RDEB, suggesting that reducing Flii in blistered skin could be a potential new approach for treating patients with EB.

  20. AtRAV1 and AtRAV2 overexpression in cotton increases fiber length differentially under drought stress and delays flowering.

    PubMed

    Mittal, Amandeep; Jiang, Yingwen; Ritchie, Glen L; Burke, John J; Rock, Christopher D

    2015-12-01

    There is a longstanding problem of an inverse relationship between cotton fiber qualities versus high yields. To better understand drought stress signaling and adaptation in cotton (Gossypium hirsutum) fiber development, we expressed the Arabidopsis transcription factors RELATED_TO_ABA-INSENSITIVE3/VIVIPAROUS1/(RAV1) and AtRAV2, which encode APETALA2-Basic3 domain proteins shown to repress transcription of FLOWERING_LOCUS_T (FT) and to promote stomatal opening cell-autonomously. In three years of field trials, we show that AtRAV1 and AtRAV2-overexpressing cotton had ∼5% significantly longer fibers with only marginal decreases in yields under well-watered or drought stress conditions that resulted in 40-60% yield penalties and 3-7% fiber length penalties in control plants. The longer transgenic fibers from drought-stressed transgenics could be spun into yarn which was measurably stronger and more uniform than that from well-watered control fibers. The transgenic AtRAV1 and AtRAV2 lines flowered later and retained bolls at higher nodes, which correlated with repression of endogenous GhFT-Like (FTL) transcript accumulation. Elevated expression early in development of ovules was observed for GhRAV2L, GhMYB25-Like (MYB25L) involved in fiber initiation, and GhMYB2 and GhMYB25 involved in fiber elongation. Altered expression of RAVs controlling critical nodes in developmental and environmental signaling hierarchies has the potential for phenotypic modification of crops. PMID:26706061

  1. Decrease of D2 receptor binding but increase in D2-stimulated G-protein activation, dopamine transporter binding and behavioural sensitization in brains of mice treated with a chronic escalating dose 'binge' cocaine administration paradigm.

    PubMed

    Bailey, A; Metaxas, A; Yoo, J H; McGee, T; Kitchen, I

    2008-08-01

    Understanding the neurobiology of the transition from initial drug use to excessive drug use has been a challenge in drug addiction. We examined the effect of chronic 'binge' escalating dose cocaine administration, which mimics human compulsive drug use, on behavioural responses and the dopaminergic system of mice and compared it with a chronic steady dose (3 x 15 mg/kg/day) 'binge' cocaine administration paradigm. Male C57BL/6J mice were injected with saline or cocaine in an escalating dose paradigm for 14 days. Locomotor and stereotypy activity were measured and quantitative autoradiographic mapping of D(1) and D(2) receptors, dopamine transporters and D(2)-stimulated [(35)S]GTPgammaS binding was performed in the brains of mice treated with this escalating and steady dose paradigm. An initial sensitization to the locomotor effects of cocaine followed by a dose-dependent increase in the duration of the locomotor effect of cocaine was observed in the escalating but not the steady dose paradigm. Sensitization to the stereotypy effect of cocaine and an increase in cocaine-induced stereotypy score was observed from 3 x 20 to 3 x 25 mg/kg/day cocaine. There was a significant decrease in D(2) receptor density, but an increase in D(2)-stimulated G-protein activity and dopamine transporter density in the striatum of cocaine-treated mice, which was not observed in our steady dose paradigm. Our results document that chronic 'binge' escalating dose cocaine treatment triggers profound behavioural and neurochemical changes in the dopaminergic system, which might underlie the transition from drug use to compulsive drug use associated with addiction, which is a process of escalation.

  2. Disruption of the ErbB signaling in adolescence increases striatal dopamine levels and affects learning and hedonic-like behavior in the adult mouse.

    PubMed

    Golani, Idit; Tadmor, Hagar; Buonanno, Andres; Kremer, Ilana; Shamir, Alon

    2014-11-01

    The ErbB signaling pathway has been genetically and functionally implicated in schizophrenia. Numerous findings support the dysregulation of Neuregulin (NRG) and epidermal growth factor (EGF) signaling in schizophrenia. However, it is unclear whether alterations of these pathways in the adult brain or during development are involved in the pathophysiology of schizophrenia. Herein we characterized the behavioral profile and molecular changes resulting from pharmacologically blocking the ErbB signaling pathway during a critical period in the development of decision making, planning, judgments, emotions, social cognition and cognitive skills, namely adolescence. We demonstrate that chronic administration of the pan-ErbB kinase inhibitor JNJ-28871063 (JNJ) to adolescent mice elevated striatal dopamine levels and reduced preference for sucrose without affecting locomotor activity and exploratory behavior. In adulthood, adolescent JNJ-treated mice continue to consume less sucrose and needed significantly more correct-response trials to reach the learning criterion during the discrimination phase of the T-maze reversal learning task than their saline-injected controls. In addition, JNJ mice exhibited deficit in reference memory but not in working memory as measured in the radial arm maze. Inhibition of the pathway during adolescence did not affect exploratory behavior and locomotor activity in the open field, social interaction, social memory, and reversal learning in adult mice. Our data suggest that alteration of ErbB signaling during adolescence resulted in changes in the dopaminergic systems that emerge in pathological learning and hedonic behavior in adulthood, and pinpoints the possible role of the pathway in the development of cognitive skills and motivated behavior. PMID:25451700

  3. let-7 Overexpression Leads to an Increased Fraction of Cells in G2/M, Direct Down-regulation of Cdc34, and Stabilization of Wee1 Kinase in Primary Fibroblasts*S⃞

    PubMed Central

    Legesse-Miller, Aster; Elemento, Olivier; Pfau, Sarah J.; Forman, Joshua J.; Tavazoie, Saeed; Coller, Hilary A.

    2009-01-01

    microRNAs play a critically important role in a wide array of biological processes including those implicated in cancer, neuro-degenerative and metabolic disorders, and viral infection. Although we have begun to understand microRNA biogenesis and function, experimental demonstration of their functional effects and the molecular mechanisms by which they function remains a challenge. Members of the let-7/miR-98 family play a critical role in cell cycle control with respect to differentiation and tumorigenesis. In this study, we show that exogenous addition of pre-let-7 in primary human fibroblasts results in a decrease in cell number and an increased fraction of cells in the G2/M cell cycle phase. Combining microarray techniques with DNA sequence analysis to identify potential let-7 targets, we discovered 838 genes with a let-7 binding site in their 3′-untranslated region that were down-regulated upon overexpression of let-7b. Among these genes is cdc34, the ubiquitin-conjugating enzyme of the Skp1/cullin/F-box (SCF) complex. Cdc34 protein levels are strongly down-regulated by let-7 overexpression. Reporter assays demonstrated direct regulation of the cdc34 3′-untranslated region by let-7. We hypothesized that low Cdc34 levels would result in decreased SCF activity, stabilization of the SCF target Wee1, and G2/M accumulation. Consistent with this hypothesis, small interfering RNA-mediated down-regulation of Wee1 reversed the G2/M phenotype induced by let-7 overexpression. We conclude that Cdc34 is a functional target of let-7 and that let-7 induces down-regulation of Cdc34, stabilization of the Wee1 kinase, and an increased fraction of cells in G2/M in primary fibroblasts. PMID:19126550

  4. Prolonged survival in pancreatic cancer patients with increased regucalcin gene expression: Overexpression of regucalcin suppresses the proliferation in human pancreatic cancer MIA PaCa-2 cells in vitro.

    PubMed

    Yamaguchi, Masayoshi; Osuka, Satoru; Weitzmann, M Neale; El-Rayes, Bassel F; Shoji, Mamoru; Murata, Tomiyasu

    2016-05-01

    Approximately 90% of all pancreatic cancers are pancreatic ductal adenocarcinomas (PDAC). PDAC is a highly aggressive malignancy and is one of the deadliest. This poor clinical outcome is due to the prominent resistance of pancreatic cancer to drug and radiation therapies. Regucalcin plays a pivotal role as a suppressor protein in signal transduction in various types of cells including tumor tissues. We demonstrated that the prolonged survival is induced in PDAC patients with increased regucalcin gene expression using a dataset of PDAC obtained from GEO database (GSE17891) together with the clinical annotation data file. Moreover, overexpression of regucalcin with full length was demonstrated to suppress the proliferation, cell death and migration in human pancreatic cancer MIA PaCa-2 (K-ras mutated) cells that possess resistance to drug and radiation therapies. Suppressive effects of regucalcin on cell proliferation and death were not seen in the cells overexpressed with regucalcin cDNA alternatively spliced variants (deleted exon 4 or deleted exon 4 and 5). Regucalcin was suggested to induce G1 and G2/M phase cell cycle arrest in MIA PaCa-2 cells. Suppressive effects of regucalcin on cell proliferation were independent of cell death. Overexpression of regucalcin was found to suppress signaling pathways including Akt, MAP kinase and SAPK/JNK, to increase the protein levels of p53, a tumor suppresser, and to decrease K-ras, c-fos and c-jun, a oncogene, by suppressing signaling pathways that are related to signaling of K-ras. Regucalcin may play a potential role as a suppressor protein in human pancreatic cancer. PMID:26935290

  5. Increased regucalcin gene expression extends survival in breast cancer patients: Overexpression of regucalcin suppresses the proliferation and metastatic bone activity in MDA-MB-231 human breast cancer cells in vitro.

    PubMed

    Yamaguchi, Masayoshi; Osuka, Satoru; Weitzmann, M Neale; Shoji, Mamoru; Murata, Tomiyasu

    2016-08-01

    Human breast cancer is highly metastatic to bone and drives bone turnover. Breast cancer metastases cause osteolytic lesions and skeletal damage that leads to bone fractures. Regucalcin, which plays a pivotal role as an inhibitor of signal transduction and transcription activity, has been suggested to act as a suppressor of human cancer. In the present study, we compared the clinical outcome between 44 breast cancer patients with higher regucalcin expression and 43 patients with lower regucalcin expression. Prolonged relapse-free survival was identified in the patients with increased regucalcin gene expression. We further demonstrated that overexpression of full length, but not alternatively spliced variants of regucalcin, induces G1 and G2/M phase cell cycle arrest, suppressing the proliferation of MDA-MB-231 cells, a commonly used in vitro model of human breast cancer that metastasize to bone causing osteolytic lesions. Overexpression of regucalcin was found to suppress multiple signaling pathways including Akt, MAP kinase and SAPK/JNK, and NF-κB p65 and β-catenin along with increased p53, a tumor suppressor, and decreased K-ras, c-fos and c-jun. Moreover, we found that co-culture of regucalcin-overexpressing MDA-MB-231 cells with mouse bone marrow cells prevented enhanced osteoclastogenesis and suppressed mineralization in mouse bone marrow cells in vitro. Taken together, the present study suggests that regucalcin may have important anticancer properties in human breast cancer patients. Mechanistically, these effects are likely mediated through suppression of multiple signaling pathways, upregulation of p53 and downregulation of oncogenes leading to anti-proliferative effects and reduced metastases to bone, a phenotype associated with poor clinical outcome. PMID:27221776

  6. Overexpression of miR-7-1 increases efficacy of green tea polyphenols for induction of apoptosis in human malignant neuroblastoma SH-SY5Y and SK-N-DZ cells.

    PubMed

    Chakrabarti, Mrinmay; Ai, Walden; Banik, Naren L; Ray, Swapan K

    2013-02-01

    Neuroblastoma is an extracranial solid tumor that usually occurs in infants and children. Malignant neuroblastomas remain mostly refractory to currently available chemotherapeutic agents. So, new therapeutic agents and their molecular mechanisms for induction of cell death must be explored for successful treatment of human malignant neuroblastomas. Two polyphenolic compounds, which are abundant in green tea, are (-)-epigallocatechin (EGC) and (-)-epigallocatechin-3-gallate (EGCG) that possess impressive anti-cancer properties. It is not known yet whether EGC and EGCG can modulate the levels of expression of specific microRNAs (miRs) for induction of apoptosis in human malignant neuroblastomas. In this investigation, we revealed that treatment with EGC or EGCG caused induction of apoptosis with significant changes in expression of specific oncogenic miRs (OGmiRs) and tumor suppressor miRs (TSmiRs) in human malignant neuroblastoma SH-SY5Y and SK-N-DZ cell lines. Treatment of both cell lines with either 50 μM EGC or 50 μM EGCG decreased expression of the OGmiRs (miR-92, miR-93, and miR-106b) and increased expression of the TSmiRs (miR-7-1, miR-34a, and miR-99a) leading to induction of extrinsic and intrinsic pathways of apoptosis. Our data also demonstrated that overexpression of miR-93 decreased efficacy while overexpression of miR-7-1 increased efficacy of the green tea polyphenols for induction of apoptosis in both cell lines. In conclusion, our current investigation clearly indicates that overexpression of miR-7-1 can highly potentiate efficacy of EGCG for induction of apoptosis in human malignant neuroblastoma cells.

  7. Dopamine, uncertainty and TD learning

    PubMed Central

    Niv, Yael; Duff, Michael O; Dayan, Peter

    2005-01-01

    Substantial evidence suggests that the phasic activities of dopaminergic neurons in the primate midbrain represent a temporal difference (TD) error in predictions of future reward, with increases above and decreases below baseline consequent on positive and negative prediction errors, respectively. However, dopamine cells have very low baseline activity, which implies that the representation of these two sorts of error is asymmetric. We explore the implications of this seemingly innocuous asymmetry for the interpretation of dopaminergic firing patterns in experiments with probabilistic rewards which bring about persistent prediction errors. In particular, we show that when averaging the non-stationary prediction errors across trials, a ramping in the activity of the dopamine neurons should be apparent, whose magnitude is dependent on the learning rate. This exact phenomenon was observed in a recent experiment, though being interpreted there in antipodal terms as a within-trial encoding of uncertainty. PMID:15953384

  8. Dopamine in motivational control: rewarding, aversive, and alerting

    PubMed Central

    Bromberg-Martin, Ethan S.; Matsumoto, Masayuki; Hikosaka, Okihide

    2010-01-01

    SUMMARY Midbrain dopamine neurons are well known for their strong responses to rewards and their critical role in positive motivation. It has become increasingly clear, however, that dopamine neurons also transmit signals related to salient but non-rewarding experiences such as aversive and alerting events. Here we review recent advances in understanding the reward and non-reward functions of dopamine. Based on this data, we propose that dopamine neurons come in multiple types that are connected with distinct brain networks and have distinct roles in motivational control. Some dopamine neurons encode motivational value, supporting brain networks for seeking, evaluation, and value learning. Others encode motivational salience, supporting brain networks for orienting, cognition, and general motivation. Both types of dopamine neurons are augmented by an alerting signal involved in rapid detection of potentially important sensory cues. We hypothesize that these dopaminergic pathways for value, salience, and alerting cooperate to support adaptive behavior. PMID:21144997

  9. Alcohol-induced alterations in dopamine modulation of prefrontal activity.

    PubMed

    Trantham-Davidson, Heather; Chandler, L Judson

    2015-12-01

    Long-term alcohol use leads to persistent cognitive deficits that may be associated with maladaptive changes in the neurocircuitry that mediates executive functions. Impairments caused by these changes can persist well into abstinence and have a negative impact on quality of life and job performance, and can increase the probability of relapse. Many of the changes that affect cognitive function appear to involve dysregulation of the mesocortical dopamine system. This includes changes in dopamine release and alterations in dopamine receptor expression and function in the medial prefrontal cortex (PFC). This review summarizes the cellular effects of acute and chronic ethanol exposure on dopamine release and dopamine receptor function in the PFC with the goal of providing greater understanding of the effects of alcohol-use disorders on the dopamine system and how this relates to deficits in the executive function of the PFC. PMID:26558348

  10. Genome-Wide Analysis of the AP2/ERF Gene Family in Physic Nut and Overexpression of the JcERF011 Gene in Rice Increased Its Sensitivity to Salinity Stress.

    PubMed

    Tang, Yuehui; Qin, Shanshan; Guo, Yali; Chen, Yanbo; Wu, Pingzhi; Chen, Yaping; Li, Meiru; Jiang, Huawu; Wu, Guojiang

    2016-01-01

    The AP2/ERF transcription factors play crucial roles in plant growth, development and responses to biotic and abiotic stresses. A total of 119 AP2/ERF genes (JcAP2/ERFs) have been identified in the physic nut genome; they include 16 AP2, 4 RAV, 1 Soloist, and 98 ERF genes. Phylogenetic analysis indicated that physic nut AP2 genes could be divided into 3 subgroups, while ERF genes could be classed into 11 groups or 43 subgroups. The AP2/ERF genes are non-randomly distributed across the 11 linkage groups of the physic nut genome and retain many duplicates which arose from ancient duplication events. The expression patterns of several JcAP2/ERF duplicates in the physic nut showed differences among four tissues (root, stem, leaf, and seed), and 38 JcAP2/ERF genes responded to at least one abiotic stressor (drought, salinity, phosphate starvation, and nitrogen starvation) in leaves and/or roots according to analysis of digital gene expression tag data. The expression of JcERF011 was downregulated by salinity stress in physic nut roots. Overexpression of the JcERF011 gene in rice plants increased its sensitivity to salinity stress. The increased expression levels of several salt tolerance-related genes were impaired in the JcERF011-overexpressing plants under salinity stress. PMID:26943337

  11. Genome-Wide Analysis of the AP2/ERF Gene Family in Physic Nut and Overexpression of the JcERF011 Gene in Rice Increased Its Sensitivity to Salinity Stress

    PubMed Central

    Tang, Yuehui; Qin, Shanshan; Guo, Yali; Chen, Yanbo; Wu, Pingzhi; Chen, Yaping; Li, Meiru; Jiang, Huawu; Wu, Guojiang

    2016-01-01

    The AP2/ERF transcription factors play crucial roles in plant growth, development and responses to biotic and abiotic stresses. A total of 119 AP2/ERF genes (JcAP2/ERFs) have been identified in the physic nut genome; they include 16 AP2, 4 RAV, 1 Soloist, and 98 ERF genes. Phylogenetic analysis indicated that physic nut AP2 genes could be divided into 3 subgroups, while ERF genes could be classed into 11 groups or 43 subgroups. The AP2/ERF genes are non-randomly distributed across the 11 linkage groups of the physic nut genome and retain many duplicates which arose from ancient duplication events. The expression patterns of several JcAP2/ERF duplicates in the physic nut showed differences among four tissues (root, stem, leaf, and seed), and 38 JcAP2/ERF genes responded to at least one abiotic stressor (drought, salinity, phosphate starvation, and nitrogen starvation) in leaves and/or roots according to analysis of digital gene expression tag data. The expression of JcERF011 was downregulated by salinity stress in physic nut roots. Overexpression of the JcERF011 gene in rice plants increased its sensitivity to salinity stress. The increased expression levels of several salt tolerance-related genes were impaired in the JcERF011-overexpressing plants under salinity stress. PMID:26943337

  12. Overexpression of a miR393-resistant form of transport inhibitor response protein 1 (mTIR1) enhances salt tolerance by increased osmoregulation and Na+ exclusion in Arabidopsis thaliana.

    PubMed

    Chen, Zhehao; Hu, Lingzhi; Han, Ning; Hu, Jiangqin; Yang, Yanjun; Xiang, Taihe; Zhang, Xujia; Wang, Lilin

    2015-01-01

    Soil salinity is a common environmental stress factor that limits agricultural production worldwide. Plants have evolved different strategies to achieve salt tolerance. miR393 has been identified as closely related to biotic and abiotic stresses, and targets F-box genes that encode auxin receptors. The miR393-TIR1/AFB2/AFB3 regulatory module was discovered to have multiple functions that manipulate the auxin response. This study focused on miR393 and one of its targets, TIR1, and found that they played potential roles in response to salt stress. Our results showed that overexpression of a miR393-resistant TIR1 gene (mTIR1) in Arabidopsis clearly enhanced salt stress tolerance, which led to a higher germination rate, less water loss, reduced inhibition of root elongation, delayed senescence, decreased death rate and stabilized Chl content. These plants accumulated more proline and anthocyanin, and displayed enhanced osmotic stress tolerance. The expression of some salt stress-related genes was altered, and sodium content can be reduced in these plants under salt stress. We proposed that highly increased auxin signaling by overexpression of mTIR1 may trigger auxin-mediated downstream pathways to enhance plant salt stress resistance by osmoregulation and increased Na(+) exclusion.

  13. Dopamine release in rat striatum - Physiological coupling to tyrosine supply

    NASA Technical Reports Server (NTRS)

    During, Matthew J.; Acworth, Ian N.; Wurtman, Richard J.

    1989-01-01

    Intracerebral microdialysis was used to monitor dopamine release in rat striatal extracellular fluid following the intraperitoneal administration of dopamine's precursor amino acid, L-tyrosine. Dopamine concentrations in dialysates increased transiently after tyrosine (50-100 mg/kg) administration. Pretreatment with haloperidol or the partial lesioning of nigrostriatal neurons enhanced the effect of tyrosine on dopamine release, and haloperidol also prolonged this effect. These data suggest that nigrostriatal dopaminergic neurons are responsive to changes in precursor availability under basal conditions, but that receptor-mediated feedback mechanisms limit the magnitude and duration of this effect.

  14. Dopamine Gene Profiling to Predict Impulse Control and Effects of Dopamine Agonist Ropinirole.

    PubMed

    MacDonald, Hayley J; Stinear, Cathy M; Ren, April; Coxon, James P; Kao, Justin; Macdonald, Lorraine; Snow, Barry; Cramer, Steven C; Byblow, Winston D

    2016-07-01

    Dopamine agonists can impair inhibitory control and cause impulse control disorders for those with Parkinson disease (PD), although mechanistically this is not well understood. In this study, we hypothesized that the extent of such drug effects on impulse control is related to specific dopamine gene polymorphisms. This double-blind, placebo-controlled study aimed to examine the effect of single doses of 0.5 and 1.0 mg of the dopamine agonist ropinirole on impulse control in healthy adults of typical age for PD onset. Impulse control was measured by stop signal RT on a response inhibition task and by an index of impulsive decision-making on the Balloon Analogue Risk Task. A dopamine genetic risk score quantified basal dopamine neurotransmission from the influence of five genes: catechol-O-methyltransferase, dopamine transporter, and those encoding receptors D1, D2, and D3. With placebo, impulse control was better for the high versus low genetic risk score groups. Ropinirole modulated impulse control in a manner dependent on genetic risk score. For the lower score group, both doses improved response inhibition (decreased stop signal RT) whereas the lower dose reduced impulsiveness in decision-making. Conversely, the higher score group showed a trend for worsened response inhibition on the lower dose whereas both doses increased impulsiveness in decision-making. The implications of the present findings are that genotyping can be used to predict impulse control and whether it will improve or worsen with the administration of dopamine agonists. PMID:26942320

  15. A new dopamine-β-hydroxylase inhibitor

    PubMed Central

    Andén, N. -E.; Fuxe, K.

    1971-01-01

    1. The dopamine-β-hydroxylase inhibitor bis(4-methyl-1-homopiperazinyl-thiocarbonyl) disulphide (FLA-63; 25 mg/kg i.p.) caused within 4 h a 65% loss of noradrenaline throughout the intact rat spinal cord and also cranial to a transection of the cut spinal cord. Caudal to the lesion, there was only an insignificant depletion of 17% indicating the importance of nerve impulses for the disappearance of noradrenaline. 2. Dopamine accumulated in the spinal cord after treatment with FLA-63 although the amounts were not sufficient to replace the missing noradrenaline. Even after treatment with L-3,4-dihydroxyphenylalanine (L-DOPA), the catecholamine store was incompletely replenished by dopamine. 3. After a large depletion of the noradrenaline stores, induced by repeated doses of FLA-63 or by reserpine plus FLA-63, the L-DOPA-induced increase in flexor reflex activity of the hind limbs of spinal rats was inhibited much more than after pretreatment with α-methyl-tyrosine or reserpine. FLA-63 blocked the formation of noradrenaline but not of dopamine from L-DOPA. 4. The increase in flexor reflex activity induced by the noradrenaline receptor stimulating agent clonidine was not changed by FLA-63, indicating that the noradrenaline receptor sensitivity was not influenced. 5. After depletion of the noradrenaline stores, the small formation of noradrenaline from L-DOPA may be of greater functional significance for the noradrenaline receptor stimulation than the greater formation of dopamine, but the dopamine formed also has a slight action. With intact noradrenaline stores, displacement of endogenous noradrenaline by newly formed dopamine contributes, at least after monoamine oxidase inhibition, to the increase in the flexor reflex activity caused by L-DOPA. PMID:4339882

  16. Dopamine triggers heterosynaptic plasticity.

    PubMed

    Ishikawa, Masago; Otaka, Mami; Huang, Yanhua H; Neumann, Peter A; Winters, Bradley D; Grace, Anthony A; Schlüter, Oliver M; Dong, Yan

    2013-04-17

    As a classic neuromodulator, dopamine has long been thought to modulate, rather than trigger, synaptic plasticity. In contrast, our present results demonstrate that within the parallel projections of dopaminergic and GABAergic terminals from the ventral tegmental area to the nucleus accumbens core (NAcCo), action-potential-activated release of dopamine heterosynaptically triggers LTD at GABAergic synapses, which is likely mediated by activating presynaptically located dopamine D1 class receptors and expressed by inhibiting presynaptic release of GABA. Moreover, this dopamine-mediated heterosynaptic LTD is abolished after withdrawal from cocaine exposure. These results suggest that action-potential-dependent dopamine release triggers very different cellular consequences from those induced by volume release or pharmacological manipulation. Activation of the ventral tegmental area to NAcCo projections is essential for emotional and motivational responses. This dopamine-mediated LTD allows a flexible output of NAcCo neurons, whereas disruption of this LTD may contribute to the rigid emotional and motivational state observed in addicts during cocaine withdrawal.

  17. Overexpression of a Novel NAC Domain-Containing Transcription Factor Gene (AaNAC1) Enhances the Content of Artemisinin and Increases Tolerance to Drought and Botrytis cinerea in Artemisia annua.

    PubMed

    Lv, Zongyou; Wang, Shu; Zhang, Fangyuan; Chen, Lingxian; Hao, Xiaolong; Pan, Qifang; Fu, Xueqing; Li, Ling; Sun, Xiaofen; Tang, Kexuan

    2016-09-01

    The NAC (NAM, ATAF and CUC) superfamily is one of the largest plant-specific transcription factor families. NAC transcription factors always play important roles in response to various abiotic stresses. A NAC transcription factor gene AaNAC1 containing a complete open reading frame (ORF) of 864 bp was cloned from Artemisia annua. The expression of AaNAC1 could be induced by dehydration, cold, salicylic acid (SA) and methyl jasmonate (MJ), suggesting that it might be a key regulator of stress signaling pathways in A. annua. AaNAC1 was shown to be localized to the nuclei by transforming tobacco leaf epidermal cells. When AaNAC1 was overexpressed in A. annua, the content of artemisinin and dihydroartemisinic acid was increased by 79% and 150%, respectively. The expression levels of artemisinin biosynthetic pathway genes, i.e. amorpha-4,11-diene synthase (ADS), artemisinic aldehyde Δ11(13) reductase (DBR2) and aldehyde dehydrogenase 1 (ALDH1), were increased. Dual luciferase (dual-LUC) assays showed that AaNAC1 could activate the transcription of ADS in vivo. The transgenic A. annua exhibited increased tolerance to drought and resistance to Botrytis cinerea. When AaNAC1 was overexpressed in Arabidopsis, the transgenic Arabidopsis were markedly more tolerant to drought. The transgenic Arabidopsis showed increased resistance to B. cinerea. These results indicate that AaNAC1 can potentially be used in transgenic breeding for improving the content of artemisinin and drought tolerance in A. annua. PMID:27388340

  18. Absence of NMDA receptors in dopamine neurons attenuates dopamine release but not conditioned approach during Pavlovian conditioning.

    PubMed

    Parker, Jones G; Zweifel, Larry S; Clark, Jeremy J; Evans, Scott B; Phillips, Paul E M; Palmiter, Richard D

    2010-07-27

    During Pavlovian conditioning, phasic dopamine (DA) responses emerge to reward-predictive stimuli as the subject learns to anticipate reward delivery. This observation has led to the hypothesis that phasic dopamine signaling is important for learning. To assess the ability of mice to develop anticipatory behavior and to characterize the contribution of dopamine, we used a food-reinforced Pavlovian conditioning paradigm. As mice learned the cue-reward association, they increased their head entries to the food receptacle in a pattern that was consistent with conditioned anticipatory behavior. D1-receptor knockout (D1R-KO) mice had impaired acquisition, and systemic administration of a D1R antagonist blocked both the acquisition and expression of conditioned approach in wild-type mice. To assess the specific contribution of phasic dopamine transmission, we tested mice lacking NMDA-type glutamate receptors (NMDARs) exclusively in dopamine neurons (NR1-KO mice). Surprisingly, NR1-KO mice learned at the same rate as their littermate controls. To evaluate the contribution of NMDARs to phasic dopamine release in this paradigm, we performed fast-scan cyclic voltammetry in the nucleus accumbens of awake mice. Despite having significantly attenuated phasic dopamine release following reward delivery, KO mice developed cue-evoked dopamine release at the same rate as controls. We conclude that NMDARs in dopamine neurons enhance but are not critical for phasic dopamine release to behaviorally relevant stimuli; furthermore, their contribution to phasic dopamine signaling is not necessary for the development of cue-evoked dopamine or anticipatory activity in a D1R-dependent Pavlovian conditioning paradigm.

  19. Overexpression of VMAT-2 and DT-diaphorase protects substantia nigra-derived cells against aminochrome neurotoxicity

    PubMed Central

    Muñoz, Patricia; Paris, Irmgard; Sanders, Laurie H.; Greenamyre, J. Timothy; Segura-Aguilar, Juan

    2013-01-01

    We tested the hypothesis that both VMAT-2 and DT-diaphorase are an important cellular defense against aminochrome-dependent neurotoxicity during dopamine oxidation. A cell line with VMAT-2 and DT-diaphorase over-expressed was created. The transfection of RCSN-3 cells with a bicistronic plasmid coding for VMAT-2 fused with GFP-IRES-DT-diaphorase cDNA induced a significant increase in protein expression of VMAT-2 (7-fold; P<0.001) and DT-diaphorase (9-fold; P<0.001), accompanied by a 4- and 5.5-fold significant increase in transport and enzyme activity, respectively. Studies with synaptic vesicles from rat substantia nigra revealed that VMAT-2 uptake of 3H-aminochrome 6.3 ± 0.4nmol/min/mg was similar to dopamine uptake 6.2 ± 0.3 nmol/min/mg that which were dependent on ATP. Interestingly, aminochrome uptake was inhibited by 2 μM lobeline but not reserpine (1 and 10 μM). Incubation of cells overexpressing VMAT-2 and DT-diaphorase with 20 μM aminochrome resulted in (i) a significant decrease in cell death (6-fold, P<0.001); (ii) normal ultra structure determined by transmission electron microscopy contrasting with a significant increase of autophagosome and a dramatic remodeling of the mitochondrial inner membrane in wild type cells; (iii) normal level of ATP (256 ± 11 μM) contrasting with a significant decrease in wild type cells (121 ± 11 μM, P<0.001); and (iv) a significant decrease in DNA laddering (21 ± 8 pixels, P<0.001) cells in comparison with wild type cells treated with 20 μM aminochrome (269 ± 9). These results support our hypothesis that VMAT-2 and DT-diaphorase are an important defense system against aminochrome formed during dopamine oxidation. PMID:22483869

  20. Constitutive Ret activity in knock-in multiple endocrine neoplasia type B mice induces profound elevation of brain dopamine concentration via enhanced synthesis and increases the number of TH-positive cells in the substantia nigra.

    PubMed

    Mijatovic, Jelena; Airavaara, Mikko; Planken, Anu; Auvinen, Petri; Raasmaja, Atso; Piepponen, T Petteri; Costantini, Frank; Ahtee, Liisa; Saarma, Mart

    2007-05-01

    Ret is the common signaling receptor for glial cell line-derived neurotrophic factor (GDNF) and other ligands of the GDNF family that have potent effects on brain dopaminergic neurons. The Met918Thr mutation leads to constitutive activity of Ret receptor tyrosine kinase, causing the cancer syndrome called multiple endocrine neoplasia type B (MEN2B). We used knock-in MEN2B mice with the Ret-MEN2B mutation to study the effects of constitutive Ret activity on the brain dopaminergic system and found robustly increased concentrations of dopamine (DA) and its metabolites in the striatum, cortex, and hypothalamus. The concentrations of brain serotonin were not affected and those of noradrenaline were slightly increased only in the lower brainstem. Tyrosine hydroxylase (TH) protein levels were increased in the striatum and substantia nigra/ventral tegmental area (SN/VTA), and TH mRNA levels were increased in SN/VTA of MEN2B mice, suggesting that constitutive Ret activity increases DA levels by increasing its synthesis. Also, the striatal DA transporter protein levels in the MEN2B mice were increased, which agrees with increased sensitivity of these mice to the stimulatory effects of cocaine. In the SN pars compacta of homozygous MEN2B mice, we found a 26% increase in the number of TH-positive cells, but no differences were found in the VTA. Thus, we show here that the constitutive Ret activity in mice is sufficient to increase the number of dopaminergic neurons and leads to profound elevation of brain DA concentration. These data clearly suggest that Ret activity per se can have a direct biological function that actively changes and shapes the brain dopaminergic system. PMID:17475787

  1. Transgenic over-expression of slit2 enhances disruption of blood-brain barrier and increases cell death after traumatic brain injury in mice.

    PubMed

    Li, Shuai; Li, Hang; He, Xiao-Fei; Li, Ge; Zhang, Qun; Liang, Feng-Ying; Jia, Huan-Huan; Li, Jiang-Chao; Huang, Ren; Pei, Zhong; Wang, Li-Jing; Zhang, Yu

    2016-09-19

    Traumatic brain injury (TBI) is the leading cause of mortality and disability among male adolescents and young adults; and mild traumatic brain injury is the most common type of traumatic brain injury. The disruption of blood-brain barrier (BBB) plays an important role in brain trauma. Previously, we have found that slit2, a member of slit protein family, increases permeability of BBB. In the present study, we examined the role of slit2 in the pathogenesis of mild TBI in a mouse model of micro TBI. Rhodamine BandPI (PropidiumIodide) staining were used to detect the permeability of BBB and cell death, respectively. The leakage of Rhodamine B and cell death were significantly increased in Slit2-Tg mice than in C57 control mice after micro TBI. The present results suggest that over expression of slit2 plays a detrimental role in the pathophysiology of mild TBI.

  2. Increases of ferrous iron oxidation activity and arsenic stressed cell growth by overexpression of Cyc2 in Acidithiobacillus ferrooxidans ATCC19859.

    PubMed

    Liu, Wei; Lin, Jianqun; Pang, Xin; Mi, Shuang; Cui, Shuang; Lin, Jianqiang

    2013-01-01

    Acidithiobacillus ferrooxidans plays an important role in bioleaching in reproducing the mineral oxidant of ferric iron (Fe(3+) ) by oxidization of ferrous iron (Fe(2+) ). The high-molecular-weight c-type cytochrome Cyc2 that is located in the external membrane is postulated as the first electron carrier in the Fe(2+) oxidation respiratory pathway of A. ferrooxidans. To increase ferrous iron oxidation activity, a recombinant plasmid pTCYC2 containing cyc2 gene under the control of Ptac promoter was constructed and transferred into A. ferrooxidans ATCC19859. The transcriptional level of cyc2 gene was increased by 2.63-fold and Cyc2 protein expression was observed in the recombinant strain compared with the control. The ferrous iron oxidation activity and the arsenic stressed cell growth of the recombinant strain were also elevated.

  3. Overexpression of D-xylose reductase (xyl1) gene and antisense inhibition of D-xylulokinase (xyiH) gene increase xylitol production in Trichoderma reesei.

    PubMed

    Hong, Yuanyuan; Dashtban, Mehdi; Kepka, Greg; Chen, Sanfeng; Qin, Wensheng

    2014-01-01

    T. reesei is an efficient cellulase producer and biomass degrader. To improve xylitol production in Trichoderma reesei strains by genetic engineering, two approaches were used in this study. First, the presumptive D-xylulokinase gene in T. reesei (xyiH), which has high homology to known fungi D-xylulokinase genes, was silenced by transformation of T. reesei QM9414 strain with an antisense construct to create strain S6-2-2. The expression of the xyiH gene in the transformed strain S6-2-2 decreased at the mRNA level, and D-xylulokinase activity decreased after 48 h of incubation. This led to an increase in xylitol production from undetectable levels in wild-type T. reesei QM9414 to 8.6 mM in S6-2-2. The T. reesei Δxdh is a xylose dehydrogenase knockout strain with increased xylitol production compared to the wild-type T. reesei QM9414 (22.8 mM versus undetectable). The copy number of the xylose reductase gene (xyl1) in T. reesei Δxdh strain was increased by genetic engineering to create a new strain Δ9-5-1. The Δ9-5-1 strain showed a higher xyl1 expression and a higher yield of xylose reductase, and xylitol production was increased from 22.8 mM to 24.8 mM. Two novel strains S6-2-2 and Δ9-5-1 are capable of producing higher yields of xylitol. T. reesei has great potential in the industrial production of xylitol.

  4. Vascular α1D-adrenoceptors are overexpressed in aorta of the aryl hydrocarbon receptor null mouse: role of increased angiotensin II

    PubMed Central

    Vázquez-Cuevas, F. G.; López-Guerrero, J. J.; Figueroa-García, M. C.; Gallardo-Ortiz, I. A.; Ibarra, M.; Rodríguez-Sosa, M.; Gonzalez, F. J.; Elizondo, G.

    2009-01-01

    Summary The hypothesis that α1D-adrenoceptors may mediate the pro-hypertensive actions of angiotensin II (Ang II) was tested in isolated aorta (α1D-adrenoceptor bearing tissue) of the aryl hydrocarbon receptor null mouse (AhR−/−), which shows increased levels of Ang II, cardiac hypertrophy and hypertension. The effect of captopril (an angiotensin converting enzyme inhibitor) on both blood pressure and aortic α1D-adrenoceptor expression and function in mice were determined. Basal blood pressure was higher in AhR−/− mice, while captopril therapy decreased it to wild-type (WT) values. Aortas of adult WT and AhR−/− mice were stimulated by phenylephrine or noradrenaline to induce contraction; the maximal effect was higher in AhR−/− mice, without a significant change in pEC50. PA2 values for the selective α1D-adrenoceptor antagonist BMY 7378 (8-[2-[4-(2-methoxyphenyl)-1-piperazynil]ethyl]-8-azaspiro [4.5]decane-7,9-dione) were 9.19 and 8.94 for WT and AhR−/−, respectively; while Schild slopes were not different from 1. PCR experiments showed c. 77% increase in AhR−/− α1D-adrenoceptors cDNA compared with WT mice; while western blot analysis demonstrated c. 88% increase in α1D-adrenoceptor protein in AhR−/− mice. Captopril therapy decreased α1D-adrenoceptor-induced contraction and protein in AhR−/− mice to WT levels. These data support the hypothesis that under conditions where Ang II is elevated, vascular α1D-adrenoceptors are increased, and further suggest that both Ang II and vascular α1D-adrenoceptors could be related in the onset of hypertension. PMID:18598287

  5. Increased oxidative DNA damage and hepatocyte overexpression of specific cytochrome P450 isoforms in hepatitis of mice infected with Helicobacter hepaticus.

    PubMed Central

    Sipowicz, M. A.; Chomarat, P.; Diwan, B. A.; Anver, M. A.; Awasthi, Y. C.; Ward, J. M.; Rice, J. M.; Kasprzak, K. S.; Wild, C. P.; Anderson, L. M.

    1997-01-01

    A recently discovered bacterium, Helicobacter hepaticus, infects the intrahepatic bile canaliculi of mice, causing a severe chronic hepatitis culminating in liver cancer. Thus, it affords an animal model for study of bacteria-associated tumorigenesis including H. pylori-related gastric cancer. Reactive oxygen species are often postulated to contribute to this process. We now report that hepatitis of male mice infected with H. hepaticus show significant increases in the oxidatively damaged DNA deoxynucleoside 8-hydroxydeoxyguanosine, with the degree of damage increasing with progression of the disease. Perfusion of infected livers with nitro blue tetrazolium revealed that superoxide was produced in the cytoplasm of hepatocytes, especially in association with plasmacytic infiltrates near portal triads. Contrary to expectations, Kupffer cells, macrophages, and neutrophils were rarely involved. However, levels of cytochrome P450 (CYP) isoforms 1A2 and 2A5 in hepatocytes appeared to be greatly increased, as indicated by the number of cells positive in immunohistochemistry and the intensity of staining in many cells, concomitant with severe hepatitis. The CYP2A5 immunohistochemical staining co-localized with formazan deposits resulting from nitro blue tetrazolium reduction and occurred in nuclei as well as cytoplasm. These findings suggest that CYP2A5 contributes to the superoxide production and 8-hydroxydeoxyguanosine formation, although reactive oxygen species from an unknown source in the hepatocytes leading to CYP2A5 induction or coincidental occurrence of these events are also possibilities. Three glutathione S-transferase isoforms, mGSTP1-1 (pi), mGSTA1-1 (YaYa), and mGSTA4-4, also showed striking increases evidencing major oxidative stress in these livers. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:9327726

  6. Sustained N-methyl-D-aspartate receptor hypofunction remodels the dopamine system and impairs phasic signaling

    PubMed Central

    Ferris, Mark J.; Milenkovic, Marija; Liu, Shuai; Mielnik, Catharine A.; Beerepoot, Pieter; John, Carrie E.; España, Rodrigo A.; Sotnikova, Tatyana D.; Gainetdinov, Raul R.; Borgland, Stephanie L.; Jones, Sara R.; Ramsey, Amy J.

    2014-01-01

    Chronic N-methyl-D-aspartate receptor (NMDAR) hypofunction has been proposed as a contributing factor to symptoms of schizophrenia. However, it is unclear how sustained NMDAR hypofunction throughout development affects other neurotransmitter systems that have been implicated in the disease. Dopamine neuron biochemistry and activity were examined to determine whether sustained NMDAR hypofunction causes a state of hyperdopaminergia. We report that a global, genetic reduction in NMDARs led to a remodeling of dopamine neurons, substantially affecting two key regulators of dopamine homeostasis, i.e. tyrosine hydroxylase and the dopamine transporter. In NR1 knockdown mice, dopamine synthesis and release were attenuated, and dopamine clearance was increased. Although these changes would have the effect of reducing dopamine transmission, we demonstrated that a state of hyperdopaminergia existed in these mice because dopamine D2 autoreceptors were desensitized. In support of this conclusion, NR1 knockdown dopamine neurons have higher tonic firing rates. Although the tonic firing rates are higher, phasic signaling is impaired, and dopamine overflow cannot be achieved with exogenous high-frequency stimulation that models phasic firing. Through the examination of several parameters of dopamine neurotransmission, we provide evidence that chronic NMDAR hypofunction leads to a state of elevated synaptic dopamine. Compensatory mechanisms to attenuate hyperdopaminergia also impact the ability to generate dopamine surges through phasic firing. PMID:24754704

  7. Intrarenal dopamine inhibits progression of diabetic nephropathy.

    PubMed

    Zhang, Ming-Zhi; Yao, Bing; Yang, Shilin; Yang, Haichun; Wang, Suwan; Fan, Xiaofeng; Yin, Huiyong; Fogo, Agnes B; Moeckel, Gilbert W; Harris, Raymond C

    2012-10-01

    The kidney has a local intrarenal dopaminergic system, and in the kidney, dopamine modulates renal hemodynamics, inhibits salt and fluid reabsorption, antagonizes the renin-angiotensin system, and inhibits oxidative stress. The current study examined the effects of alterations in the intrarenal dopaminergic system on kidney structure and function in models of type 1 diabetes. We studied catechol-O-methyl-transferase (COMT)(-/-) mice, which have increased renal dopamine production due to decreased dopamine metabolism, and renal transplantation was used to determine whether the effects seen with COMT deficiency were kidney-specific. To determine the effects of selective inhibition of intrarenal dopamine production, we used mice with proximal tubule deletion of aromatic amino acid decarboxylase (ptAADC(-/-)). Compared with wild-type diabetic mice, COMT(-/-) mice had decreased hyperfiltration, decreased macula densa cyclooxygenase-2 expression, decreased albuminuria, decreased glomerulopathy, and inhibition of expression of markers of inflammation, oxidative stress, and fibrosis. These differences were also seen in diabetic mice with a transplanted kidney from COMT(-/-) mice. In contrast, diabetic ptAADC(-/-) mice had increased nephropathy. Our study demonstrates an important role of the intrarenal dopaminergic system to modulate the development and progression of diabetic kidney injury and indicate that the decreased renal dopamine production may have important consequences in the underlying pathogenesis of diabetic nephropathy. PMID:22688335

  8. Over-expression of JcDGAT1 from Jatropha curcas increases seed oil levels and alters oil quality in transgenic Arabidopsis thaliana.

    PubMed

    Misra, Aparna; Khan, Kasim; Niranjan, Abhishek; Nath, Pravendra; Sane, Vidhu A

    2013-12-01

    The increasing consumption of fossil fuels and petroleum products is leading to their rapid depletion and is a matter of concern around the globe. Substitutes of fossil fuels are required to sustain the pace of economic development. In this context, oil from the non food crops (biofuel) has shown potential to substitute fossil fuels. Jatropha curcas is an excellent shrub spread and naturalized across the globe. Its oil contains a high percentage of unsaturated fatty acids (about 78-84% of total fatty acid content) making the oil suitable for biodiesel production. Despite its high oil content, it has been poorly studied in terms of important enzymes/genes responsible for oil biosynthesis. Here, we describe the isolation of the full length cDNA clone of JcDGAT1, a key enzyme involved in oil biosynthesis, from J. curcas seeds and manipulation of oil content and composition in transgenic Arabidopsis plants by its expression. Transcript analysis of JcDGAT1 reveals a gradual increase from early seed development to its maturation. Homozygous transgenic Arabidopsis lines expressing JcDGAT1 both under CaMV35S promoter and a seed specific promoter show an enhanced level of total oil content (up by 30-41%) in seeds but do not show any phenotypic differences. In addition, our studies also show alterations in the oil composition through JcDGAT1 expression. While the levels of saturated FAs such as palmitate and stearate in the oil do not change, there is significant reproducible decrease in the levels of oleic acid and a concomitant increase in levels of linolenic acid both under the CaMV35S promoter as well as the seed specific promoter. Our studies thus confirm that DGAT is involved in flux control in oil biosynthesis and show that JcDGAT1 could be used specifically to manipulate and improve oil content and composition in plants. PMID:24125179

  9. Overexpression of the rat inducible 70-kD heat stress protein in a transgenic mouse increases the resistance of the heart to ischemic injury.

    PubMed Central

    Marber, M S; Mestril, R; Chi, S H; Sayen, M R; Yellon, D M; Dillmann, W H

    1995-01-01

    Myocardial protection and changes in gene expression follow whole body heat stress. Circumstantial evidence suggests that an inducible 70-kD heat shock protein (hsp70i), increased markedly by whole body heat stress, contributes to the protection. Transgenic mouse lines were constructed with a cytomegalovirus enhancer and beta-actin promoter driving rat hsp70i expression in heterozygote animals. Unstressed, transgene positive mice expressed higher levels of myocardial hsp70i than transgene negative mice after whole body heat stress. This high level of expression occurred without apparent detrimental effect. The hearts harvested from transgene positive mice and transgene negative littermates were Langendorff perfused and subjected to 20 min of warm (37 degrees C) zero-flow ischemia and up to 120 min of reflow while contractile recovery and creatine kinase efflux were measured. Myocardial infarction was demarcated by triphenyltetrazolium. In transgene positive compared with transgene negative hearts, the zone of infarction was reduced by 40%, contractile function at 30 min of reflow was doubled, and efflux of creatine kinase was reduced by approximately 50%. Our findings suggest for the first time that increased myocardial hsp70i expression results in protection of the heart against ischemic injury and that the antiischemic properties of hsp70i have possible therapeutic relevance. Images PMID:7706448

  10. Electroanalysis of dopamine at a gold electrode modified with N-acetylcysteine self-assembled monolayer.

    PubMed

    Liu, Ting; Li, Meixian; Li, Qianyuan

    2004-07-01

    Voltammetric behavior of dopamine (DA) on a gold electrode modified with the self-assembled monolayer (SAM) of N-acetylcysteine has been investigated, and one pair of well-defined redox peaks of dopamine is obtained at the SAM modified gold electrode. The oxidation peak current increases linearly with the concentration of dopamine in the range of 1.0x10 (-6)to 2.0x10 (-4)moll(-1). The detection limit is 8.0x10(-7)moll(-1). This method will be applicable to the determination of dopamine in injection of dopamine hydrochloride, and the good recovery of dopamine is obtained. Furthermore, The SAM modified gold electrode can resolve well the voltammetric responses of dopamine and ascorbic acid (AA), so it can also be applied to the determination of dopamine in the presence of ascorbic acid.

  11. Resolving the Role of Plant NAD-Glutamate Dehydrogenase: III. Overexpressing Individually or Simultaneously the Two Enzyme Subunits Under Salt Stress Induces Changes in the Leaf Metabolic Profile and Increases Plant Biomass Production.

    PubMed

    Tercé-Laforgue, Thérèse; Clément, Gilles; Marchi, Laura; Restivo, Francesco M; Lea, Peter J; Hirel, Bertrand

    2015-10-01

    NAD-dependent glutamate dehydrogenase (NAD-GDH) of higher plants has a central position at the interface between carbon and nitrogen metabolism due to its ability to carry out the deamination of glutamate. In order to obtain a better understanding of the physiological function of NAD-GDH under salt stress conditions, transgenic tobacco (Nicotiana tabacum L.) plants that overexpress two genes from Nicotiana plumbaginifolia individually (GDHA and GDHB) or simultaneously (GDHA/B) were grown in the presence of 50 mM NaCl. In the different GDH overexpressors, the NaCl treatment induced an additional increase in GDH enzyme activity, indicating that a post-transcriptional mechanism regulates the final enzyme activity under salt stress conditions. A greater shoot and root biomass production was observed in the three types of GDH overexpressors following growth in 50 mM NaCl, when compared with the untransformed plants subjected to the same salinity stress. Changes in metabolites representative of the plant carbon and nitrogen status were also observed. They were mainly characterized by an increased amount of starch present in the leaves of the GDH overexpressors as compared with the wild type when plants were grown in 50 mM NaCl. Metabolomic analysis revealed that overexpressing the two genes GDHA and GDHB, individually or simultaneously, induced a differential accumulation of several carbon- and nitrogen-containing molecules involved in a variety of metabolic, developmental and stress-responsive processes. An accumulation of digalactosylglycerol, erythronate and porphyrin was found in the GDHA, GDHB and GDHA/B overexpressors, suggesting that these molecules could contribute to the improved performance of the transgenic plants under salinity stress conditions.

  12. Resolving the Role of Plant NAD-Glutamate Dehydrogenase: III. Overexpressing Individually or Simultaneously the Two Enzyme Subunits Under Salt Stress Induces Changes in the Leaf Metabolic Profile and Increases Plant Biomass Production.

    PubMed

    Tercé-Laforgue, Thérèse; Clément, Gilles; Marchi, Laura; Restivo, Francesco M; Lea, Peter J; Hirel, Bertrand

    2015-10-01

    NAD-dependent glutamate dehydrogenase (NAD-GDH) of higher plants has a central position at the interface between carbon and nitrogen metabolism due to its ability to carry out the deamination of glutamate. In order to obtain a better understanding of the physiological function of NAD-GDH under salt stress conditions, transgenic tobacco (Nicotiana tabacum L.) plants that overexpress two genes from Nicotiana plumbaginifolia individually (GDHA and GDHB) or simultaneously (GDHA/B) were grown in the presence of 50 mM NaCl. In the different GDH overexpressors, the NaCl treatment induced an additional increase in GDH enzyme activity, indicating that a post-transcriptional mechanism regulates the final enzyme activity under salt stress conditions. A greater shoot and root biomass production was observed in the three types of GDH overexpressors following growth in 50 mM NaCl, when compared with the untransformed plants subjected to the same salinity stress. Changes in metabolites representative of the plant carbon and nitrogen status were also observed. They were mainly characterized by an increased amount of starch present in the leaves of the GDH overexpressors as compared with the wild type when plants were grown in 50 mM NaCl. Metabolomic analysis revealed that overexpressing the two genes GDHA and GDHB, individually or simultaneously, induced a differential accumulation of several carbon- and nitrogen-containing molecules involved in a variety of metabolic, developmental and stress-responsive processes. An accumulation of digalactosylglycerol, erythronate and porphyrin was found in the GDHA, GDHB and GDHA/B overexpressors, suggesting that these molecules could contribute to the improved performance of the transgenic plants under salinity stress conditions. PMID:26251210

  13. Differential influence of dopamine transport rate on the potencies of cocaine, amphetamine, and methylphenidate.

    PubMed

    Calipari, Erin S; Ferris, Mark J; Siciliano, Cody A; Jones, Sara R

    2015-01-21

    Dopamine transporter (DAT) levels vary across brain regions and individuals, and are altered by drug history and disease states; however, the impact of altered DAT expression on psychostimulant effects in brain has not been systematically explored. Using fast scan cyclic voltammetry, we measured the effects of elevated DAT levels on presynaptic dopamine parameters as well as the uptake inhibition potency of the blockers cocaine and methylphenidate (MPH) and the releaser amphetamine (AMPH) in the nucleus accumbens core. Here we found that increases in DAT levels, resulting from either genetic overexpression or MPH self-administration, caused markedly increased maximal rates of uptake (Vmax) that were positively correlated with the uptake inhibition potency of AMPH and MPH, but not cocaine. AMPH and MPH were particularly sensitive to DAT changes, with a 100% increase in Vmax resulting in a 200% increase in potency. The relationship between Vmax and MPH potency was the same as that for AMPH, but was different from that for cocaine, indicating that MPH more closely resembles a releaser with regard to uptake inhibition. Conversely, the effects of MPH on stimulated dopamine release were similar to those of cocaine, with inverted U-shaped increases in release over a concentration-response curve. This was strikingly different from the release profile of AMPH, which showed only reductions at high concentrations, indicating that MPH is not a pure releaser. These data indicate that although MPH is a DAT blocker, its uptake-inhibitory actions are affected by DAT changes in a similar manner to releasers. Together, these data show that fluctuations in DAT levels alter the potency of releasers and MPH but not blockers and suggest an integral role of the DAT in the addictive potential of AMPH and related compounds. PMID:25474655

  14. Differential Influence of Dopamine Transport Rate on the Potencies of Cocaine, Amphetamine, and Methylphenidate

    PubMed Central

    2015-01-01

    Dopamine transporter (DAT) levels vary across brain regions and individuals, and are altered by drug history and disease states; however, the impact of altered DAT expression on psychostimulant effects in brain has not been systematically explored. Using fast scan cyclic voltammetry, we measured the effects of elevated DAT levels on presynaptic dopamine parameters as well as the uptake inhibition potency of the blockers cocaine and methylphenidate (MPH) and the releaser amphetamine (AMPH) in the nucleus accumbens core. Here we found that increases in DAT levels, resulting from either genetic overexpression or MPH self-administration, caused markedly increased maximal rates of uptake (Vmax) that were positively correlated with the uptake inhibition potency of AMPH and MPH, but not cocaine. AMPH and MPH were particularly sensitive to DAT changes, with a 100% increase in Vmax resulting in a 200% increase in potency. The relationship between Vmax and MPH potency was the same as that for AMPH, but was different from that for cocaine, indicating that MPH more closely resembles a releaser with regard to uptake inhibition. Conversely, the effects of MPH on stimulated dopamine release were similar to those of cocaine, with inverted U-shaped increases in release over a concentration–response curve. This was strikingly different from the release profile of AMPH, which showed only reductions at high concentrations, indicating that MPH is not a pure releaser. These data indicate that although MPH is a DAT blocker, its uptake-inhibitory actions are affected by DAT changes in a similar manner to releasers. Together, these data show that fluctuations in DAT levels alter the potency of releasers and MPH but not blockers and suggest an integral role of the DAT in the addictive potential of AMPH and related compounds. PMID:25474655

  15. Increased furfural tolerance due to overexpression of NADH-dependent oxidoreductase FucO in Escherichia coli strains engineered for the production of ethanol and lactate.

    PubMed

    Wang, X; Miller, E N; Yomano, L P; Zhang, X; Shanmugam, K T; Ingram, L O

    2011-08-01

    Furfural is an important fermentation inhibitor in hemicellulose sugar syrups derived from woody biomass. The metabolism of furfural by NADPH-dependent oxidoreductases, such as YqhD (low K(m) for NADPH), is proposed to inhibit the growth and fermentation of xylose in Escherichia coli by competing with biosynthesis for NADPH. The discovery that the NADH-dependent propanediol oxidoreductase (FucO) can reduce furfural provided a new approach to improve furfural tolerance. Strains that produced ethanol or lactate efficiently as primary products from xylose were developed. These strains included chromosomal mutations in yqhD expression that permitted the fermentation of xylose broths containing up to 10 mM furfural. Expression of fucO from plasmids was shown to increase furfural tolerance by 50% and to permit the fermentation of 15 mM furfural. Product yields with 15 mM furfural were equivalent to those of control strains without added furfural (85% to 90% of the theoretical maximum). These two defined genetic traits can be readily transferred to enteric biocatalysts designed to produce other products. A similar strategy that minimizes the depletion of NADPH pools by native detoxification enzymes may be generally useful for other inhibitory compounds in lignocellulosic sugar streams and with other organisms.

  16. Strong increase of foliar inulin occurs in transgenic lettuce plants (Lactuca sativa L.) overexpressing the Asparagine Synthetase A gene from Escherichia coli.

    PubMed

    Sobolev, Anatoli P; Segre, Anna L; Giannino, Donato; Mariotti, Domenico; Nicolodi, Chiara; Brosio, Elvino; Amato, Maria E

    2007-12-26

    Transgenic lettuce (Lactuca sativa L. cv. 'Cortina') lines expressing the asparagine synthetase A gene from Escherichia coli were produced to alter the plant nitrogen status and eventually enhance growth. The relative molecular abundance of water-soluble metabolites was measured by 1H NMR in transgenic and conventional plants at early developmental stages and grown under the same conditions. NMR metabolic profiles assessed that a transgenic line and the wild-type counterpart shared the same compounds, but it also revealed side effects on the carbon metabolism following genetic modification. Concerning the nitrogen status, the amino acid content did not vary significantly, except for glutamic acid and gamma-aminobutyric acid, which diminished in the transgenics. As for the carbon metabolism, in transgenic leaves the contents of sucrose, glucose, and fructose decreased, whereas that of inulin increased up to 30 times, accompanied by the alteration of most Krebs's cycle organic acids and the rise of tartaric acid compared to nontransformed controls. Lettuce leaf inulins consisted of short oligomeric chains made of one glucose unit bound to two/four fructose units. Inulins are beneficial for human health, and they are extracted from plants and commercialized as long-chain types, whereas the short forms are synthesized chemically. Hence, lettuce genotypes with high content of foliar short-chain inulin represent useful materials for breeding strategies and a potential source for low molecular weight inulin.

  17. Increased furfural tolerance due to overexpression of NADH-dependent oxidoreductase FucO in Escherichia coli strains engineered for the production of ethanol and lactate.

    PubMed

    Wang, X; Miller, E N; Yomano, L P; Zhang, X; Shanmugam, K T; Ingram, L O

    2011-08-01

    Furfural is an important fermentation inhibitor in hemicellulose sugar syrups derived from woody biomass. The metabolism of furfural by NADPH-dependent oxidoreductases, such as YqhD (low K(m) for NADPH), is proposed to inhibit the growth and fermentation of xylose in Escherichia coli by competing with biosynthesis for NADPH. The discovery that the NADH-dependent propanediol oxidoreductase (FucO) can reduce furfural provided a new approach to improve furfural tolerance. Strains that produced ethanol or lactate efficiently as primary products from xylose were developed. These strains included chromosomal mutations in yqhD expression that permitted the fermentation of xylose broths containing up to 10 mM furfural. Expression of fucO from plasmids was shown to increase furfural tolerance by 50% and to permit the fermentation of 15 mM furfural. Product yields with 15 mM furfural were equivalent to those of control strains without added furfural (85% to 90% of the theoretical maximum). These two defined genetic traits can be readily transferred to enteric biocatalysts designed to produce other products. A similar strategy that minimizes the depletion of NADPH pools by native detoxification enzymes may be generally useful for other inhibitory compounds in lignocellulosic sugar streams and with other organisms. PMID:21685167

  18. Control of lactotrop proliferation by dopamine: Essential role of signaling through D2 receptors and ERKs

    PubMed Central

    Iaccarino, Ciro; Samad, Tarek A.; Mathis, Carole; Kercret, Henri; Picetti, Roberto; Borrelli, Emiliana

    2002-01-01

    Dopamine is thought to exert a negative control on lactotrop cell proliferation and prolactin production. Indeed, mice lacking the D2 receptor develop pituitary tumors of lactotrop origin. Because lactotrops express two isoforms of D2R, D2L, and D2S, in a specific ratio, we decided to explore the physiological importance of their relative abundance in vivo. Thus, we generated transgenic animals overexpressing either D2L or D2S in lactotrops. Increased expression of D2S, but not of D2L, leads to mitogen-activated protein kinase (MAPK) induction, which results in pituitary hypoplasia. On the other hand, levels of phosphorylated MAPKs are drastically reduced in pituitary tumors generated by the absence of D2-dependent signaling. These results underline a critical role of D2-mediated MAPK activation in lactotrop proliferation. Furthermore, whereas D2S overexpression results to a drastic reduction of prolactin, D2L overexpression elevates it. Our findings underscore a different role of the two D2R isoforms in the pituitary gland physiology. PMID:12391292

  19. MPA-capped CdTe quantum dots exposure causes neurotoxic effects in nematode Caenorhabditis elegans by affecting the transporters and receptors of glutamate, serotonin and dopamine at the genetic level, or by increasing ROS, or both

    NASA Astrophysics Data System (ADS)

    Wu, Tianshu; He, Keyu; Zhan, Qinglin; Ang, Shengjun; Ying, Jiali; Zhang, Shihan; Zhang, Ting; Xue, Yuying; Tang, Meng

    2015-12-01

    As quantum dots (QDs) are widely used in biomedical applications, the number of studies focusing on their biological properties is increasing. While several studies have attempted to evaluate the toxicity of QDs towards neural cells, the in vivo toxic effects on the nervous system and the molecular mechanisms are unclear. The aim of the present study was to investigate the neurotoxic effects and the underlying mechanisms of water-soluble cadmium telluride (CdTe) QDs capped with 3-mercaptopropionic acid (MPA) in Caenorhabditis elegans (C. elegans). Our results showed that exposure to MPA-capped CdTe QDs induced behavioral defects, including alterations to body bending, head thrashing, pharyngeal pumping and defecation intervals, as well as impaired learning and memory behavior plasticity, based on chemotaxis or thermotaxis, in a dose-, time- and size-dependent manner. Further investigations suggested that MPA-capped CdTe QDs exposure inhibited the transporters and receptors of glutamate, serotonin and dopamine in C. elegans at the genetic level within 24 h, while opposite results were observed after 72 h. Additionally, excessive reactive oxygen species (ROS) generation was observed in the CdTe QD-treated worms, which confirmed the common nanotoxicity mechanism of oxidative stress damage, and might overcome the increased gene expression of neurotransmitter transporters and receptors in C. elegans induced by long-term QD exposure, resulting in more severe behavioral impairments.

  20. MPA-capped CdTe quantum dots exposure causes neurotoxic effects in nematode Caenorhabditis elegans by affecting the transporters and receptors of glutamate, serotonin and dopamine at the genetic level, or by increasing ROS, or both.

    PubMed

    Wu, Tianshu; He, Keyu; Zhan, Qinglin; Ang, Shengjun; Ying, Jiali; Zhang, Shihan; Zhang, Ting; Xue, Yuying; Tang, Meng

    2015-12-28

    As quantum dots (QDs) are widely used in biomedical applications, the number of studies focusing on their biological properties is increasing. While several studies have attempted to evaluate the toxicity of QDs towards neural cells, the in vivo toxic effects on the nervous system and the molecular mechanisms are unclear. The aim of the present study was to investigate the neurotoxic effects and the underlying mechanisms of water-soluble cadmium telluride (CdTe) QDs capped with 3-mercaptopropionic acid (MPA) in Caenorhabditis elegans (C. elegans). Our results showed that exposure to MPA-capped CdTe QDs induced behavioral defects, including alterations to body bending, head thrashing, pharyngeal pumping and defecation intervals, as well as impaired learning and memory behavior plasticity, based on chemotaxis or thermotaxis, in a dose-, time- and size-dependent manner. Further investigations suggested that MPA-capped CdTe QDs exposure inhibited the transporters and receptors of glutamate, serotonin and dopamine in C. elegans at the genetic level within 24 h, while opposite results were observed after 72 h. Additionally, excessive reactive oxygen species (ROS) generation was observed in the CdTe QD-treated worms, which confirmed the common nanotoxicity mechanism of oxidative stress damage, and might overcome the increased gene expression of neurotransmitter transporters and receptors in C. elegans induced by long-term QD exposure, resulting in more severe behavioral impairments.

  1. Putting desire on a budget: dopamine and energy expenditure, reconciling reward and resources

    PubMed Central

    Beeler, Jeff A.; Frazier, Cristianne R. M.; Zhuang, Xiaoxi

    2012-01-01

    Accumulating evidence indicates integration of dopamine function with metabolic signals, highlighting a potential role for dopamine in energy balance, frequently construed as modulating reward in response to homeostatic state. Though its precise role remains controversial, the reward perspective of dopamine has dominated investigation of motivational disorders, including obesity. In the hypothesis outlined here, we suggest instead that the primary role of dopamine in behavior is to modulate activity to adapt behavioral energy expenditure to the prevailing environmental energy conditions, with the role of dopamine in reward and motivated behaviors derived from its primary role in energy balance. Dopamine has long been known to modulate activity, exemplified by psychostimulants that act via dopamine. More recently, there has been nascent investigation into the role of dopamine in modulating voluntary activity, with some investigators suggesting that dopamine may serve as a final common pathway that couples energy sensing to regulated voluntary energy expenditure. We suggest that interposed between input from both the internal and external world, dopamine modulates behavioral energy expenditure along two axes: a conserve-expend axis that regulates generalized activity and an explore-exploit axes that regulates the degree to which reward value biases the distribution of activity. In this view, increased dopamine does not promote consumption of tasty food. Instead increased dopamine promotes energy expenditure and exploration while decreased dopamine favors energy conservation and exploitation. This hypothesis provides a mechanistic interpretation to an apparent paradox: the well-established role of dopamine in food seeking and the findings that low dopaminergic functions are associated with obesity. Our hypothesis provides an alternative perspective on the role of dopamine in obesity and reinterprets the “reward deficiency hypothesis” as a perceived energy deficit

  2. Syntaxin 1A interaction with the dopamine transporter promotes amphetamine-induced dopamine efflux.

    PubMed

    Binda, Francesca; Dipace, Concetta; Bowton, Erica; Robertson, Sabrina D; Lute, Brandon J; Fog, Jacob U; Zhang, Minjia; Sen, Namita; Colbran, Roger J; Gnegy, Margaret E; Gether, Ulrik; Javitch, Jonathan A; Erreger, Kevin; Galli, Aurelio

    2008-10-01

    The soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein syntaxin 1A (SYN1A) interacts with and regulates the function of transmembrane proteins, including ion channels and neurotransmitter transporters. Here, we define the first 33 amino acids of the N terminus of the dopamine (DA) transporter (DAT) as the site of direct interaction with SYN1A. Amphetamine (AMPH) increases the association of SYN1A with human DAT (hDAT) in a heterologous expression system (hDAT cells) and with native DAT in murine striatal synaptosomes. Immunoprecipitation of DAT from the biotinylated fraction shows that the AMPH-induced increase in DAT/SYN1A association occurs at the plasma membrane. In a superfusion assay of DA efflux, cells overexpressing SYN1A exhibited significantly greater AMPH-induced DA release with respect to control cells. By combining the patch-clamp technique with amperometry, we measured DA release under voltage clamp. At -60 mV, a physiological resting potential, AMPH did not induce DA efflux in hDAT cells and DA neurons. In contrast, perfusion of exogenous SYN1A (3 microM) into the cell with the whole-cell pipette enabled AMPH-induced DA efflux at -60 mV in both hDAT cells and DA neurons. It has been shown recently that Ca2+/calmodulin-dependent protein kinase II (CaMKII) is activated by AMPH and regulates AMPH-induced DA efflux. Here, we show that AMPH-induced association between DAT and SYN1A requires CaMKII activity and that inhibition of CaMKII blocks the ability of exogenous SYN1A to promote DA efflux. These data suggest that AMPH activation of CaMKII supports DAT/SYN1A association, resulting in a mode of DAT capable of DA efflux.

  3. Glucocerebrosidase gene therapy prevents α-synucleinopathy of midbrain dopamine neurons.

    PubMed

    Rocha, Emily M; Smith, Gaynor A; Park, Eric; Cao, Hongmei; Brown, Eilish; Hayes, Melissa A; Beagan, Jonathan; McLean, Jesse R; Izen, Sarah C; Perez-Torres, Eduardo; Hallett, Penelope J; Isacson, Ole

    2015-10-01

    Diminished lysosomal function can lead to abnormal cellular accumulation of specific proteins, including α-synuclein, contributing to disease pathogenesis of vulnerable neurons in Parkinson's disease (PD) and related α-synucleinopathies. GBA1 encodes for the lysosomal hydrolase glucocerebrosidase (GCase), and mutations in GBA1 are a prominent genetic risk factor for PD. Previous studies showed that in sporadic PD, and in normal aging, GCase brain activity is reduced and levels of corresponding glycolipid substrates are increased. The present study tested whether increasing GCase through AAV-GBA1 intra-cerebral gene delivery in two PD rodent models would reduce the accumulation of α-synuclein and protect midbrain dopamine neurons from α-synuclein-mediated neuronal damage. In the first model, transgenic mice overexpressing wildtype α-synuclein throughout the brain (ASO mice) were used, and in the second model, a rat model of selective dopamine neuron degeneration was induced by AAV-A53T mutant α-synuclein. In ASO mice, intra-cerebral AAV-GBA1 injections into several brain regions increased GCase activity and reduced the accumulation of α-synuclein in the substantia nigra and striatum. In rats, co-injection of AAV-GBA1 with AAV-A53T α-synuclein into the substantia nigra prevented α-synuclein-mediated degeneration of nigrostriatal dopamine neurons by 6 months. These neuroprotective effects were associated with altered protein expression of markers of autophagy. These experiments demonstrate, for the first time, the neuroprotective effects of increasing GCase against dopaminergic neuron degeneration, and support the development of therapeutics targeting GCase or other lysosomal genes to improve neuronal handling of α-synuclein.

  4. Recovery of dopamine transporters with methamphetamine detoxification is not linked to changes in dopamine release

    DOE PAGESBeta

    Volkow, Nora D.; Wang, Gene-Jack; Smith, Lisa; Fowler, Joanna S.; Telang, Frank; Logan, Jean; Tomasi, Dardo

    2015-07-21

    Metamphetamine’s widepread abuse and concerns that it may increase Parkinson’s disease led us to assess if the reported loss of dopamine transporters (DAT) in methamphetamine abusers (MA) reflected damage to dopamine neurons. Using PET with [11C]cocaine to measure DAT, and with [11C]raclopride to measure dopamine release (assessed as changes in specific binding of [11C]raclopride between placebo and methylphenidate), which was used as marker of dopamine neuronal function, we show that MA (n=16), tested during early detoxification, had lower DAT (20-30%) but overall normal DA release in striatum (except for a small decrease in left putamen), when compared to controls (n=15).more » In controls, DAT were positively correlated with DA release (higher DAT associated with larger DA increases), consistent with DAT serving as markers of DA terminals. In contrast, MA showed a trend for a negative correlation (p=0.07) (higher DAT associated with lower DA increases), consistent with reduced DA re-uptake following DAT downregulation. MA who remained abstinent nine-months later (n=9) showed significant increases in DAT (20%) but methylphenidate-induced dopamine increases did not change. In contrast, in controls, DAT did not change when retested 9 months later but methylphenidate-induced dopamine increases in ventral striatum were reduced (p=0.05). Baseline D2/D3 receptors in caudate were lower in MA than in controls and did not change with detoxification, nor did they change in the controls upon retest. The loss of DAT in the MA, which was not associated with a concomitant reduction in dopamine release as would have been expected if DAT loss reflected DA terminal degneration; as well as the recovery of DAT after protracted detoxification, which was not associated with increased dopamine release as would have been expected if DAT increases reflected terminal regeneration, indicate that the loss of DAT in these MA does not reflect degeneration of dopamine terminals.« less

  5. Recovery of dopamine transporters with methamphetamine detoxification is not linked to changes in dopamine release

    SciTech Connect

    Volkow, Nora D.; Wang, Gene-Jack; Smith, Lisa; Fowler, Joanna S.; Telang, Frank; Logan, Jean; Tomasi, Dardo

    2015-07-21

    Metamphetamine’s widepread abuse and concerns that it may increase Parkinson’s disease led us to assess if the reported loss of dopamine transporters (DAT) in methamphetamine abusers (MA) reflected damage to dopamine neurons. Using PET with [11C]cocaine to measure DAT, and with [11C]raclopride to measure dopamine release (assessed as changes in specific binding of [11C]raclopride between placebo and methylphenidate), which was used as marker of dopamine neuronal function, we show that MA (n=16), tested during early detoxification, had lower DAT (20-30%) but overall normal DA release in striatum (except for a small decrease in left putamen), when compared to controls (n=15). In controls, DAT were positively correlated with DA release (higher DAT associated with larger DA increases), consistent with DAT serving as markers of DA terminals. In contrast, MA showed a trend for a negative correlation (p=0.07) (higher DAT associated with lower DA increases), consistent with reduced DA re-uptake following DAT downregulation. MA who remained abstinent nine-months later (n=9) showed significant increases in DAT (20%) but methylphenidate-induced dopamine increases did not change. In contrast, in controls, DAT did not change when retested 9 months later but methylphenidate-induced dopamine increases in ventral striatum were reduced (p=0.05). Baseline D2/D3 receptors in caudate were lower in MA than in controls and did not change with detoxification, nor did they change in the controls upon retest. The loss of DAT in the MA, which was not associated with a concomitant reduction in dopamine release as would have been expected if DAT loss reflected DA terminal degneration; as well as the recovery of DAT after protracted detoxification, which was not associated with increased dopamine release as would have been expected if DAT increases reflected terminal regeneration, indicate that the loss of DAT in these MA does not reflect degeneration of

  6. Dopamine encoding of Pavlovian incentive stimuli diminishes with extended training.

    PubMed

    Clark, Jeremy J; Collins, Anne L; Sanford, Christina Akers; Phillips, Paul E M

    2013-02-20

    Dopamine is highly implicated both as a teaching signal in reinforcement learning and in motivating actions to obtain rewards. However, theoretical disconnects remain between the temporal encoding properties of dopamine neurons and the behavioral consequences of its release. Here, we demonstrate in rats that dopamine evoked by Pavlovian cues increases during acquisition, but dissociates from stable conditioned appetitive behavior as this signal returns to preconditioning levels with extended training. Experimental manipulation of the statistical parameters of the behavioral paradigm revealed that this attenuation of cue-evoked dopamine release during the postasymptotic period was attributable to acquired knowledge of the temporal structure of the task. In parallel, conditioned behavior became less dopamine dependent after extended training. Thus, the current work demonstrates that as the presentation of reward-predictive stimuli becomes anticipated through the acquisition of task information, there is a shift in the neurobiological substrates that mediate the motivational properties of these incentive stimuli. PMID:23426680

  7. A causal link between prediction errors, dopamine neurons and learning.

    PubMed

    Steinberg, Elizabeth E; Keiflin, Ronald; Boivin, Josiah R; Witten, Ilana B; Deisseroth, Karl; Janak, Patricia H

    2013-07-01

    Situations in which rewards are unexpectedly obtained or withheld represent opportunities for new learning. Often, this learning includes identifying cues that predict reward availability. Unexpected rewards strongly activate midbrain dopamine neurons. This phasic signal is proposed to support learning about antecedent cues by signaling discrepancies between actual and expected outcomes, termed a reward prediction error. However, it is unknown whether dopamine neuron prediction error signaling and cue-reward learning are causally linked. To test this hypothesis, we manipulated dopamine neuron activity in rats in two behavioral procedures, associative blocking and extinction, that illustrate the essential function of prediction errors in learning. We observed that optogenetic activation of dopamine neurons concurrent with reward delivery, mimicking a prediction error, was sufficient to cause long-lasting increases in cue-elicited reward-seeking behavior. Our findings establish a causal role for temporally precise dopamine neuron signaling in cue-reward learning, bridging a critical gap between experimental evidence and influential theoretical frameworks.

  8. Decreased brain dopamine cell numbers in human cocaine users.

    PubMed

    Little, Karley Y; Ramssen, Eric; Welchko, Ryan; Volberg, Vitaly; Roland, Courtney J; Cassin, Bader

    2009-08-15

    Cocaine use diminishes striatal and midbrain dopamine neuronal components in both post-mortem and in vivo human experiments. The diffuse nature of these declines suggests the possibility that cocaine use might cause a loss of dopamine neurons in humans. Previous rodent studies have not detected cocaine-induced dopamine cell damage. The present experiment involved counting midbrain dopamine neurons utilizing both melanin and tyrosine hydroxylase immunoreactivity. Well-preserved blocks ranging from +38 mm obex to +45 mm obex were examined in 10 cocaine users and 9 controls. Sections were also examined for signs of acute pathological injury by counting activated macrophages and microglia. Melanized cells at six midbrain levels were significantly reduced in cocaine users by both drug exposures. The estimated total number of melanized dopamine cells in the anterior midbrain was significantly reduced in cocaine users by 16%. Results with tyrosine hydroxylase immunoreactivity were less conclusive because of variability in staining. Both activated macrophages and activated microglia were significantly increased among cocaine users. Cocaine exposure may have neurotoxic effects on dopamine neurons in humans. The infiltration of phagocytic cells suggests that the lower number of dopamine cells found in cocaine users was a relatively recent effect. The loss of dopamine cells could contribute to and intensify cocaine dependence, as well as anhedonic and depressive symptoms, in some cocaine users. Further efforts at clarifying the pathophysiological mechanisms involved may help explain treatment refractoriness, and identify targets for therapeutic intervention. PMID:19233481

  9. Dopamine modulates novelty seeking behavior during decision making.

    PubMed

    Costa, Vincent D; Tran, Valery L; Turchi, Janita; Averbeck, Bruno B

    2014-10-01

    Novelty seeking refers to the tendency of humans and animals to explore novel and unfamiliar stimuli and environments. The idea that dopamine modulates novelty seeking is supported by evidence that novel stimuli excite dopamine neurons and activate brain regions receiving dopaminergic input. In addition, dopamine is shown to drive exploratory behavior in novel environments. It is not clear whether dopamine promotes novelty seeking when it is framed as the decision to explore novel options versus the exploitation of familiar options. To test this hypothesis, we administered systemic injections of saline or GBR-12909, a selective dopamine transporter (DAT) inhibitor, to monkeys and assessed their novelty seeking behavior during a probabilistic decision making task. The task involved pseudorandom introductions of novel choice options. This allowed monkeys the opportunity to explore novel options or to exploit familiar options that they had already sampled. We found that DAT blockade increased the monkeys' preference for novel options. A reinforcement learning (RL) model fit to the monkeys' choice data showed that increased novelty seeking after DAT blockade was driven by an increase in the initial value the monkeys assigned to novel options. However, blocking DAT did not modulate the rate at which the monkeys learned which cues were most predictive of reward or their tendency to exploit that knowledge. These data demonstrate that dopamine enhances novelty-driven value and imply that excessive novelty seeking-characteristic of impulsivity and behavioral addictions-might be caused by increases in dopamine, stemming from less reuptake.

  10. Dopamine D3 Receptors Modulate the Ability of Win-Paired Cues to Increase Risky Choice in a Rat Gambling Task.

    PubMed

    Barrus, Michael M; Winstanley, Catharine A

    2016-01-20

    Similar to other addiction disorders, the cues inherent in many gambling procedures are thought to play an important role in mediating their addictive nature. Animal models of gambling-related behavior, while capturing dimensions of economic decision making, have yet to address the impact that these salient cues may have in promoting maladaptive choice. Here, we determined whether adding win-associated audiovisual cues to a rat gambling task (rGT) would influence decision making. Thirty-two male Long-Evans rats were tested on either the cued or uncued rGT. In these tasks, animals chose between four options associated with different magnitudes and frequencies of reward and punishing time-out periods. As in the Iowa Gambling Task, favoring options associated with smaller per-trial rewards but smaller losses and avoiding the tempting "high-risk, high-reward" decks maximized profits. Although the reinforcement contingencies were identical in both task versions, rats' choice of the disadvantageous risky options was significantly greater on the cued task. Furthermore, a D3 receptor agonist increased choice of the disadvantageous options, whereas a D3 antagonist had the opposite effects, only on the cued task. These findings are consistent with the reported role of D3 receptors in mediating the facilitatory effects of cues in addiction. Collectively, these results indicate that the cued rGT is a valuable model with which to study the mechanism by which salient cues can invigorate maladaptive decision making, an important and understudied component of both gambling and substance use disorders. Significance statement: We used a rodent analog of the Iowa Gambling Task to determine whether the addition of audiovisual cues would affect choice preferences. Adding reward-concurrent cues significantly increased risky choice. This is the first clear demonstration that reward-paired cues can bias cost/benefit decision making against a subject's best interests in a manner concordant

  11. Dopamine D3 Receptors Modulate the Ability of Win-Paired Cues to Increase Risky Choice in a Rat Gambling Task.

    PubMed

    Barrus, Michael M; Winstanley, Catharine A

    2016-01-20

    Similar to other addiction disorders, the cues inherent in many gambling procedures are thought to play an important role in mediating their addictive nature. Animal models of gambling-related behavior, while capturing dimensions of economic decision making, have yet to address the impact that these salient cues may have in promoting maladaptive choice. Here, we determined whether adding win-associated audiovisual cues to a rat gambling task (rGT) would influence decision making. Thirty-two male Long-Evans rats were tested on either the cued or uncued rGT. In these tasks, animals chose between four options associated with different magnitudes and frequencies of reward and punishing time-out periods. As in the Iowa Gambling Task, favoring options associated with smaller per-trial rewards but smaller losses and avoiding the tempting "high-risk, high-reward" decks maximized profits. Although the reinforcement contingencies were identical in both task versions, rats' choice of the disadvantageous risky options was significantly greater on the cued task. Furthermore, a D3 receptor agonist increased choice of the disadvantageous options, whereas a D3 antagonist had the opposite effects, only on the cued task. These findings are consistent with the reported role of D3 receptors in mediating the facilitatory effects of cues in addiction. Collectively, these results indicate that the cued rGT is a valuable model with which to study the mechanism by which salient cues can invigorate maladaptive decision making, an important and understudied component of both gambling and substance use disorders. Significance statement: We used a rodent analog of the Iowa Gambling Task to determine whether the addition of audiovisual cues would affect choice preferences. Adding reward-concurrent cues significantly increased risky choice. This is the first clear demonstration that reward-paired cues can bias cost/benefit decision making against a subject's best interests in a manner concordant

  12. Enhanced striatal dopamine release during food stimulation in binge eating disorder.

    PubMed

    Wang, Gene-Jack; Geliebter, Allan; Volkow, Nora D; Telang, Frank W; Logan, Jean; Jayne, Millard C; Galanti, Kochavi; Selig, Peter A; Han, Hao; Zhu, Wei; Wong, Christopher T; Fowler, Joanna S

    2011-08-01

    Subjects with binge eating disorder (BED) regularly consume large amounts of food in short time periods. The neurobiology of BED is poorly understood. Brain dopamine, which regulates motivation for food intake, is likely to be involved. We assessed the involvement of brain dopamine in the motivation for food consumption in binge eaters. Positron emission tomography (PET) scans with [(11)C]raclopride were done in 10 obese BED and 8 obese subjects without BED. Changes in extracellular dopamine in the striatum in response to food stimulation in food-deprived subjects were evaluated after placebo and after oral methylphenidate (MPH), a drug that blocks the dopamine reuptake transporter and thus amplifies dopamine signals. Neither the neutral stimuli (with or without MPH) nor the food stimuli when given with placebo increased extracellular dopamine. The food stimuli when given with MPH significantly increased dopamine in the caudate and putamen in the binge eaters but not in the nonbinge eaters. Dopamine increases in the caudate were significantly correlated with the binge eating scores but not with BMI. These results identify dopamine neurotransmission in the caudate as being of relevance to the neurobiology of BED. The lack of correlation between BMI and dopamine changes suggests that dopamine release per se does not predict BMI within a group of obese individuals but that it predicts binge eating.

  13. Enhanced Striatal Dopamine Release During Food Stimulation in Binge Eating Disorder

    PubMed Central

    Wang, Gene-Jack; Geliebter, Allan; Volkow, Nora D.; Telang, Frank W.; Logan, Jean; Jayne, Millard C.; Galanti, Kochavi; Selig, Peter A.; Han, Hao; Zhu, Wei; Wong, Christopher T.; Fowler, Joanna S.

    2011-01-01

    Subjects with binge eating disorder (BED) regularly consume large amounts of food in short time periods. The neurobiology of BED is poorly understood. Brain dopamine, which regulates motivation for food intake, is likely to be involved. We assessed the involvement of brain dopamine in the motivation for food consumption in binge eaters. Positron emission tomography (PET) scans with [11C]raclopride were done in 10 obese BED and 8 obese subjects without BED. Changes in extracellular dopamine in the striatum in response to food stimulation in food-deprived subjects were evaluated after placebo and after oral methylphenidate (MPH), a drug that blocks the dopamine reuptake transporter and thus amplifies dopamine signals. Neither the neutral stimuli (with or without MPH) nor the food stimuli when given with placebo increased extracellular dopamine. The food stimuli when given with MPH significantly increased dopamine in the caudate and putamen in the binge eaters but not in the nonbinge eaters. Dopamine increases in the caudate were significantly correlated with the binge eating scores but not with BMI. These results identify dopamine neurotransmission in the caudate as being of relevance to the neurobiology of BED. The lack of correlation between BMI and dopamine changes suggests that dopamine release per se does not predict BMI within a group of obese individuals but that it predicts binge eating. PMID:21350434

  14. Enhanced striatal dopamine release during food stimulation in binge eating disorder

    SciTech Connect

    Wang, g.j.; Wang, G.-J.; Geliebter, A.; Volkow, N.D.; Telang, F.W.; Logan, Jaynbe, M.C.; Galanti, K.; Selig, P.A.; Han, H.; Zhu, W.; Wong, C.T.; Fowler, J.S.

    2011-01-13

    Subjects with binge eating disorder (BED) regularly consume large amounts of food in short time periods. The neurobiology of BED is poorly understood. Brain dopamine, which regulates motivation for food intake, is likely to be involved. We assessed the involvement of brain dopamine in the motivation for food consumption in binge eaters. Positron emission tomography (PET) scans with [{sup 11}C]raclopride were done in 10 obese BED and 8 obese subjects without BED. Changes in extracellular dopamine in the striatum in response to food stimulation in food-deprived subjects were evaluated after placebo and after oral methylphenidate (MPH), a drug that blocks the dopamine reuptake transporter and thus amplifies dopamine signals. Neither the neutral stimuli (with or without MPH) nor the food stimuli when given with placebo increased extracellular dopamine. The food stimuli when given with MPH significantly increased dopamine in the caudate and putamen in the binge eaters but not in the nonbinge eaters. Dopamine increases in the caudate were significantly correlated with the binge eating scores but not with BMI. These results identify dopamine neurotransmission in the caudate as being of relevance to the neurobiology of BED. The lack of correlation between BMI and dopamine changes suggests that dopamine release per se does not predict BMI within a group of obese individuals but that it predicts binge eating.

  15. Amphetamine elevates nucleus accumbens dopamine via an action potential-dependent mechanism that is modulated by endocannabinoids.

    PubMed

    Covey, Dan P; Bunner, Kendra D; Schuweiler, Douglas R; Cheer, Joseph F; Garris, Paul A

    2016-06-01

    The reinforcing effects of abused drugs are mediated by their ability to elevate nucleus accumbens dopamine. Amphetamine (AMPH) was historically thought to increase dopamine by an action potential-independent, non-exocytotic type of release called efflux, involving reversal of dopamine transporter function and driven by vesicular dopamine depletion. Growing evidence suggests that AMPH also acts by an action potential-dependent mechanism. Indeed, fast-scan cyclic voltammetry demonstrates that AMPH activates dopamine transients, reward-related phasic signals generated by burst firing of dopamine neurons and dependent on intact vesicular dopamine. Not established for AMPH but indicating a shared mechanism, endocannabinoids facilitate this activation of dopamine transients by broad classes of abused drugs. Here, using fast-scan cyclic voltammetry coupled to pharmacological manipulations in awake rats, we investigated the action potential and endocannabinoid dependence of AMPH-induced elevations in nucleus accumbens dopamine. AMPH increased the frequency, amplitude and duration of transients, which were observed riding on top of slower dopamine increases. Surprisingly, silencing dopamine neuron firing abolished all AMPH-induced dopamine elevations, identifying an action potential-dependent origin. Blocking cannabinoid type 1 receptors prevented AMPH from increasing transient frequency, similar to reported effects on other abused drugs, but not from increasing transient duration and inhibiting dopamine uptake. Thus, AMPH elevates nucleus accumbens dopamine by eliciting transients via cannabinoid type 1 receptors and promoting the summation of temporally coincident transients, made more numerous, larger and wider by AMPH. Collectively, these findings are inconsistent with AMPH eliciting action potential-independent dopamine efflux and vesicular dopamine depletion, and support endocannabinoids facilitating phasic dopamine signalling as a common action in drug reinforcement.

  16. Desensitization, phosphorylation and palmitoylation of the human dopamine D1 receptor.

    PubMed

    Ng, G Y; Mouillac, B; George, S R; Caron, M; Dennis, M; Bouvier, M; O'Dowd, B F

    1994-03-15

    The regulation and post-translational modifications of the human dopamine D1 receptor were studied in the baculovirus-eukaryotic cell expression system. Baculovirus constructs containing either the DNA encoding the dopamine D1 receptor or a DNA encoding a c-myc epitope tagged dopamine D1 receptor (c-myc-dopamine D1 receptor) were used to infect Spodoptera frugiperda (Sf9) insect cells. Expressed dopamine D1 and c-myc-dopamine D1 receptors bound agonists and antagonists with affinities and a rank order of potency characteristic of a classical dopamine D1 receptor pharmacological profile. In membrane preparations from cells expressing c-myc-dopamine D1 receptor, the photoaffinity label [125I](3-methyl-2-[4'-azidophenyl]-2,3,5-tetrahydro-2H-3-benzazepine) ([125I]MAB) bound specifically upon photolysis. A major broad band of approximately 48 kDa was detected. This species was identified in immunoblots by the monoclonal antibody raised against the c-myc epitope of c-myc-dopamine D1 receptor was isolated by immunoprecipitation from whole cells and was shown to be post-translationally modified by phosphorylation and palmitoylation. Exposure of cells expressing c-myc-dopamine D1 receptor to dopamine for 15 min resulted in a reduction in the maximal dopamine stimulated adenylyl cyclase activity, which was accompanied by an increased phosphorylation of the receptor and a rapid redistribution of surface c-myc-dopamine D1 receptor as detected by in situ immunofluorescence. Dopamine exposure also resulted in an increased level of incorporation of [3H]palmitic acid into the receptor. Thus, we provide the first evidence that the human dopamine D1 receptor undergoes agonist-dependent desensitization, phosphorylation and palmitoylation.

  17. The dopamine theory of addiction: 40 years of highs and lows.

    PubMed

    Nutt, David J; Lingford-Hughes, Anne; Erritzoe, David; Stokes, Paul R A

    2015-05-01

    For several decades, addiction has come to be viewed as a disorder of the dopamine neurotransmitter system; however, this view has not led to new treatments. In this Opinion article, we review the origins of the dopamine theory of addiction and discuss the ability of addictive drugs to elicit the release of dopamine in the human striatum. There is robust evidence that stimulants increase striatal dopamine levels and some evidence that alcohol may have such an effect, but little evidence, if any, that cannabis and opiates increase dopamine levels. Moreover, there is good evidence that striatal dopamine receptor availability and dopamine release are diminished in individuals with stimulant or alcohol dependence but not in individuals with opiate, nicotine or cannabis dependence. These observations have implications for understanding reward and treatment responses in various addictions. PMID:25873042

  18. The dopamine theory of addiction: 40 years of highs and lows.

    PubMed

    Nutt, David J; Lingford-Hughes, Anne; Erritzoe, David; Stokes, Paul R A

    2015-05-01

    For several decades, addiction has come to be viewed as a disorder of the dopamine neurotransmitter system; however, this view has not led to new treatments. In this Opinion article, we review the origins of the dopamine theory of addiction and discuss the ability of addictive drugs to elicit the release of dopamine in the human striatum. There is robust evidence that stimulants increase striatal dopamine levels and some evidence that alcohol may have such an effect, but little evidence, if any, that cannabis and opiates increase dopamine levels. Moreover, there is good evidence that striatal dopamine receptor availability and dopamine release are diminished in individuals with stimulant or alcohol dependence but not in individuals with opiate, nicotine or cannabis dependence. These observations have implications for understanding reward and treatment responses in various addictions.

  19. Effect of dopamine on viability of BHK-21 cells.

    PubMed

    Moshkov, D A; Abramova, M B; Shubina, V S; Lavrovskaya, V P; Pavlik, L L; Lezhnev, E I

    2010-09-01

    We studied the effects of dopamine added to culture medium on survival of floating or adherent BHK-21 cells differing by organization of actin cytoskeleton. The viability of floating cells more drastically decreased with increasing dopamine concentration and duration of exposure than that of adherent cells. The cells worse adhered to the substrate and formed a monolayer. The formed monolayer degrades, cell borders become blurred, cells, polygonal in the control, are rounded. Preliminary blockade of dopamine receptors with haloperidol, inessential for cell survival and morphology, does not prevent the destructive effect of dopamine on the cells. Ultrastructural study revealed increased density of filamentous actin threads in deep compartments of cell cytoplasm after dopamine treatment, this increase being more pronounced in cells grown in suspension. Bearing in mind the polymerizing effect of dopamine on globular actin in vitro and the fact that the content of this protein in floating cells is higher than in adherent cells, we can conclude that the decrease in viability of BHK-21 cells is caused by interaction of dopamine with cytoplasmic globular actin. PMID:21246101

  20. Dopamine D1 and D2 receptor immunoreactivities in the arcuate-median eminence complex and their link to the tubero-infundibular dopamine neurons.

    PubMed

    Romero-Fernandez, W; Borroto-Escuela, D O; Vargas-Barroso, V; Narváez, M; Di Palma, M; Agnati, L F; Larriva Sahd, J; Fuxe, K

    2014-07-18

    Dopamine D1 and D2 receptor immunohistochemistry and Golgi techniques were used to study the structure of the adult rat arcuate-median eminence complex, and determine the distribution of the dopamine D1 and D2 receptor immunoreactivities therein, particularly in relation to the tubero-infundibular dopamine neurons. Punctate dopamine D1 and D2 receptor immunoreactivities, likely located on nerve terminals, were enriched in the lateral palisade zone built up of nerve terminals, while the densities were low to modest in the medial palisade zone. A codistribution of dopamine D1 receptor or dopamine D2 receptor immunoreactive puncta with tyrosine hydroxylase immunoreactive nerve terminals was demonstrated in the external layer. Dopamine D1 receptor but not dopamine D2 receptor immnunoreactivites nerve cell bodies were found in the ventromedial part of the arcuate nucleus and in the lateral part of the internal layer of the median eminence forming a continuous cell mass presumably representing neuropeptide Y immunoreactive nerve cell bodies. The major arcuate dopamine/ tyrosine hydroxylase nerve cell group was found in the dorsomedial part. A large number of tyrosine hydroxylase immunoreactive nerve cell bodies in this region demonstrated punctate dopamine D1 receptor immunoreactivity but only a few presented dopamine D2 receptor immunoreactivity which were mainly found in a substantial number of tyrosine hydroxylase cell bodies of the ventral periventricular hypothalamic nucleus, also belonging to the tubero-infundibular dopamine neurons. Structural evidence for projections of the arcuate nerve cells into the median eminence was also obtained. Distal axons formed horizontal axons in the internal layer issuing a variable number of collaterals classified into single or multiple strands located in the external layer increasing our understanding of the dopamine nerve terminal networks in this region.  Dopamine D1 and D2 receptors may therefore directly and differentially

  1. Dopamine D1 and D2 Receptor Immunoreactivities in the Arcuate-Median Eminence Complex and their Link to the Tubero-Infundibular Dopamine Neurons

    PubMed Central

    Romero-Fernandez, W.; Borroto-Escuela, D.O.; Vargas-Barroso, V.; Narváez, M.; Di Palma, M.; Agnati, L.F.; Sahd, J. Larriva

    2014-01-01

    Dopamine D1 and D2 receptor immunohistochemistry and Golgi techniques were used to study the structure of the adult rat arcuate-median eminence complex, and determine the distribution of the dopamine D1 and D2 receptor immunoreactivities therein, particularly in relation to the tubero-infundibular dopamine neurons. Punctate dopamine D1 and D2 receptor immunoreactivities, likely located on nerve terminals, were enriched in the lateral palisade zone built up of nerve terminals, while the densities were low to modest in the medial palisade zone. A codistribution of dopamine D1 receptor or dopamine D2 receptor immunoreactive puncta with tyrosine hydroxylase immunoreactive nerve terminals was demonstrated in the external layer. Dopamine D1 receptor but not dopamine D2 receptor immnunoreactivites nerve cell bodies were found in the ventromedial part of the arcuate nucleus and in the lateral part of the internal layer of the median eminence forming a continuous cell mass presumably representing neuropeptide Y immunoreactive nerve cell bodies. The major arcuate dopamine/ tyrosine hydroxylase nerve cell group was found in the dorsomedial part. A large number of tyrosine hydroxylase immunoreactive nerve cell bodies in this region demonstrated punctate dopamine D1 receptor immunoreactivity but only a few presented dopamine D2 receptor immunoreactivity which were mainly found in a substantial number of tyrosine hydroxylase cell bodies of the ventral periventricular hypothalamic nucleus, also belonging to the tuberoinfundibular dopamine neurons. Structural evidence for projections of the arcuate nerve cells into the median eminence was also obtained. Distal axons formed horizontal axons in the internal layer issuing a variable number of collaterals classified into single or multiple strands located in the external layer increasing our understanding of the dopamine nerve terminal networks in this region. Dopamine D1 and D2 receptors may therefore directly and differentially

  2. Overexpression of two cambium-abundant Chinese fir (Cunninghamia lanceolata) α-expansin genes ClEXPA1 and ClEXPA2 affect growth and development in transgenic tobacco and increase the amount of cellulose in stem cell walls.

    PubMed

    Wang, Guifeng; Gao, Yan; Wang, Jinjun; Yang, Liwei; Song, Rentao; Li, Xiaorong; Shi, Jisen

    2011-05-01

    Expansins are unique plant cell wall proteins that possess the ability to induce immediately cell wall extension in vitro and cell expansion in vivo. To investigate the biological functions of expansins that are abundant in wood-forming tissues, we cloned two expansin genes from the differentiating xylem of Chinese fir (Cunninghamia lanceolata (Lamb.) Hook). Phylogenetic reconstruction indicated that they belong to α-expansin (EXPA), named ClEXPA1 and ClEXPA2. Expression pattern analysis demonstrated that they are preferentially expressed in the cambium region. Overexpression of ClEXPA1 and ClEXPA2 in tobacco plants yielded pleiotropic phenotypes of plant height, stem diameter, leaf number and seed pod. The height and diameter growth of the 35S(pro) :ClEXPA1 and 35S(pro) :ClEXPA2 transgenic plants were increased drastically, exhibiting an enlargement of pith parenchyma cell size. Isolated cell walls of ClEXPA1 and ClEXPA2 overexpressors contained 30%-50% higher cellulose contents than the wild type, accompanied by a thickening of the cell walls in the xylem region. Both ClEXPA1 and ClEXPA2 are involved in plant growth and development, with a partially functional overlap. Expansins are not only able to induce cell expansion in different tissues/organs in vivo, but they also can act as a potential activator during secondary wall formation by directly or indirectly affecting cellulose metabolism, probably in a cell type-dependent manner.

  3. Excessive cocaine use results from decreased phasic dopamine signaling in the striatum

    PubMed Central

    Willuhn, Ingo; Burgeno, Lauren M.; Groblewski, Peter A.; Phillips, Paul E. M.

    2014-01-01

    Drug addiction is a neuropsychiatric disorder marked by escalating drug use. Dopamine neurotransmission in the ventromedial striatum (VMS) mediates acute reinforcing effects of abused drugs, but with protracted use the dorsolateral striatum (DLS) is thought to assume control over drug seeking. We measured striatal dopamine release during a cocaine self-administration regimen that produced escalation of drug taking in rats. Surprisingly, we found that phasic dopamine decreased in both regions as the rate of cocaine intake increased; with the decrement in dopamine in the VMS significantly correlated with the rate of escalation. Administration of the dopamine precursor L-DOPA at a dose that replenished dopamine signaling in the VMS reversed escalation, thereby demonstrating the causal relationship between diminished dopamine transmission and excessive drug use. Thus, together these data provide mechanistic and therapeutic insight into the excessive drug intake that emerges following protracted use. PMID:24705184

  4. The Transfection of BDNF to Dopamine Neurons Potentiates the Effect of Dopamine D3 Receptor Agonist Recovering the Striatal Innervation, Dendritic Spines and Motor Behavior in an Aged Rat Model of Parkinson’s Disease

    PubMed Central

    Razgado-Hernandez, Luis F.; Espadas-Alvarez, Armando J.; Reyna-Velazquez, Patricia; Sierra-Sanchez, Arturo; Anaya-Martinez, Veronica; Jimenez-Estrada, Ismael; Bannon, Michael J.; Martinez-Fong, Daniel; Aceves-Ruiz, Jorge

    2015-01-01

    The progressive degeneration of the dopamine neurons of the pars compacta of substantia nigra and the consequent loss of the dopamine innervation of the striatum leads to the impairment of motor behavior in Parkinson’s disease. Accordingly, an efficient therapy of the disease should protect and regenerate the dopamine neurons of the substantia nigra and the dopamine innervation of the striatum. Nigral neurons express Brain Derived Neurotropic Factor (BDNF) and dopamine D3 receptors, both of which protect the dopamine neurons. The chronic activation of dopamine D3 receptors by their agonists, in addition, restores, in part, the dopamine innervation of the striatum. Here we explored whether the over-expression of BDNF by dopamine neurons potentiates the effect of the activation of D3 receptors restoring nigrostriatal innervation. Twelve-month old Wistar rats were unilaterally injected with 6-hydroxydopamine into the striatum. Five months later, rats were treated with the D3 agonist 7-hydroxy-N,N-di-n-propy1-2-aminotetralin (7-OH-DPAT) administered i.p. during 4½ months via osmotic pumps and the BDNF gene transfection into nigral cells using the neurotensin-polyplex nanovector (a non-viral transfection) that selectively transfect the dopamine neurons via the high-affinity neurotensin receptor expressed by these neurons. Two months after the withdrawal of 7-OH-DPAT when rats were aged (24 months old), immunohistochemistry assays were made. The over-expression of BDNF in rats receiving the D3 agonist normalized gait and motor coordination; in addition, it eliminated the muscle rigidity produced by the loss of dopamine. The recovery of motor behavior was associated with the recovery of the nigral neurons, the dopamine innervation of the striatum and of the number of dendritic spines of the striatal neurons. Thus, the over-expression of BDNF in dopamine neurons associated with the chronic activation of the D3 receptors appears to be a promising strategy for restoring

  5. Updating dopamine reward signals.

    PubMed

    Schultz, Wolfram

    2013-04-01

    Recent work has advanced our knowledge of phasic dopamine reward prediction error signals. The error signal is bidirectional, reflects well the higher order prediction error described by temporal difference learning models, is compatible with model-free and model-based reinforcement learning, reports the subjective rather than physical reward value during temporal discounting and reflects subjective stimulus perception rather than physical stimulus aspects. Dopamine activations are primarily driven by reward, and to some extent risk, whereas punishment and salience have only limited activating effects when appropriate controls are respected. The signal is homogeneous in terms of time course but heterogeneous in many other aspects. It is essential for synaptic plasticity and a range of behavioural learning situations.

  6. Growth of dopamine crystals

    NASA Astrophysics Data System (ADS)

    Patil, Vidya; Patki, Mugdha

    2016-05-01

    Many nonlinear optical (NLO) crystals have been identified as potential candidates in optical and electro-optical devices. Use of NLO organic crystals is expected in photonic applications. Hence organic nonlinear optical materials have been intensely investigated due to their potentially high nonlinearities, and rapid response in electro-optic effect compared to inorganic NLO materials. There are many methods to grow organic crystals such as vapor growth method, melt growth method and solution growth method. Out of these methods, solution growth method is useful in providing constraint free crystal. Single crystals of Dopamine have been grown by evaporating the solvents from aqueous solution. Crystals obtained were of the size of orders of mm. The crystal structure of dopamine was determined using XRD technique. Images of crystals were obtained using FEG SEM Quanta Series under high vacuum and low KV.

  7. Role of Histidine 547 of Human Dopamine Transporter in Molecular Interaction with HIV-1 Tat and Dopamine Uptake

    PubMed Central

    Yuan, Yaxia; Quizon, Pamela M.; Sun, Wei-Lun; Yao, Jianzhuang; Zhu, Jun; Zhan, Chang-Guo

    2016-01-01

    HIV-1 Tat plays an important role in HIV-associated neurocognitive disorders (HAND) by disrupting neurotransmission including dopamine uptake by human dopamine transporter (hDAT). Previous studies have demonstrated that HIV-1 Tat directly binds to hDAT and some amino-acid mutations that attenuate the hDAT-Tat binding also significantly decreased dopamine uptake activity of hDAT. This combined computational-experimental study demonstrates that histidine-547 (H547) of hDAT plays a crucial role in the hDAT-Tat binding and dopamine uptake by hDAT, and that the H547A mutation can not only considerably attenuate Tat-induced inhibition of dopamine uptake, but also significantly increase the Vmax of hDAT for dopamine uptake. The finding of such an unusual hDAT mutant capable of both increasing the Vmax of hDAT for dopamine uptake and disrupting the hDAT-Tat binding may provide an exciting knowledge basis for development of novel concepts for therapeutic treatment of the HAND. PMID:27250920

  8. Dopamine Receptor Antagonists Enhance Proliferation and Neurogenesis of Midbrain Lmx1a-expressing Progenitors

    PubMed Central

    Hedlund, Eva; Belnoue, Laure; Theofilopoulos, Spyridon; Salto, Carmen; Bye, Chris; Parish, Clare; Deng, Qiaolin; Kadkhodaei, Banafsheh; Ericson, Johan; Arenas, Ernest; Perlmann, Thomas; Simon, András

    2016-01-01

    Degeneration of dopamine neurons in the midbrain causes symptoms of the movement disorder, Parkinson disease. Dopamine neurons are generated from proliferating progenitor cells localized in the embryonic ventral midbrain. However, it remains unclear for how long cells with dopamine progenitor character are retained and if there is any potential for reactivation of such cells after cessation of normal dopamine neurogenesis. We show here that cells expressing Lmx1a and other progenitor markers remain in the midbrain aqueductal zone beyond the major dopamine neurogenic period. These cells express dopamine receptors, are located in regions heavily innervated by midbrain dopamine fibres and their proliferation can be stimulated by antagonizing dopamine receptors, ultimately leading to increased neurogenesis in vivo. Furthermore, treatment with dopamine receptor antagonists enhances neurogenesis in vitro, both from embryonic midbrain progenitors as well as from embryonic stem cells. Altogether our results indicate a potential for reactivation of resident midbrain cells with dopamine progenitor potential beyond the normal period of dopamine neurogenesis. PMID:27246266

  9. Nicotinic receptors regulate the dynamic range of dopamine release in vivo.

    PubMed

    Koranda, Jessica L; Cone, Jackson J; McGehee, Daniel S; Roitman, Mitchell F; Beeler, Jeff A; Zhuang, Xiaoxi

    2014-01-01

    Nicotinic acetylcholine receptors (nAChRs) are expressed presynaptically on dopamine axon terminals, and their activation by endogenous acetylcholine from striatal cholinergic interneurons enhances dopamine release both independently of and in concert with dopamine neuron activity. Acute nAChR inactivation is believed to enhance the contrast between low- and high-frequency dopamine cell activity. Although these studies reveal a key role for acute activation and inactivation of nAChRs in striatal microcircuitry, it remains unknown if chronic inactivation/desensitization of nAChRs can alter dopamine release dynamics. Using in vivo cyclic voltammetry in anaesthetized mice, we examined whether chronic inactivation of nAChRs modulates dopamine release across a parametric range of stimulation, varying both frequency and pulse number. Deletion of β2*nAChRs and chronic nicotine exposure greatly diminished dopamine release across the entire range of stimulation parameters. In addition, we observed a facilitation of dopamine release at low frequency and pulse number in wild-type mice that is absent in the β2* knockout and chronic nicotine mice. These data suggest that deletion or chronic desensitization of nAChRs reduces the dynamic range of dopamine release in response to dopamine cell activity, decreasing rather than increasing contrast between high and low dopamine activity.

  10. Cannabinoid receptor activation shifts temporally engendered patterns of dopamine release.

    PubMed

    Oleson, Erik B; Cachope, Roger; Fitoussi, Aurelie; Tsutsui, Kimberly; Wu, Sharon; Gallegos, Jacqueline A; Cheer, Joseph F

    2014-05-01

    The ability to discern temporally pertinent environmental events is essential for the generation of adaptive behavior in conventional tasks, and our overall survival. Cannabinoids are thought to disrupt temporally controlled behaviors by interfering with dedicated brain timing networks. Cannabinoids also increase dopamine release within the mesolimbic system, a neural pathway generally implicated in timing behavior. Timing can be assessed using fixed-interval (FI) schedules, which reinforce behavior on the basis of time. To date, it remains unknown how cannabinoids modulate dopamine release when responding under FI conditions, and for that matter, how subsecond dopamine release is related to time in these tasks. In the present study, we hypothesized that cannabinoids would accelerate timing behavior in an FI task while concurrently augmenting a temporally relevant pattern of dopamine release. To assess this possibility, we measured subsecond dopamine concentrations in the nucleus accumbens while mice responded for food under the influence of the cannabinoid agonist WIN 55,212-2 in an FI task. Our data reveal that accumbal dopamine concentrations decrease proportionally to interval duration--suggesting that dopamine encodes time in FI tasks. We further demonstrate that WIN 55,212-2 dose-dependently increases dopamine release and accelerates a temporal behavioral response pattern in a CB1 receptor-dependent manner--suggesting that cannabinoid receptor activation modifies timing behavior, in part, by augmenting time-engendered patterns of dopamine release. Additional investigation uncovered a specific role for endogenous cannabinoid tone in timing behavior, as elevations in 2-arachidonoylglycerol, but not anandamide, significantly accelerated the temporal response pattern in a manner akin to WIN 55,212-2. PMID:24345819

  11. Maternal Immune Activation Disrupts Dopamine System in the Offspring

    PubMed Central

    Luchicchi, Antonio; Lecca, Salvatore; Melis, Miriam; De Felice, Marta; Cadeddu, Francesca; Frau, Roberto; Muntoni, Anna Lisa; Fadda, Paola; Devoto, Paola

    2016-01-01

    Background: In utero exposure to maternal viral infections is associated with a higher incidence of psychiatric disorders with a supposed neurodevelopmental origin, including schizophrenia. Hence, immune response factors exert a negative impact on brain maturation that predisposes the offspring to the emergence of pathological phenotypes later in life. Although ventral tegmental area dopamine neurons and their target regions play essential roles in the pathophysiology of psychoses, it remains to be fully elucidated how dopamine activity and functionality are disrupted in maternal immune activation models of schizophrenia. Methods: Here, we used an immune-mediated neurodevelopmental disruption model based on prenatal administration of the polyriboinosinic-polyribocytidilic acid in rats, which mimics a viral infection and recapitulates behavioral abnormalities relevant to psychiatric disorders in the offspring. Extracellular dopamine levels were measured by brain microdialysis in both the nucleus accumbens shell and the medial prefrontal cortex, whereas dopamine neurons in ventral tegmental area were studied by in vivo electrophysiology. Results: Polyriboinosinic-polyribocytidilic acid-treated animals, at adulthood, displayed deficits in sensorimotor gating, memory, and social interaction and increased baseline extracellular dopamine levels in the nucleus accumbens, but not in the prefrontal cortex. In polyriboinosinic-polyribocytidilic acid rats, dopamine neurons showed reduced spontaneously firing rate and population activity. Conclusions: These results confirm that maternal immune activation severely impairs dopamine system and that the polyriboinosinic-polyribocytidilic acid model can be considered a proper animal model of a psychiatric condition that fulfills a multidimensional set of validity criteria predictive of a human pathology. PMID:26819283

  12. Dopamine uptake dynamics are preserved under isoflurane anesthesia.

    PubMed

    Brodnik, Zachary D; España, Rodrigo A

    2015-10-01

    Fast scan cyclic voltammetry is commonly used for measuring the kinetics of dopamine release and uptake. For experiments using an anesthetized preparation, urethane is preferentially used because it does not alter dopamine uptake kinetics compared to freely moving animals. Unfortunately, urethane is highly toxic, can induce premature death during experiments, and cannot be used for recovery surgeries. Isoflurane is an alternative anesthetic that is less toxic than urethane, produces a stable level of anesthesia over extended periods, and is often used for recovery surgeries. Despite these benefits, the effects of isoflurane on dopamine release and uptake have not been directly characterized. In the present studies, we assessed the utility of isoflurane for voltammetry experiments by testing dopamine signaling parameters under baseline conditions, after treatment with the dopamine uptake inhibitor cocaine, and after exposure to increasing concentrations of isoflurane. Our results indicate that surgical levels of isoflurane do not significantly alter terminal mechanisms of dopamine release and uptake over prolonged periods of time. Consequently, we propose that isoflurane is an acceptable anesthetic for voltammetry experiments, which in turn permits the design of studies in which dopamine signaling is examined under anesthesia prior to recovery and subsequent experimentation in the same animals. PMID:26321152

  13. Optogenetic stimulation of VTA dopamine neurons reveals that tonic but not phasic patterns of dopamine transmission reduce ethanol self-administration.

    PubMed

    Bass, Caroline E; Grinevich, Valentina P; Gioia, Dominic; Day-Brown, Jonathan D; Bonin, Keith D; Stuber, Garret D; Weiner, Jeff L; Budygin, Evgeny A

    2013-01-01

    There is compelling evidence that acute ethanol exposure stimulates ventral tegmental area (VTA) dopamine cell activity and that VTA-dependent dopamine release in terminal fields within the nucleus accumbens plays an integral role in the regulation of ethanol drinking behaviors. Unfortunately, due to technical limitations, the specific temporal dynamics linking VTA dopamine cell activation and ethanol self-administration are not known. In fact, establishing a causal link between specific patterns of dopamine transmission and ethanol drinking behaviors has proven elusive. Here, we sought to address these gaps in our knowledge using a newly developed viral-mediated gene delivery strategy to selectively express Channelrhodopsin-2 (ChR2) on dopamine cells in the VTA of wild-type rats. We then used this approach to precisely control VTA dopamine transmission during voluntary ethanol drinking sessions. The results confirmed that ChR2 was selectively expressed on VTA dopamine cells and delivery of blue light pulses to the VTA induced dopamine release in accumbal terminal fields with very high temporal and spatial precision. Brief high frequency VTA stimulation induced phasic patterns of dopamine release in the nucleus accumbens. Lower frequency stimulation, applied for longer periods mimicked tonic increases in accumbal dopamine. Notably, using this optogenetic approach in rats engaged in an intermittent ethanol drinking procedure, we found that tonic, but not phasic, stimulation of VTA dopamine cells selectively attenuated ethanol drinking behaviors. Collectively, these data demonstrate the effectiveness of a novel viral targeting strategy that can be used to restrict opsin expression to dopamine cells in standard outbred animals and provide the first causal evidence demonstrating that tonic activation of VTA dopamine neurons selectively decreases ethanol self-administration behaviors.

  14. Overexpression of Grain Amaranth (Amaranthus hypochondriacus) AhERF or AhDOF Transcription Factors in Arabidopsis thaliana Increases Water Deficit- and Salt-Stress Tolerance, Respectively, via Contrasting Stress-Amelioration Mechanisms

    PubMed Central

    Massange-Sánchez, Julio A.; Palmeros-Suárez, Paola A.; Espitia-Rangel, Eduardo; Rodríguez-Arévalo, Isaac; Sánchez-Segura, Lino; Martínez-Gallardo, Norma A.; Alatorre-Cobos, Fulgencio; Tiessen, Axel; Délano-Frier, John P.

    2016-01-01

    Two grain amaranth transcription factor (TF) genes were overexpressed in Arabidopsis plants. The first, coding for a group VII ethylene response factor TF (i.e., AhERF-VII) conferred tolerance to water-deficit stress (WS) in transgenic Arabidopsis without affecting vegetative or reproductive growth. A significantly lower water-loss rate in detached leaves coupled to a reduced stomatal opening in leaves of plants subjected to WS was associated with this trait. WS tolerance was also associated with an increased antioxidant enzyme activity and the accumulation of putative stress-related secondary metabolites. However, microarray and GO data did not indicate an obvious correlation between WS tolerance, stomatal closure, and abscisic acid (ABA)-related signaling. This scenario suggested that stomatal closure during WS in these plants involved ABA-independent mechanisms, possibly involving reactive oxygen species (ROS). WS tolerance may have also involved other protective processes, such as those employed for methyl glyoxal detoxification. The second, coding for a class A and cluster I DNA binding with one finger TF (i.e., AhDof-AI) provided salt-stress (SS) tolerance with no evident fitness penalties. The lack of an obvious development-related phenotype contrasted with microarray and GO data showing an enrichment of categories and genes related to developmental processes, particularly flowering. SS tolerance also correlated with increased superoxide dismutase activity but not with augmented stomatal closure. Additionally, microarray and GO data indicated that, contrary to AhERF-VII, SS tolerance conferred by AhDof-AI in Arabidopsis involved ABA-dependent and ABA-independent stress amelioration mechanisms. PMID:27749893

  15. Atypical Dopamine Uptake Inhibitors that Provide Clues About Cocaine's Mechanism at the Dopamine Transporter

    NASA Astrophysics Data System (ADS)

    Hauck Newman, Amy; Katz, Jonathan L.

    The dopamine transporter (DAT) has been a primary target for cocaine abuse/addiction medication discovery. However predicted addiction liability and limited clinical evaluation has provided a formidable challenge for development of these agents for human use. The unique and atypical pharmacological profile of the benztropine (BZT) class of dopamine uptake inhibitors, in preclinical models of cocaine effects and abuse, has encouraged further development of these agents. Moreover, in vivo studies have challenged the original DAT hypothesis and demonstrated that DAT occupancy and subsequent increases in dopamine produced by BZT analogues are significantly delayed and long lasting, as compared to cocaine. These important and distinctive elements are critical to the lack of abuse liability among BZT analogues, and improve their potential for development as treatments for cocaine abuse and possibly other neuropsychiatric disorders.

  16. Dopamine transporters are involved in the onset of hypoxia-induced dopamine efflux in striatum as revealed by in vivo microdialysis.

    PubMed

    Orset, Cyrille; Parrot, Sandrine; Sauvinet, Valérie; Cottet-Emard, Jean-Marie; Bérod, Anne; Pequignot, Jean-Marc; Denoroy, Luc

    2005-06-01

    Although many studies have revealed alterations in neurotransmission during ischaemia, few works have been devoted to the neurochemical effects of mild hypoxia, a situation encountered during life in altitude or in several pathologies. In that context, the present work was undertaken to determine the in vivo mechanisms underlying the striatal dopamine efflux induced by mild hypoxaemic hypoxia. For that purpose, the extracellular concentrations of dopamine and its metabolite 3,4-dihydroxyphenyl acetic acid were simultaneously measured using brain microdialysis during acute hypoxic exposure (10% O(2), 1h) in awake rats. Hypoxia induced a +80% increase in dopamine. Application of the dopamine transporters inhibitor, nomifensine (10 microM), just before the hypoxia prevented the rise in dopamine during the early part of hypoxia; in contrast the application of nomifensine after the beginning of hypoxia, failed to alter the increase in dopamine. Application of the voltage-dependent Na(+) channel blocker tetrodotoxin abolished the increase in dopamine, whether administered just before or after the beginning of hypoxia. These data show that the neurochemical mechanisms of the dopamine efflux may change over the course of the hypoxic exposure, dopamine transporters being involved only at the beginning of hypoxia.

  17. Pharmacological differences between the D-2 autoreceptor and the D-1 dopamine receptor in rabbit retina

    SciTech Connect

    Dubocovich, M.L.; Weiner, N.

    1985-06-01

    The effect of dopamine receptor agonists and antagonists was studied on the calcium-dependent release of (/sup 3/H)dopamine elicited by field stimulation at 3 Hz for a duration of 1 min (20 mA, 2 msec) from the rabbit retina in vitro and on adenylate cyclase activity in homogenates of rabbit retina. The relative order of potency of dopamine receptor agonists to inhibit the stimulation-evoked (/sup 3/H)dopamine release was pergolide greater than bromocriptine greater than apomorphine greater than LY 141865 greater than N,N-di-n-propyldopamine greater than or equal to dopamine. The relative order of potencies of dopamine receptor antagonists to increase (/sup 3/H)dopamine release was: S-sulpiride greater than or equal to domperidone greater than or equal to spiroperidol greater than metoclopramide greater than fluphenazine greater than or equal to R-sulpiride. alpha-Flupenthixol (0.01-1 microM) and (+)-butaclamol (0.01-1 microM) did not increase (/sup 3/H)dopamine overflow when added alone, but they antagonized the concentration-dependent inhibitory effect of apomorphine (0.1-10 microM). These results suggest that the dopamine inhibitory autoreceptor involved in the modulation of dopamine release from the rabbit retina possesses the pharmacological characteristics of a D-2 dopamine receptor. Maximal stimulation by 30 microM dopamine resulted in a 3-fold increase in adenylate cyclase activity with half-maximal stimulation occurring at a concentration of 2.46 microM. Apomorphine and pergolide elicited a partial stimulation of adenylate cyclase activity. However, at low concentrations both compounds were more potent than dopamine.

  18. Dopamine-prolactin pathway potentially contributes to the schizophrenia and type 2 diabetes comorbidity.

    PubMed

    Gragnoli, C; Reeves, G M; Reazer, J; Postolache, T T

    2016-01-01

    Schizophrenia (SCZ) and type 2 diabetes (T2D) are clinically associated, and common knowledge attributes this association to side effects of antipsychotic treatment. However, even drug-naive patients with SCZ are at increased risk for T2D. Dopamine dysfunction has a central role in SCZ. It is well-known that dopamine constitutively inhibits prolactin (PRL) secretion via the dopamine receptor 2 (DR2D). If dopamine is increased or if dopamine receptors hyperfunction, PRL may be reduced. During the first SCZ episode, low PRL levels are associated with worse symptoms. PRL is essential in human and social bonding, as well as it is implicated in glucose homeostasis. Dopamine dysfunction, beyond contributing to SCZ symptoms, may lead to altered appetite and T2D. To our knowledge, there are no studies of the genetics of the SCZ-T2D comorbidity focusing jointly on the dopamine and PRL pathway in the attempt to capture molecular heterogeneity correlated to possible disease manifestation heterogeneity. In this dopamine-PRL pathway-focused-hypothesis-driven review on the association of SCZ with T2D, we report a specific revision of what it is known about PRL and dopamine in relation to what we theorize is one of the missing links between the two disorders. We suggest that new studies are necessary to establish the genetic role of PRL and dopamine pathway in SCZ-T2D comorbidity. PMID:27093067

  19. Delta 9-tetrahydrocannabinol induces dopamine release in the human striatum.

    PubMed

    Bossong, Matthijs G; van Berckel, Bart N M; Boellaard, Ronald; Zuurman, Lineke; Schuit, Robert C; Windhorst, Albert D; van Gerven, Joop M A; Ramsey, Nick F; Lammertsma, Adriaan A; Kahn, René S

    2009-02-01

    The influence of cannabis on mental health receives growing scientific and political attention. An increasing demand for treatment of cannabis dependence has refueled the discussion about the addictive potential of cannabis. A key feature of all addictive drugs is the ability to increase synaptic dopamine levels in the striatum, a mechanism involved in their rewarding and motivating effects. However, it is currently unknown if cannabis can stimulate striatal dopamine neurotransmission in humans. Here we show that Delta 9-tetrahydrocannabinol (THC), the main psychoactive component in cannabis, induces dopamine release in the human striatum. Using the dopamine D(2)/D(3) receptor tracer [(11)C]raclopride and positron emission tomography in seven healthy subjects, we demonstrate that THC inhalation reduces [(11)C]raclopride binding in the ventral striatum and the precommissural dorsal putamen but not in other striatal subregions. This is consistent with an increase in dopamine levels in these regions. These results suggest that THC shares a potentially addictive property with other drugs of abuse. Further, it implies that the endogenous cannabinoid system is involved in regulating striatal dopamine release. This allows new directions in research on the effects of THC in neuropsychiatric disorders, such as schizophrenia. PMID:18754005

  20. Neuronal release of endogenous dopamine from corpus of guinea pig stomach.

    PubMed

    Shichijo, K; Sakurai-Yamashita, Y; Sekine, I; Taniyama, K

    1997-11-01

    Neuronal release of endogenous dopamine was identified in mucosa-free preparations (muscle layer including intramural plexus) from guinea pig stomach corpus by measuring tissue dopamine content and dopamine release and by immunohistochemical methods using a dopamine antiserum. Dopamine content in mucosa-free preparations of guinea pig gastric corpus was one-tenth of norepinephrine content. Electrical transmural stimulation of mucosa-free preparations of gastric corpus increased the release of endogenous dopamine in a frequency-dependent (3-20 Hz) manner. The stimulated release of dopamine was prevented by either removal of external Ca2+ or treatment with tetrodotoxin. Dopamine-immunopositive nerve fibers surrounding choline acetyltransferase-immunopositive ganglion cells were seen in the myenteric plexus of whole mount preparations of gastric corpus even after bilateral transection of the splanchnic nerve proximal to the junction with the vagal nerve (section of nerves between the celiac ganglion and stomach). Domperidone and sulpiride potentiated the stimulated release of acetylcholine and reversed the dopamine-induced inhibition of acetylcholine release from mucosa-free preparations. These results indicate that dopamine is physiologically released from neurons and from possible dopaminergic nerve terminals and regulates cholinergic neuronal activity in the corpus of guinea pig stomach. PMID:9374701

  1. The impact of a parkinsonian lesion on dynamic striatal dopamine transmission depends on nicotinic receptor activation

    PubMed Central

    Jennings, Katie A.; Platt, Nicola J.; Cragg, Stephanie J.

    2015-01-01

    Dopamine function is disturbed in Parkinson's disease (PD), but whether and how release of dopamine from surviving neurons is altered has long been debated. Nicotinic acetylcholine receptors (nAChRs) on dopamine axons powerfully govern dopamine release and could be critical contributing factors. We revisited whether fundamental properties of dopamine transmission are changed in a parkinsonian brain and tested the potentially profound masking effects of nAChRs. Using real-time detection of dopamine in mouse striatum after a partial 6-hydroxydopamine lesion and under nAChR inhibition, we reveal that dopamine signals show diminished sensitivity to presynaptic activity. This effect manifested as diminished contrast between DA release evoked by the lowest versus highest frequencies. This reduced activity-dependence was underpinned by loss of short-term facilitation of dopamine release, consistent with an increase in release probability (Pr). With nAChRs active, the reduced activity-dependence of dopamine release after a parkinsonian lesion was masked. Consequently, moment-by-moment variation in activity of nAChRs may lead to dynamic co-variation in dopamine signal impairments in PD. PMID:26117304

  2. Neuronal release of endogenous dopamine from corpus of guinea pig stomach.

    PubMed

    Shichijo, K; Sakurai-Yamashita, Y; Sekine, I; Taniyama, K

    1997-11-01

    Neuronal release of endogenous dopamine was identified in mucosa-free preparations (muscle layer including intramural plexus) from guinea pig stomach corpus by measuring tissue dopamine content and dopamine release and by immunohistochemical methods using a dopamine antiserum. Dopamine content in mucosa-free preparations of guinea pig gastric corpus was one-tenth of norepinephrine content. Electrical transmural stimulation of mucosa-free preparations of gastric corpus increased the release of endogenous dopamine in a frequency-dependent (3-20 Hz) manner. The stimulated release of dopamine was prevented by either removal of external Ca2+ or treatment with tetrodotoxin. Dopamine-immunopositive nerve fibers surrounding choline acetyltransferase-immunopositive ganglion cells were seen in the myenteric plexus of whole mount preparations of gastric corpus even after bilateral transection of the splanchnic nerve proximal to the junction with the vagal nerve (section of nerves between the celiac ganglion and stomach). Domperidone and sulpiride potentiated the stimulated release of acetylcholine and reversed the dopamine-induced inhibition of acetylcholine release from mucosa-free preparations. These results indicate that dopamine is physiologically released from neurons and from possible dopaminergic nerve terminals and regulates cholinergic neuronal activity in the corpus of guinea pig stomach.

  3. Cytochrome P450 mediates dopamine formation in the brain in vivo.

    PubMed

    Bromek, Ewa; Haduch, Anna; Gołembiowska, Krystyna; Daniel, Władysława A

    2011-09-01

    The cytochrome P450-mediated synthesis of dopamine from tyramine has been shown in vitro. The aim of the present study was to demonstrate the ability of rat cytochrome P450 (CYP) 2D to synthesize dopamine from tyramine in the brain in vivo. We employed two experimental models using reserpinized rats with a blockade of the classical pathway of dopamine synthesis from tyrosine. Model A estimated dopamine production from endogenous tyramine in brain structures in vivo (ex vivo measurement of a tissue dopamine level), while Model B measured extracellular dopamine produced from exogenous tyramine (an in vivo microdialysis). In Model A, quinine (a CYP2D inhibitor) given intraperitoneally caused a significant decrease in dopamine level in the striatum and nucleus accumbens and tended to fall in the substantia nigra and frontal cortex. In Model B, an increase in extracellular dopamine level was observed after tyramine given intrastructurally (the striatum). After joint administration of tyramine and quinine, the amount of the dopamine formed was significantly lower compared to the group receiving tyramine only. The results of the two complementary experimental models indicate that the hydroxylation of tyramine to dopamine may take place in rat brain in vivo, and that CYP2D catalyzes this reaction.

  4. Striatal dopamine D2/3 receptor binding following dopamine depletion in subjects at Ultra High Risk for psychosis.

    PubMed

    Bloemen, Oswald J N; de Koning, Mariken B; Gleich, Tobias; Meijer, Julia; de Haan, Lieuwe; Linszen, Don H; Booij, Jan; van Amelsvoort, Thérèse A M J

    2013-02-01

    Altered striatal dopaminergic neurotransmission is thought to be fundamental to schizophrenia. Increased presynaptic dopaminergic activity ([18F]-DOPA PET) may predate the onset of psychotic symptoms and correlates to clinical symptoms in subjects at Ultra High Risk (UHR) for developing psychosis. Postsynaptic dopaminergic neurotransmission has not been investigated yet in UHR patients. We hypothesized that synaptic dopamine concentration would be increased in UHR patients, and that synaptic dopamine concentration would be related to symptom severity. 14 UHR patients and 15 age and IQ matched controls completed an [123I]-IBZM SPECT scan at baseline and again after dopamine depletion with alpha-methyl-para-tyrosine (AMPT). We measured changes in radiotracer binding potential, compared these between UHR patients and controls, and correlated these to symptom severity. The UHR group as a whole did not differ significantly from controls. AMPT significantly reduced symptom severity in the UHR group (p=0.014). Higher synaptic dopamine concentration predicted larger reduction of positive symptoms following depletion in the UHR group (p=0.01). In UHR patients, positive symptoms responded to dopamine depletion, comparable to observations in schizophrenia, suggesting a similar mechanism. Higher synaptic dopamine concentration was associated with more severe positive symptoms and a greater reduction of these symptoms following depletion.

  5. Could Dopamine Agonists Aid in Drug Development for Anorexia Nervosa?

    PubMed Central

    Frank, Guido K. W.

    2014-01-01

    Anorexia nervosa is a severe psychiatric disorder most commonly starting during the teenage-years and associated with food refusal and low body weight. Typically there is a loss of menses, intense fear of gaining weight, and an often delusional quality of altered body perception. Anorexia nervosa is also associated with a pattern of high cognitive rigidity, which may contribute to treatment resistance and relapse. The complex interplay of state and trait biological, psychological, and social factors has complicated identifying neurobiological mechanisms that contribute to the illness. The dopamine D1 and D2 neurotransmitter receptors are involved in motivational aspects of food approach, fear extinction, and cognitive flexibility. They could therefore be important targets to improve core and associated behaviors in anorexia nervosa. Treatment with dopamine antagonists has shown little benefit, and it is possible that antagonists over time increase an already hypersensitive dopamine pathway activity in anorexia nervosa. On the contrary, application of dopamine receptor agonists could reduce circuit responsiveness, facilitate fear extinction, and improve cognitive flexibility in anorexia nervosa, as they may be particularly effective during underweight and low gonadal hormone states. This article provides evidence that the dopamine receptor system could be a key factor in the pathophysiology of anorexia nervosa and dopamine agonists could be helpful in reducing core symptoms of the disorder. This review is a theoretical approach that primarily focuses on dopamine receptor function as this system has been mechanistically better described than other neurotransmitters that are altered in anorexia nervosa. However, those proposed dopamine mechanisms in anorexia nervosa also warrant further study with respect to their interaction with other neurotransmitter systems, such as serotonin pathways. PMID:25988121

  6. Mesolimbic Dopamine Encodes Prediction Errors in a State-Dependent Manner

    PubMed Central

    Papageorgiou, Georgios K.; Baudonnat, Mathieu; Cucca, Flavia; Walton, Mark E.

    2016-01-01

    Summary Mesolimbic dopamine encodes the benefits of a course of action. However, the value of an appetitive reward depends strongly on an animal’s current state. To investigate the relationship between dopamine, value, and physiological state, we monitored sub-second dopamine release in the nucleus accumbens core while rats made choices between food and sucrose solution following selective satiation on one of these reinforcers. Dopamine signals reflected preference for the reinforcers in the new state, decreasing to the devalued reward and, after satiation on food, increasing for the valued sucrose solution. These changes were rapid and selective, with dopamine release returning to pre-satiation patterns when the animals were re-tested in a standard food-restricted state. Such rapid and selective adaptation of dopamine-associated value signals could provide an important signal to promote efficient foraging for a varied diet. PMID:27050518

  7. Behavioral, neurochemical and morphological changes induced by the overexpression of munc18-1a in brain of mice: relevance to schizophrenia

    PubMed Central

    Urigüen, L; Gil-Pisa, I; Munarriz-Cuezva, E; Berrocoso, E; Pascau, J; Soto-Montenegro, M L; Gutiérrez-Adán, A; Pintado, B; Madrigal, J L M; Castro, E; Sánchez-Blázquez, P; Ortega, J E; Guerrero, M J; Ferrer-Alcon, M; García-Sevilla, J A; Micó, J A; Desco, M; Leza, J C; Pazos, Á; Garzón, J; Meana, J J

    2013-01-01

    Overexpression of the mammalian homolog of the unc-18 gene (munc18-1) has been described in the brain of subjects with schizophrenia. Munc18-1 protein is involved in membrane fusion processes, exocytosis and neurotransmitter release. A transgenic mouse strain that overexpresses the protein isoform munc18-1a in the brain was characterized. This animal displays several schizophrenia-related behaviors, supersensitivity to hallucinogenic drugs and deficits in prepulse inhibition that reverse after antipsychotic treatment. Relevant brain areas (that is, cortex and striatum) exhibit reduced expression of dopamine D1 receptors and dopamine transporters together with enhanced amphetamine-induced in vivo dopamine release. Magnetic resonance imaging demonstrates decreased gray matter volume in the transgenic animal. In conclusion, the mouse overexpressing brain munc18-1a represents a new valid animal model that resembles functional and structural abnormalities in patients with schizophrenia. The animal could provide valuable insights into phenotypic aspects of this psychiatric disorder. PMID:23340504

  8. The Role of Dopamine and Its Dysfunction as a Consequence of Oxidative Stress.

    PubMed

    Juárez Olguín, Hugo; Calderón Guzmán, David; Hernández García, Ernestina; Barragán Mejía, Gerardo

    2016-01-01

    Dopamine is a neurotransmitter that is produced in the substantia nigra, ventral tegmental area, and hypothalamus of the brain. Dysfunction of the dopamine system has been implicated in different nervous system diseases. The level of dopamine transmission increases in response to any type of reward and by a large number of strongly additive drugs. The role of dopamine dysfunction as a consequence of oxidative stress is involved in health and disease. Introduce new potential targets for the development of therapeutic interventions based on antioxidant compounds. The present review focuses on the therapeutic potential of antioxidant compounds as a coadjuvant treatment to conventional neurological disorders is discussed.

  9. The Role of Dopamine and Its Dysfunction as a Consequence of Oxidative Stress

    PubMed Central

    Juárez Olguín, Hugo; Calderón Guzmán, David; Hernández García, Ernestina; Barragán Mejía, Gerardo

    2016-01-01

    Dopamine is a neurotransmitter that is produced in the substantia nigra, ventral tegmental area, and hypothalamus of the brain. Dysfunction of the dopamine system has been implicated in different nervous system diseases. The level of dopamine transmission increases in response to any type of reward and by a large number of strongly additive drugs. The role of dopamine dysfunction as a consequence of oxidative stress is involved in health and disease. Introduce new potential targets for the development of therapeutic interventions based on antioxidant compounds. The present review focuses on the therapeutic potential of antioxidant compounds as a coadjuvant treatment to conventional neurological disorders is discussed. PMID:26770661

  10. Expression of dopamine D2 receptor in PC-12 cells and regulation of membrane conductances by dopamine.

    PubMed

    Zhu, W H; Conforti, L; Millhorn, D E

    1997-10-01

    PC-12 cells depolarize during hypoxia and release dopamine. The hypoxia-induced depolarization is due to inhibition of an O2-sensitive K+ current. The role of dopamine released during hypoxia is uncertain, but it could act as an autocrine to modulate membrane conductance during hypoxia. The current study was undertaken to investigate this possibility. Reverse transcription-polymerase chain reaction and sequence analysis revealed that the D2 isoform of the dopamine receptor is expressed in rat PC-12 cells. Exogenously applied dopamine and the D2 agonist quinpirole elicited inhibition of a voltage-dependent K+ current (I(K)) that was prevented by sulpiride, a D2 receptor antagonist. Dopamine and quinpirole applied during hypoxia potentiated the inhibitory effect of hypoxia on I(K). We also found that quinpirole caused reversible inhibition of a voltage-dependent Ca2+ current (I(Ca)) and attenuation of the increase in intracellular free Ca2+ during hypoxia. Our results indicate that dopamine released from PC-12 cells during hypoxia acts via a D2 receptor to "autoregulate" I(K) and I(Ca). PMID:9357757

  11. Addiction: Beyond dopamine reward circuitry

    SciTech Connect

    Volkow, N.D.; Wang, G.; Volkow, N.D.; Wang, G.-J.; Fowler, J.S.; Tomasi, D.; Telang, F.

    2011-09-13

    Dopamine (DA) is considered crucial for the rewarding effects of drugs of abuse, but its role in addiction is much less clear. This review focuses on studies that used PET to characterize the brain DA system in addicted subjects. These studies have corroborated in humans the relevance of drug-induced fast DA increases in striatum [including nucleus accumbens (NAc)] in their rewarding effects but have unexpectedly shown that in addicted subjects, drug-induced DA increases (as well as their subjective reinforcing effects) are markedly blunted compared with controls. In contrast, addicted subjects show significant DA increases in striatum in response to drug-conditioned cues that are associated with self-reports of drug craving and appear to be of a greater magnitude than the DA responses to the drug. We postulate that the discrepancy between the expectation for the drug effects (conditioned responses) and the blunted pharmacological effects maintains drug taking in an attempt to achieve the expected reward. Also, whether tested during early or protracted withdrawal, addicted subjects show lower levels of D2 receptors in striatum (including NAc), which are associated with decreases in baseline activity in frontal brain regions implicated in salience attribution (orbitofrontal cortex) and inhibitory control (anterior cingulate gyrus), whose disruption results in compulsivity and impulsivity. These results point to an imbalance between dopaminergic circuits that underlie reward and conditioning and those that underlie executive function (emotional control and decision making)