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Nature Protocols Aug 2014This protocol describes a method for the laboratory synthesis of enantiomerically enriched, chiral tetrahydroisoquinolines through the application of a chiral...
This protocol describes a method for the laboratory synthesis of enantiomerically enriched, chiral tetrahydroisoquinolines through the application of a chiral sulfinamido urea catalyst for the Povarov reaction. Tetrahydroisoquinolines are bicyclic organic frameworks present in a wide assortment of natural and synthetic biologically important compounds including martinelline, scoulerine and tubocurarine. The methodology involves the [4+2] cycloaddition of a N-arylimines with electron-rich olefins such as vinyl lactams and dihydropyrroles in the presence of a two-catalyst system consisting of an achiral strong Brønsted acid (o-nitrobenzenesulfonic acid), together with the chiral sulfinamido urea derivative 1. The anion-binding properties of the urea lead to the association of the ion pair that results from protonation of the imine substrate. Cycloaddition is followed by spontaneous proton loss with re-aromatization to provide the tetrahydroisoquinoline products in highly enantio-enriched form.
Topics: Alkenes; Benzenesulfonates; Cycloaddition Reaction; Molecular Conformation; Tetrahydroisoquinolines
PubMed: 25010906
DOI: 10.1038/nprot.2014.125 -
Journal of Neurogenetics 2016Cholinergic dysfunction contributes to cognitive deficits in schizophrenia. The atypical antipsychotic clozapine improves cognition in patients with schizophrenia,...
Cholinergic dysfunction contributes to cognitive deficits in schizophrenia. The atypical antipsychotic clozapine improves cognition in patients with schizophrenia, possibly through modulation of the cholinergic system. However, little is known about specific underlying mechanisms. We investigated the acute and chronic effects of clozapine on cholinergic synaptic transmission in cultured superior cervical ganglion (SCG) neurons. Spontaneous excitatory postsynaptic currents (sEPSCs) were detected and were reversibly inhibited by the nicotinic receptor antagonist d-tubocurarine, confirming that the synaptic responses were primarily mediated by nicotinic receptors. Bath application of clozapine at therapeutic concentrations rapidly and reversely inhibited both the amplitude and frequency of sEPSCs in a concentration-dependent manner, without changing either rise or decay time, suggesting that clozapine effects have both presynaptic and postsynaptic origins. The acute effects of clozapine on sEPSCs were recapitulated by chronic treatment of SCG cultures with similar concentrations of clozapine, as clozapine treatment for 4 d reduced the frequency and amplitude of sEPSCs without affecting their kinetics. Cell survival analysis indicated that SCG neuron cell counts after chronic clozapine treatment were comparable to the control group. These results demonstrate that therapeutic concentrations of clozapine suppress nicotinic synaptic transmission in SCG cholinergic synapses, a simple in vitro preparation of cholinergic transmission.
Topics: Animals; Antipsychotic Agents; Cells, Cultured; Clozapine; Excitatory Postsynaptic Potentials; Mice; Neurons; Superior Cervical Ganglion; Synaptic Transmission
PubMed: 27627024
DOI: 10.1080/01677063.2016.1229779 -
Advances in Physiology Education Jun 2017This study describes an undergraduate student laboratory activity using isolated preparations from rat gastrointestinal tissues that possess contractile profiles...
This study describes an undergraduate student laboratory activity using isolated preparations from rat gastrointestinal tissues that possess contractile profiles typically exhibited by striated and smooth muscle cells. While students are introduced to an ex vivo methodology, they can compare differences in trace experiments, twitch aspects, phasic and tonic properties, force-frequency relationships, and pharmacological responsiveness of esophageal (striated) and fundic (smooth muscle) segments. Muscle strips were subjected to electrical field stimulation (EFS) applied by platinum electrodes immersed in the physiological solution. The contractile profile of EFS responses varied between these two types of gut preparations. Atropine and tubocurarine revealed differential inhibitory influences in esophagus or fundus tissues; caffeine and procaine produced similar effects, i.e., potentiation and blockade of the EFS-induced contractile response in these tissues, respectively. Experimental results obtained during the activity helped the improvement of student learning about basic concepts previously discussed in theoretical lectures. To measure student learning with this laboratory exercise, a questionnaire was applied before and after the activity, and the number of expected correct answers, concerning the mechanisms of contraction in striated and smooth muscle, could be clearly evidenced.
Topics: Animals; Electric Stimulation; Esophagus; In Vitro Techniques; Muscle Contraction; Muscle, Smooth; Muscle, Striated; Physiology; Rats; Stomach
PubMed: 28526693
DOI: 10.1152/advan.00150.2016 -
The Journal of Organic Chemistry Jan 2017Two consecutive Cu-catalyzed Ullmann-type C-O couplings permitted the first successful entry toward the curare alkaloids (±)-tubocurine and (±)-curine. Starting from...
Two consecutive Cu-catalyzed Ullmann-type C-O couplings permitted the first successful entry toward the curare alkaloids (±)-tubocurine and (±)-curine. Starting from vanillin, the synthetic sequence comprises 15 linear steps and includes a total of 24 transformations. In addition, the total synthesis of tubocurine represents a formal total synthesis of the famous arrow poison alkaloid tubocurarine.
Topics: Benzaldehydes; Catalysis; Chemistry, Organic; Copper; Isoquinolines; Molecular Structure; Tubocurarine
PubMed: 27997804
DOI: 10.1021/acs.joc.6b02647 -
Neuropharmacology Nov 2014VUF10166 (2-chloro-3-(4-methyl piperazin-1-yl)quinoxaline) is a ligand that binds with high affinity to 5-HT3 receptors. Here we synthesise [(3)H]VUF10166 and...
VUF10166 (2-chloro-3-(4-methyl piperazin-1-yl)quinoxaline) is a ligand that binds with high affinity to 5-HT3 receptors. Here we synthesise [(3)H]VUF10166 and characterise its binding properties at 5-HT3A and 5-HT3AB receptors. At 5-HT3A receptors [(3)H]VUF10166 displayed saturable binding with a Kd of 0.18 nM. Kinetic measurements gave monophasic association (6.25 × 10(7) M(-1) min(-1)) and dissociation (0.01 min(-1)) rates that yielded a similar Kd value (0.16 nM). At 5-HT3AB receptors two association (6.15 × 10(-7), 7.23 M(-1) min(-1)) and dissociation (0.024, 0.162 min(-1)) rates were seen, yielding Kd values (0.38 nM and 22 nM) that were consistent with values obtained in saturation (Kd = 0.74 nM) and competition (Ki = 37 nM) binding experiments respectively. At both receptor types, specific binding was inhibited by classical 5-HT3 receptor-selective orthosteric ligands (5-HT, allosetron, d-tubocurarine, granisetron, mCPBG, MDL72222, quipazine), but not by non-competitive antagonists (bilobalide, ginkgolide B, picrotoxin) or competitive ligands of other Cys-loop receptors (ACh, bicuculline, glycine, gabazine). To explore VUF10166 ligand-receptor interactions we used in silico modelling and docking, and tested the predictions using site directed mutagenesis. The data suggest that VUF10166 adopts a similar orientation to 5-HT3 receptor agonists bound in AChBP (varenicline) and 5HTBP (5-HT) crystal structures.
Topics: Amino Acid Sequence; Binding, Competitive; HEK293 Cells; Humans; Molecular Docking Simulation; Molecular Sequence Data; Mutagenesis, Site-Directed; Piperidines; Quinoxalines; Radioligand Assay; Radiopharmaceuticals; Receptors, Serotonin, 5-HT3; Sequence Alignment; Tritium
PubMed: 25174552
DOI: 10.1016/j.neuropharm.2014.08.008 -
IBRO Reports Dec 2020Correlated spontaneous activity propagating over a wide region of the central nervous system is expressed during a specific period of embryonic development. We...
Correlated spontaneous activity propagating over a wide region of the central nervous system is expressed during a specific period of embryonic development. We previously demonstrated using an optical imaging technique with a voltage-sensitive dye that this wave-like activity, which we referred to as the depolarization wave, is fundamentally involved in the early process of synaptic network formation. We found that the application of bicuculline/strychnine or tubocurarine, which blocked the neurotransmitters mediating the wave, significantly reduced functional synaptic expression in the brainstem sensory nucleus. This result, particularly for -tubocurarine, an antagonist of nicotinic acetylcholine receptors, suggested that prenatal nicotine exposure associated with maternal smoking affects the development of neural circuit formation by interfering with the correlated wave. In the present study, we tested this hypothesis by examining the effects of nicotine on the correlated activity and assessing the chronic action of nicotine on functional synaptic expression along the vagal sensory pathway. observations of chick embryo behavior and electrical recording using preparations showed that the application of nicotine transiently increased embryonic movements and electrical bursts associated with the wave, but subsequently inhibited these activities, suggesting that the dominant action of the drug was to inhibit the wave. Optical imaging with the voltage-sensitive dye showed that the chronic exposure to nicotine markedly reduced functional synaptic expression in the higher-order sensory nucleus of the vagus nerve, the parabrachial nucleus. The results suggest that prenatal nicotine exposure disrupts the initial formation of the neural circuitry by inhibiting correlated spontaneous wave activity.
PubMed: 32642591
DOI: 10.1016/j.ibror.2020.06.003 -
The Journal of Toxicological Sciences 2018The increased ratio of longer amyloid-β (Aβ1-42)/shorter amyloid-β (Aβ1-40) peptides, generated from amyloid precursor protein (APP), is known to promote the...
The increased ratio of longer amyloid-β (Aβ1-42)/shorter amyloid-β (Aβ1-40) peptides, generated from amyloid precursor protein (APP), is known to promote the development of Alzheimer's disease (AD). To investigate the role of smoking in Aβ production, we determined the production of Aβ species in the presence of nicotine or methyl vinyl ketone (MVK), major components of cigarette smoke extracts, in Flp-In T-REx-293 (T-REx293) cells harboring a single copy of human APP. While treatment with nicotine or MVK did not affect the amount of APP, the levels of Aβ1-40 in the culture media were significantly increased. On the other hand, the levels of Aβ1-42 were unaltered by nicotine or MVK treatment. The Aβ1-42/Aβ1-40 ratio was therefore attenuated by cigarette smoke extracts. Similar results were obtained in T-REx293 cells harboring APP of Swedish- or London-type mutation linked to familial AD. T-REx293 cells expressed the nicotinic acetylcholine receptor (nAchR) and tubocurarine, an nAChR antagonist, completely blocked the effects of nicotine. Treatment with nicotine significantly elevated cellular levels of β-secretase that cleaves APP prior to Aβ generation. Taken together, a protective role of nicotine against AD pathology was suggested by enhanced extracellular Aβ1-40 production, which may suppress Aβ fibrillogenesis.
Topics: Alzheimer Disease; Amyloid Precursor Protein Secretases; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Butanones; Cells, Cultured; Cigarette Smoking; Depression, Chemical; Humans; Nicotine; Tobacco Products
PubMed: 29618714
DOI: 10.2131/jts.43.257 -
Comparative Biochemistry and... Dec 2014Anurans (frogs and toads) are important models for comparative studies of communication, auditory physiology, and neuroethology, but to date, most of our knowledge comes...
Anurans (frogs and toads) are important models for comparative studies of communication, auditory physiology, and neuroethology, but to date, most of our knowledge comes from in-depth studies of a relatively small number of model species. Using the well-studied green treefrog (Hyla cinerea), this study sought to develop and evaluate the use of auditory evoked potentials (AEPs) as a minimally invasive tool for investigating auditory sensitivity in a larger diversity of anuran species. The goals of the study were to assess the effects of frequency, signal level, sex, and body size on auditory brainstem response (ABR) amplitudes and latencies, characterize gross ABR morphology, and generate an audiogram that could be compared to several previously published audiograms for green treefrogs. Increasing signal level resulted in larger ABR amplitudes and shorter latencies, and these effects were frequency dependent. There was little evidence for an effect of sex or size on ABRs. Analyses consistently distinguished between responses to stimuli in the frequency ranges of the three previously-described populations of afferents that innervate the two auditory end organs in anurans. The overall shape of the audiogram shared prominent features with previously published audiograms. This study highlights the utility of AEPs as a valuable tool for the study of anuran auditory sensitivity.
Topics: Animals; Anura; Auditory Threshold; Brain Stem; Evoked Potentials, Auditory, Brain Stem; Feedback, Physiological; Female; Immobilization; Male; Models, Biological; Neuromuscular Nondepolarizing Agents; Neurons; Reproducibility of Results; Sex Characteristics; Species Specificity; Texas; Tubocurarine; Vocalization, Animal; Weight Gain
PubMed: 25151643
DOI: 10.1016/j.cbpa.2014.08.005 -
Biophysical Journal Oct 2017Tip links are thought to gate the mechanically sensitive transduction channels of hair cells, but how they form during development and regeneration remains mysterious....
Tip links are thought to gate the mechanically sensitive transduction channels of hair cells, but how they form during development and regeneration remains mysterious. In particular, it is unclear how tip links are strung between stereocilia so that they are oriented parallel to a single axis; why their polarity is uniform despite their constituent molecules' intrinsic asymmetry; and why only a single tip link is present at each tip-link position. We present here a series of simple rules that reasonably explain why these phenomena occur. In particular, our model relies on each of the two ends of the tip link having distinct Ca-dependent stability and being connected to different motor complexes. A simulation employing these rules allowed us to explore the parameter space for the model, demonstrating the importance of the feedback between transduction channels and angled links, links that are 60° off-axis with respect to mature tip links. We tested this key aspect of the model by examining angled links in chick cochlea hair cells. As implied by the assumptions used to generate the model, we found that angled links were stabilized if there was no tip link at the tip of the upper stereocilium, and appeared when transduction channels were blocked. The model thus plausibly explains how tip-link formation and pruning can occur.
Topics: Animals; Calcium; Chelating Agents; Chickens; Computer Simulation; Egtazic Acid; Epithelium; Hair Cells, Auditory; Microscopy, Electron, Scanning; Models, Biological; Neuromuscular Nondepolarizing Agents; Stereocilia; Stochastic Processes; Tissue Culture Techniques; Tubocurarine
PubMed: 29045880
DOI: 10.1016/j.bpj.2017.08.029 -
The Journal of Physiological Sciences :... Mar 2016To determine whether a capsaicin-sensitive local neural circuit constitutively modulates vagal neuromuscular transmission in the esophageal striated muscle or whether...
To determine whether a capsaicin-sensitive local neural circuit constitutively modulates vagal neuromuscular transmission in the esophageal striated muscle or whether the neural circuit operates in a stimulus-dependent manner, we compared the motility of esophageal preparations isolated from intact rats with those in which capsaicin-sensitive neurons had been destroyed. Electrical stimulation of the vagus nerve trunk evoked contractile responses in the esophagus isolated from a capsaicin-treated rat in a manner similar to those in the esophagus from a control rat. No obvious differences were observed in the inhibitory effects of D-tubocurarine on intact and capsaicin-treated rat esophageal motility. Destruction of the capsaicin-sensitive neurons did not significantly affect latency, time to peak and duration of a vagally evoked twitch-like contraction. These findings indicate that the capsaicin-sensitive neural circuit does not operate constitutively but rather is activated in response to an applied stimulus.
Topics: Animals; Capsaicin; Electric Stimulation; Esophagus; Male; Muscle Contraction; Muscle, Striated; Neurons; Rats; Rats, Sprague-Dawley; Tubocurarine; Vagus Nerve
PubMed: 26424590
DOI: 10.1007/s12576-015-0401-8