scholarly journals Brief, repeated exposure to substrates down-regulates dopamine transporter function in Xenopus oocytes in vitro and rat dorsal striatum in vivo

2002 ◽  
Vol 83 (2) ◽  
pp. 400-411 ◽  
Author(s):  
Joshua M. Gulley ◽  
Suzanne Doolen ◽  
Nancy R. Zahniser
Keyword(s):  
Dopamine Transporter ◽  
Xenopus Oocytes ◽  
Dorsal Striatum ◽  
Repeated Exposure

scholarly journals Psychomotor stimulant effects of α-pyrrolidinovalerophenone (αPVP) enantiomers correlate with drug binding kinetics at the dopamine transporter

2021 ◽  
Author(s):  
Marco Niello ◽  
Spyridon Sideromenos ◽  
Ralph Gradisch ◽  
Ronan O'Shea ◽  
Jakob Schwazer ◽  
...  
Keyword(s):  
Dopamine Transporter ◽  
In Silico ◽  
Drug Binding ◽  
Binding Kinetics ◽  
Irreversible Binding ◽  
Fast Kinetics ◽  
Slow Dissociation ◽  
Kinetics Of

Abstract α-Pyrrolidinovalerophenone (αPVP) is a psychostimulant and drug of abuse associated with severe intoxications in humans. αPVP exerts long-lasting psychostimulant effects, when compared to the classical dopamine transporter (DAT) inhibitor cocaine. Here, we compared the two enantiomeric forms of αPVP, the R- and the S-αPVP, with cocaine using a combination of in silico, in vitro and in vivo approaches. We found that αPVP enantiomers substantially differ from cocaine in their binding kinetics. The two enantiomers differ from each other in their association rates. However, they show similar slow dissociation rates leading to pseudo-irreversible binding kinetics at DAT. The pseudo-irreversible binding kinetics of αPVP is responsible for the observed non-competitive pharmacology and it correlates with persistent psychostimulant effects in mice. Thus, the slow binding kinetics of αPVP enantiomers profoundly differ from the fast kinetics of cocaine both in vitro and in vivo, suggesting drug-binding kinetics as a potential driver of psychostimulant effects in vivo.

In Vivo PET Measurements with [11C]PE2I to Evaluate Fetal Mesencephalic Transplantations to Unilateral 6-OHDA-Lesioned Rats

2005 ◽  
Vol 14 (9) ◽  
pp. 655-663 ◽  
Author(s):  
Motoki Inaji ◽  
Takahito Yoshizaki ◽  
Takashi Okauchi ◽  
Jun Maeda ◽  
Yuji Nagai ◽  
...  
Keyword(s):  
Dopamine Transporter ◽  
High Performance ◽  
Emission Tomography ◽  
Binding Potential ◽  
In Vitro Autoradiography ◽  
Positron Emission ◽  
Pet Scans ◽  
Mesencephalic Cells

Positron emission tomography (PET) is a useful tool to assess and visualize neurotransmissions in vivo. In this study, we performed repeated PET scans with [11C]PE2I, a tracer of the dopamine transporter, to evaluate the alteration of the expression of dopamine (DA) transmission component after a fetal mesencephalic transplantation. The fetal mesencephalic cells were transplanted into the striatum of unilateral 6-OHDA-lesioned rats. PET scans with [11C]PE2I were performed to evaluate the DA transporter before and 2 and 4 weeks after the transplantation. Rotation behavior tests, in vitro autoradiography, measurements of DA contents in the striatum by high-performance liquid chromatography (HPLC), and tyrosine hydroxylase (TH) immuno-histological examinations were performed at the same time points and examined for their relationship to changes in the dopamine transporter. The number of ipsilateral rotations induced by methamphetamine injections decreased. DA contents in the striatum measured with HPLC significantly increased. In the PET study, the binding potential of [11C]PE2I increased at 4 weeks. The results of the in vitro autoradiography study corresponded with those of the PET study. The degrees of the change in the binding potentials correlated with those of the numbers of rotations in the behavioral study and the DA contents in the striatum. In the histological examination, TH-positive cells with axons were observed at 2 and 4 weeks after the transplantation. As the dopamine transporter exists only in the axon terminal of DA neurons, these results suggested that PET measurements of [11C]PE2I binding indicated not only survival, but maturity and functioning of the transplanted cells. Repeated PET measurements of DA transporters are a useful tool in assessing the effectiveness of neural transplantations.

scholarly journals Striatal Syntaixin 1A Plays a Protective Role Against Iminodipropionitrile Induced Tic Disorder Through Interaction with Dopamine Transporter

2022 ◽  
Author(s):  
Xiumei Liu ◽  
Xueming Wang ◽  
Xiaoling Zhang ◽  
Aihua Cao
Keyword(s):  
Dopamine Transporter ◽  
Dopaminergic Neurons ◽  
Stereotyped Behavior ◽  
Protective Role ◽  
Snare Complex ◽  
Pathological State ◽  
Tic Disorder

Abstract An important mechanism of Tic disorder (TD) is dysfunction in the dopamine (DA) system. Our pilot observation found the expression of Syntaxin 1A (STX1A), a presynaptic SNARE complex, changed in the striatum of TD animals. The present study aimed to clarify the biological role of striatal STX1A in the pathological state of TD and the specific mechanism of its regulation of the dopaminergic system. The TD rat model was established using iminodipropionitrile (IDPN). Adenovirus was used to modulate the expression of STX1A and dopamine transporter (DAT) in vivo and vitro. Primary culture of striatal dopaminergic neurons was performed for in-vitro observation of the DA reuptake, CO-IP analysis of the interaction between STX1A and DAT. First, using immunofluorescence staining, Western blotting, and qPCR, we found that the IDPN induced TD model had reduced striatal STX1A expression. In vitro, the DA content in the supernatant was significantly lower in the STX1A overexpressed group, and the intracellular DA content was significantly higher. Overexpression of STX1A in vivo partially counteracts the IDPN-induced TD-like behaviors, including bite time and head shaking time. Meanwhile, in-vivo knockdown of STX1A can aggravates TD-like behaviors. Further, DAT was overexpressed in vivo, and the TD-like behavior was alleviated. Interestingly, overexpression of DAT in the striatum resulted in increased levels of STX1A. In order to clarify the interaction between DAT and STX1A, the CO-IP analysis was conducted based on the protein of purified striatal dopaminergic neurons. Compared to the IgG control, the blots of DAT and STX1A showed significant binding of each other. Striatal STX1A expression is decreased in TD development, and STX1A plays an anti-TD role possibly through interaction with DAT, which maintains the DA reuptake. The exorbitant DA signal caused by STX1A inhibition drives the pathological stereotyped behavior.

scholarly journals 14-3-3 is not essential for Raf-1 function: identification of Raf-1 proteins that are biologically activated in a 14-3-3- and Ras-independent manner.

1995 ◽  
Vol 15 (6) ◽  
pp. 3390-3397 ◽  
Author(s):  
N R Michaud ◽  
J R Fabian ◽  
K D Mathes ◽  
D K Morrison
Keyword(s):  
Enzymatic Activity ◽  
Xenopus Oocytes ◽  
Serine Phosphorylation ◽  
General Mechanism ◽  
Independent Manner ◽  
293 Cells ◽  
Presence And Absence ◽  
A Site

Recent reports have demonstrated the in vivo association of Raf-1 with members of the 14-3-3 protein family. To address the significance of the Raf-1-14-3-3 interaction, we investigated the enzymatic activity and biological function of Raf-1 in the presence and absence of associated 14-3-3. The interaction between these two molecules was disrupted in vivo and in vitro with a combination of molecular and biochemical techniques. Biochemical studies demonstrated that the enzymatic activities of Raf-1 were equivalent in the presence and absence of 14-3-3. Furthermore, mixing of purified Raf-1 and 14-3-3 in vitro was not sufficient to activate Raf-1. With a molecular approach, Cys-165 and Cys-168 as well as Ser-259 were identified as residues of Raf-1 required for the interaction with 14-3-3. Cys-165 and Cys-168 are located within the conserved cysteine-rich region of the CR1 domain, and Ser-259 is a conserved site of serine phosphorylation found within the CR2 domain. Mutation of either Cys-165 and Cys-168 or Ser-259 prevented the stable interaction of Raf-1 with 14-3-3 in vivo. Consistent with the model in which a site of serine phosphorylation is involved in the Raf-1-14-3-3 interaction, dephosphorylated Raf-1 was unable to associate with 14-3-3 in vitro. Phosphorylation may represent a general mechanism mediating 14-3-3 binding, because dephosphorylation of the Bcr kinase (known to interact with 14-3-3) also eliminated its association with 14-3-3. Finally, mutant Raf-1 proteins unable to stably interact with 14-3-3 exhibited enhanced enzymatic activity in human 293 cells and Xenopus oocytes and were biologically activated, as demonstrated by their ability to induced meiotic maturation of Xenopus oocytes. However, in contrast to wild-type Raf-1, activation of these mutants was independent of Ras. Our results therefore indicate that interaction with 14-3-3 is not essential for Raf-1 function.

Development and Application of In Vitro 3D Liver Models for more Physiologically Relevant Hazard Assessment of Engineered Nanomaterials

2021 ◽  
Author(s):  
◽  
Samantha V. Llewellyn
Keyword(s):  
Hazard Assessment ◽  
Prolonged Exposure ◽  
Repeated Exposure ◽  
Engineered Nanomaterials ◽  
Liver Model ◽  
Significant Difference ◽  
Pre Treatment ◽  
Exposure Routes

Due to the expanding use of nanotechnology in consumer applications, human and environmental exposure to engineered nanomaterials (ENM) is inevitable. Hepatic toxicology is important when considering ENM exposure, as the liver is the major site of ENM secondary deposition and accumulation post exposure, as well as being vital in metabolic homeostasis and detoxification. The vast range of ENMs available deems it untenable to rely on in vivo based methods to elucidate the immediate and lasting effects of ENM exposure. Therefore, this research project aimed to develop an advanced 3D in vitro liver model with enhanced physiological relevance to better understand the human health hazards, specifically genotoxicity, associated with ENM exposure. The in vitro model developed was a HepG2 3D liver spheroid model with 14-day viability and liver-like functionality, as well as proliferating capabilities required to support the evaluation of fixed DNA damage endpoints. Utilising this model, the next objective was to evaluate several toxicological endpoints (e.g. liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) for a variety of ENMs (TiO2, ZnO, Ag, BaSO4 and CeO2) under different exposure regimes designed to better mimic human exposure routes. To achieve this, the ENM were 1) pre-treated in a series of biological simulant fluids to mimic inhalation and ingestion exposure routes, and 2) applied to the 3D liver model for both short- (24hr) and prolonged (120hr) single-bolus, and repeated-fractionated daily ENM exposure regimes, prior to hazard characterisation. The effects of material biotransformation upon reactivity, cytotoxicity, (pro-)inflammatory and genotoxic potential of Ag and TiO2 was demonstrated, and illustrated that the necessity of ENM pre-treatment prior to in vitro hazard assessment should be reserved for ENM that exhibit high degrees of physico-chemical transformation and reactivity (i.e. a tiered testing strategy). When comparing dosing durations, no cytotoxicity or significant reduction in liver-like functionality was observed across either acute, prolonged or repeated exposure regimes. Acute exposure to all ENMs induced a significant increase (p≤0.05) in genotoxicity, albeit not dose-dependently. ZnO, which rapidly dissolves into ions, was the only material to exhibit genotoxicity at both an acute and prolonged exposure. For the materials selected in this study, there was no significant difference between prolonged, bolus or repeated exposure regimes, indicating that the added complexity of fractionated dosing was not necessary. In conclusion, 3D in vitro hepatic spheroid models have the capacity to be utilised for evaluating more realistic ENM exposures, thereby providing a future approach to better support in vitro ENM hazard assessment in a routine and easily accessible manner.

scholarly journals Cyclin B/cdc2 Induces c-Mos Stability by Direct Phosphorylation in Xenopus Oocytes

2001 ◽  
Vol 12 (9) ◽  
pp. 2660-2671 ◽  
Author(s):  
Anna Castro ◽  
Marion Peter ◽  
Laura Magnaghi-Jaulin ◽  
Suzanne Vigneron ◽  
Simon Galas ◽  
...  
Keyword(s):  
Xenopus Oocytes ◽  
Phosphorylation Site ◽  
Cyclin B ◽  
Mobility Shift ◽  
Cdc2 Kinase ◽  
Phosphopeptide Analysis ◽  
Vertebrate Eggs ◽  
The One

The c-Mos proto-oncogene product plays an essential role during meiotic divisions in vertebrate eggs. In Xenopus, it is required for progression of oocyte maturation and meiotic arrest of unfertilized eggs. Its degradation after fertilization is essential to early embryogenesis. In this study we investigated the mechanisms involved in c-Mos degradation. We present in vivo evidence for ubiquitin-dependent degradation of c-Mos in activated eggs. We found that c-Mos degradation is not directly dependent on the anaphase-promoting factor activator Fizzy/cdc20 but requires cyclin degradation. We demonstrate that cyclin B/cdc2 controls in vivo c-Mos phosphorylation and stabilization. Moreover, we show that cyclin B/cdc2 is capable of directly phosphorylating c-Mos in vitro, inducing a similar mobility shift to the one observed in vivo. Tryptic phosphopeptide analysis revealed a practically identical in vivo and in vitro phosphopeptide map and allowed identification of serine-3 as the largely preferential phosphorylation site as previously described ( Freeman et al., 1992 ). Altogether, these results demonstrate that, in vivo, stability of c-Mos is directly regulated by cyclin B/cdc2 kinase activity.

High Affinity Hydroxypiperidine Analogues of 4-(2-Benzhydryloxyethyl)-1-(4-fluorobenzyl)piperidine for the Dopamine Transporter:  Stereospecific Interactions in Vitro and in Vivo

2003 ◽  
Vol 46 (7) ◽  
pp. 1220-1228 ◽  
Author(s):  
Sujit K. Ghorai ◽  
Charles Cook ◽  
Matthew Davis ◽  
Sylesh K. Venkataraman ◽  
Clifford George ◽  
...  
Keyword(s):  
Dopamine Transporter ◽  
High Affinity

scholarly journals Microtubule assembly in cytoplasmic extracts of Xenopus oocytes and eggs.

1987 ◽  
Vol 105 (5) ◽  
pp. 2191-2201 ◽  
Author(s):  
D L Gard ◽  
M W Kirschner
Keyword(s):  
Xenopus Oocytes ◽  
Critical Concentration ◽  
Specific Inhibitor ◽  
Bovine Brain ◽  
High Rate ◽  
Microtubule Assembly ◽  
Microtubule Networks ◽  
Brain Tubulin

We have investigated the differences in microtubule assembly in cytoplasm from Xenopus oocytes and eggs in vitro. Extracts of activated eggs could be prepared that assembled extensive microtubule networks in vitro using Tetrahymena axonemes or mammalian centrosomes as nucleation centers. Assembly occurred predominantly from the plus-end of the microtubule with a rate constant of 2 microns.min-1.microM-1 (57 s-1.microM-1). At the in vivo tubulin concentration, this corresponds to the extraordinarily high rate of 40-50 microns.min-1. Microtubule disassembly rates in these extracts were -4.5 microns.min-1 (128 s-1) at the plus-end and -6.9 microns.min-1 (196 s-1) at the minus-end. The critical concentration for plus-end microtubule assembly was 0.4 microM. These extracts also promoted the plus-end assembly of microtubules from bovine brain tubulin, suggesting the presence of an assembly promoting factor in the egg. In contrast to activated eggs, assembly was never observed in extracts prepared from oocytes, even at tubulin concentrations as high as 20 microM. Addition of oocyte extract to egg extracts or to purified brain tubulin inhibited microtubule assembly. These results suggest that there is a plus-end-specific inhibitor of microtubule assembly in the oocyte and a plus-end-specific promoter of assembly in the eggs. These factors may serve to regulate microtubule assembly during early development in Xenopus.

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