The temporal-spatial expression and functional analysis of three gustatory receptor genes in Solenopsis invicta using sweet and bitter compounds.

The insect gustatory system participates in identifying potential food sources and avoiding toxic compounds. During this process, gustatory receptors (GRs) recognize feeding stimulant and deterrent compounds. However, the GRs involved in recognizing stimulant and deterrent compounds in the red imported fire ant, Solenopsis invicta, remain unknown. Therefore, we conducted a study on the genes SinvGR1, SinvGR32b, and SinvGR28a to investigate the roles of GRs in detecting feeding stimulant and deterrent compounds. In this current study, we found that sucrose and fructose are feeding stimulants and the bitter compound quinine is a feeding deterrent. The fire ant workers showed significant behavior changes to avoid the bitter taste in feeding stimulant compounds. Reverse transcription quantitative real-time polymerase chain reaction results from developmental stages showed that the SinvGR1, SinvGR32b, and SinvGR28a genes were highly expressed in fire ant workers. Tissue-specific expression profiles indicated that SinvGR1, SinvGR32b, and SinvGR28a were specifically expressed in the antennae and foreleg tarsi of workers, whereas SinvGR32b gene transcripts were also highly accumulated in the male antennae. Furthermore, the silencing of SinvGR1 or SinvGR32b alone and the co-silencing of both genes disrupted worker stimulation and feeding on sucrose and fructose. The results also showed that SinvGR28a is required for avoiding quinine, as workers with knockdown of the SinvGR28a gene failed to avoid and fed on quinine. This study first identified stimulant and deterrent compounds of fire ant workers and then the GRs involved in the taste recognition of these compounds. This study could provide potential target gustatory genes for the control of the fire ant.

Enhancement of muscarinic receptor-mediated excitation in spontaneously hypertensive rat adrenal medullary chromaffin cells.

One of the mechanisms for hypertension is an increase in blood catecholamines due to increased secretion from sympathetic nerve terminals and adrenal medullary chromaffin (AMC) cells. Spontaneously hypertensive rats (SHRs) are used as an animal model of hypertension. Catecholamine secretion in AMC cells occurs in response to humoral factors and neuronal inputs from the sympathetic nerve fibres. Acetylcholine (ACh) released from the nerve terminals activates nicotinic as well as muscarinic ACh receptors. The present experiment aimed to elucidate whether muscarinic receptor-mediated excitation is altered in SHR AMC cells and, if it is, how. Compared with normotensive rat AMC cells, muscarinic stimulation induced greater catecholamine secretion and larger depolarising inward currents in SHR AMC cells. In contrast to normotensive rat AMC cells, the muscarine-induced current consisted of quinine-sensitive and quinine-insensitive components. The former and the latter are possibly ascribed to nonselective cation channel activation and TWIK-related acid-sensitive K+ (TASK) channel inhibition, as noted in guinea pig AMC cells. In fact, immunoreactive material for TASK1 and several isoforms of transient receptor potential canonical (TRPC) channels was detected in SHR AMC cells. Stromal interaction molecule 1 (STIM1), which plays an essential role for heteromeric TRPC1-TRPC4 channel formation and is not expressed in normotensive rat AMC cells, was detected in the cytoplasm and co-localised with TRPC1. The expression of muscarinic M1 receptors was enhanced in SHR AMC cells compared with normotensive rats. The results indicate that muscarinic excitation is enhanced in SHR AMC cells, probably through facilitation of TRPC channel signalling.

Time and charge/pH-dependent activation of K+ channel-mediated K+ influx and K+/H+ exchange in guinea pig heart isolated mitochondria; role in bioenergetic stability.

Mitochondria play an important role not only in producing energy for the cell but also for regulating mitochondrial and cell function depending on the cell's needs and environment. Uptake of cations, anions, and substrates requires a stable, polarized transmembrane charge potential (ΔΨm). Chemiosmosis requires ion exchangers to remove Na+, K+, Ca2+, PO43-, and other charged species that enter mitochondria. Knowledge of the kinetics of mitochondrial (m) cation channels and exchangers is important in understanding their roles in regulating mitochondrial chemiosmosis and bioenergetics. The influx/efflux of K+, the most abundant mitochondrial cation, alters mitochondrial volume and shape by bringing in anions and H2O by osmosis. The effects of K+ uptake through ligand-specific mK+ channels stimulated/inhibited by agonists/antagonists on mitochondrial volume (swelling/contraction) are well known. However, a more important role for K+ influx is likely its effects on H+ cycling and bioenergetics facilitated by mitochondrial (m) K+/H+ exchange (mKHE), though the kinetics and consequences of K+ efflux by KHE are not well described. We hypothesized that a major role of K+ influx/efflux is stimulation of respiration via the influx of H+ by KHE. We proposed to modulate KHE activity by energizing guinea pig heart isolated mitochondria and by altering the mK+ cycle to capture changes in mitochondrial volume, pHm, ΔΨm, and respiration that would reflect a role for H+ influx via KHE to regulate bioenergetics. To test this, mitochondria were suspended in a 150 mM K+ buffer at pH 6.9, or in a 140 mM Cs+ buffer at pH 7.6 or 6.9 with added 10 mM K+, minimal Ca2+ and free of Na+. O2 content was measured by a Clark electrode, and pHm, ΔΨm, and volume, were measured by fluorescence spectrophotometry and light-scattering. Adding pyruvic acid (PA) alone caused increases in volume and respiration and a rapid decrease in the transmembrane pH gradient (ΔpHm = pHin-pHext) at pHext 6.9> > 7.6, so that ΔΨm was charged and maintained. BKCa agonist NS1619 and antagonist paxilline modified these effects, and KHE inhibitor quinine and K+ ionophore valinomycin depolarized ΔΨm. We postulate that K+ efflux-induced H+ influx via KHE causes an inward H+ leak that stimulates respiration, but at buffer pH 6.9 also utilizes the energy of ΔpHm, the smaller component of the overall proton motive force, ΔµH+. Thus ΔpHm establishes and maintains the ΔΨm required for utilization of substrates, entry of all cations, and for oxidative phosphorylation. Thus, K+ influx/efflux appears to play a pivotal role in regulating energetics while maintaining mitochondrial ionic balance and volume homeostasis.

PQQ Dietary Supplementation Prevents Alkylating Agent-Induced Ovarian Dysfunction in Mice.

Alkylating agents (AAs) that are commonly used for cancer therapy cause great damage to the ovary. Pyrroloquinoline-quinine (PQQ), which was initially identified as a redox cofactor for bacterial dehydrogenases, has been demonstrated to benefit the fertility of females. The aim of this study was to investigate whether PQQ dietary supplementation plays a protective role against alkylating agent-induced ovarian dysfunction. A single dose of busulphan (20 mg/kg) and cyclophosphamide (CTX, 120 mg/kg) were used to establish a mouse model of ovarian dysfunction. Feed containing PQQNa2 (5 mg/kg) was provided starting 1 week before the establishment of the mouse model until the date of sacrifice. One month later, estrous cycle period of mice were examined and recorded for consecutive 30 days. Three months later, some mice were mated with fertile male mice for fertility test. The remaining mice were sacrificed to collect serum samples and ovaries. One day before sacrifice, some mice received a single injection of BrdU to label proliferating cells. Serum samples were used for test hormonal levels. Ovaries were weighted and used to detect follicle counts, cell proliferation, cell apoptosis and cell senescence. In addition, the levels of inflammation, oxidative damage and Pgc1α expression were detected in ovaries. Results showed that PQQ treatment increased the ovarian weight and size, partially normalized the disrupted estrous cycle period and prevented the loss of follicles of mice treated with AAs. More importantly, we found that PQQ treatment significantly increased the pregnancy rate and litter size per delivery of mice treated with AAs. The protective effects of PQQ appeared to be directly mediated by promoting cell proliferation of granulosa, and inhibiting cell apoptosis of granulosa and cell senescence of ovarian stromal cells. The underlying mechanisms may attribute to the anti-oxidative stress, anti-inflammation and pro-mitochondria biogenesis effects of PQQ.Our study highlights the therapeutic potential of PQQ against ovarian dysfunction caused by alkylating agents.

Perturbation of amygdala/somatostatin-nucleus of the solitary tract projections reduces sensitivity to quinine in a brief-access test.

Neural processing in the nucleus of the solitary tract (NST) is critical for concentration-dependent intake of normally preferred and avoided taste stimuli (e.g. affective responding); and is influenced by descending input from numerous forebrain regions. In one region, the central nucleus of the amygdala (CeA), a subpopulation of neurons that project to the NST express the neuropeptide somatostatin (Sst). The present study investigated whether this CeA/Sst-to-NST pathway contributes to concentration-dependent intake of sucrose and quinine hydrochloride (QHCl) solutions using brief-access lick trials (5 s). In both female and male mice, we used virus-based optogenetic tools and laser light illumination to manipulate the activity of CeA/Sst neurons that project to the NST. During light-induced inhibition of CeA/Sst-to-NST neurons, mice licked significantly more to our three highest concentrations of QHCl compared to control mice, while sucrose intake was unaffected. Interestingly, light-induced activation of this descending pathway did not influence licking of either sucrose or QHCl. These findings suggest that the CeA/Sst-to-NST pathway must be active for normal affective responding to an exemplary aversive taste stimulus.

Inhibition of Connexin 36 attenuates HMGB1-mediated depressive-like behaviors induced by chronic unpredictable mild stress.

BACKGROUND: High mobility group box 1 (HMGB1) released by neurons and microglia was demonstrated to be an important mediator in depressive-like behaviors induced by chronic unpredictable mild stress (CUMS), which could lead to the imbalance of two different metabolic approaches in kynurenine pathway (KP), thus enhancing glutamate transmission and exacerbating depressive-like behaviors. Evidence showed that HMGB1 signaling might be regulated by Connexin (Cx) 36 in inflammatory diseases of central nervous system (CNS). Our study aimed to further explore the role of Cx36 in depressive-like behaviors and its relationship with HMGB1. METHODS: After 4-week chronic stress, behavioral tests were conducted to evaluate depressive-like behaviors, including sucrose preference test (SPT), tail suspension test (TST), forced swimming test (FST), and open field test (OFT). Western blot analysis and immunofluorescence staining were used to observe the expression and location of Cx36. Enzyme-linked immunosorbent assay (ELISA) was adopted to detect the concentrations of inflammatory cytokines. And the excitability and inward currents of hippocampal neurons were recorded by whole-cell patch clamping. RESULTS: The expression of Cx36 was significantly increased in hippocampal neurons of mice exposed to CUMS, while treatment with glycyrrhizinic acid (GZA) or quinine could both down-regulate Cx36 and alleviate depressive-like behaviors. The proinflammatory cytokines like HMGB1, tumor necrosis factor alpha (TNF-α), and interleukin-1ß (IL-1ß) were all elevated by CUMS, and application of GZA and quinine could decrease them. In addition, the enhanced excitability and inward currents of hippocampal neurons induced by lipopolysaccharide (LPS) could be reduced by either GZA or quinine. CONCLUSIONS: Inhibition of Cx36 in hippocampal neurons might attenuates HMGB1-mediated depressive-like behaviors induced by CUMS through down-regulation of the proinflammatory cytokines and reduction of the excitability and intracellular ion overload.

Early and Late Steps of Quinine Biosynthesis.

The enzymatic basis for quinine 1 biosynthesis was investigated. Transcriptomic data from the producing plant led to the discovery of three enzymes involved in the early and late steps of the pathway. A medium-chain alcohol dehydrogenase (CpDCS) and an esterase (CpDCE) yielded the biosynthetic intermediate dihydrocorynantheal 2 from strictosidine aglycone 3. Additionally, the discovery of an O-methyltransferase specific for 6'-hydroxycinchoninone 4 suggested the final step order to be cinchoninone 16/17 hydroxylation, methylation, and keto-reduction.

Impact of alkaloids in food consumption, metabolism and survival in a blood-sucking insect.

The sense of taste provides information about the "good" or "bad" quality of a food source, which may be potentially nutritious or toxic. Most alkaloids taste bitter to humans, and because bitter taste is synonymous of noxious food, they are generally rejected. This response may be due to an innate low palatability or due to a malaise that occurs after food ingestion, which could even lead to death. We investigated in the kissing bug Rhodnius prolixus, whether alkaloids such as quinine, caffeine and theophylline, are merely distasteful, or if anti-appetitive responses are caused by a post-ingestion physiological effect, or both of these options. Although anti-appetitive responses were observed for the three alkaloids, only caffeine and theophylline affect metabolic and respiratory parameters that reflected an underlying physiological stress following their ingestion. Furthermore, caffeine caused the highest mortality. In contrast, quinine appears to be a merely unpalatable compound. The sense of taste helps insects to avoid making wrong feeding decisions, such as the intake of bitter/toxic foods, and thus avoid potentially harmful effects on health, a mechanism preserved in obligate hematophagous insects.

Role of P-glycoprotein in the brain disposition of seletalisib: Evaluation of the potential for drug-drug interactions.

Seletalisib is an orally bioavailable selective inhibitor of phosphoinositide 3-kinase delta (PI3Kδ) in clinical development for the treatment of immune-mediated inflammatory diseases. The present study investigated the role of P-gp in seletalisib disposition, especially brain distribution, and the associated risks of interactions. Seletalisib was found to be actively transported by rodent and human P-gp in vitro (transfected LLC-PK1 cells; Km of ca. 20 µM), with minimal or no affinity for the other tested transporters. A distribution study in knockout rats (single oral dosing at 750 mg kg-1) showed that P-gp restricts the brain disposition of seletalisib while having minimal effect on its intestinal absorption. Restricted brain penetration was also observed in cynomolgus monkeys (single oral dosing at 30 mg kg-1) using brain microdialysis and cerebrospinal fluid sampling (Kp,uu of 0.09 and 0.24, respectively). These findings opened the question of potential pharmacokinetic interaction between seletalisib and P-gp inhibitors. In vitro, CsA inhibited the active transport of seletalisib with an IC50 of 0.13 µM. In rats, co-administration of high doses of CsA (bolus iv followed by continuous infusion) increased the brain distribution of seletalisib (single oral dosing at 5 mg kg-1). The observed data were found aligned with those predicted by in vitro-in vivo extrapolation. Based on the same extrapolation method combined with literature data, only very few P-gp inhibitors (i.e. CsA, quinine, quinidine) were predicted to increase the brain disposition of seletalisib in the clinical setting (maximal 3-fold changes).

Repeatability of Taste Recognition Threshold Measurements with QUEST and Quick Yes-No.

Taste perception, although vital for nutrient sensing, has long been overlooked in sensory assessments. This can, at least in part, be attributed to challenges associated with the handling of liquid, perishable stimuli, but also with scarce efforts to optimize testing procedures to be more time-efficient. We have previously introduced an adaptive, QUEST-based procedure to measure taste sensitivity thresholds that was quicker than other existing approaches, yet similarly reliable. Despite its advantages, the QUEST procedure lacks experimental control of false alarms (i.e., response bias) and psychometric function slope. Variations of these parameters, however, may also influence the threshold estimate. This raises the question as to whether a procedure that simultaneously assesses threshold, false-alarm rate, and slope might be able to produce threshold estimates with higher repeatability, i.e., smaller variation between repeated measurements. Here, we compared the performance of QUEST with a method that allows measurement of false-alarm rates and slopes, quick Yes-No (qYN), in a test-retest design for citric acid, sodium chloride, quinine hydrochloride, and sucrose recognition thresholds. We used complementary measures of repeatability, namely test-retest correlations and coefficients of repeatability. Both threshold procedures yielded largely overlapping thresholds with good repeatability between measurements. Together the data suggest that participants used a conservative response criterion. Furthermore, we explored the link between taste sensitivity and taste liking or which we found, however, no clear association.

A bioelectronic taste sensor based on bioengineered Escherichia coli cells combined with ITO-constructed electrochemical sensors.

In this study, we developed a novel bioelectronic taste sensor for the detection of specific bitter substances. A human bitter taste receptor, hT2R4, was efficiently expressed in Escherichia coli (E. coli), which was used as the primary recognition element. A simple and low-cost electrochemical device based on ITO-based electrolyte-semiconductor (ES) structure was innovatively employed as the transducer to assess bacterial metabolic consequences of receptor activation in real time. An apparent increase in extracellular acidification rate was observed, which was resulted from the triggering of hT2R4 receptors by their target ligand of denatonium. The sensor showed dose-dependent responses to denatonuim ranging from 50 nM to 500 nM, while non-bioengineered bacteria without hT2R4 receptors exhibited negligible responses to the same stimulus. In addition, the specificity of the proposed taste biosensor was verified using other typical bitter substances such as quinine and alpha-naphthylthiourea (ANTU). This research provides a simple and inexpensive approach for the construction of bioelectronic taste sensors.

Erlotinib treatment induces cytochrome P450 3A activity in non-small cell lung cancer patients.

AIMS: Erlotinib is a tyrosine kinase inhibitor used in the treatment of non-small cell lung cancer highly metabolized by the cytochrome P450 (CYP) 3A. Hence, CYP3A4 activity might be a useful predictor of erlotinib pharmacokinetics in personalized medicine. The effect of erlotinib on CYP3A activity was therefore studied in non-small cell lung cancer patients. METHODS: The study included 32 patients scheduled for erlotinib monotherapy. CYP3A activity was assessed using quinine as a probe before and during erlotinib treatment. Plasma from blood samples drawn 16 hours post quinine administration were analysed using HPLC with fluorescence detection to determine the quinine/3-OH-quinine ratio. RESULTS: Matched samples, available from 13 patients, showed an induction of CYP3A activity (P = 0.003, Wilcoxon's signed rank test) after 2 months of treatment. The quinine/3-OH-quinine ratio decreased from 20.2 (± 13.4) at baseline to 11.0 (± 4.34). Single-point samples, available from 19 patients, supported the decrease in ratio (P = 0.007, Mann-Whitney U-test). Generally, females had a higher CYP3A activity both at baseline and after two months of treatment. Statistical analysis by gender also showed significant increase in CYP3A activity (males, n = 10, P = 0.001, and females, n = 22, P = 0.001). CONCLUSIONS: An induction of CYP3A activity was observed after 2 months of erlotinib treatment which was also seen when subdividing based on gender. It could be important to take this into consideration for patients co-administering other CYP3A-metabolizing drugs during erlotinib treatment and also makes it difficult to use baseline CYP3A activity to predict erlotinib pharmacokinetics.

Sweet and bitter taste stimuli activate VTA projection neurons in the parabrachial nucleus.

This study investigated neural projections from the parabrachial nucleus (PBN), a gustatory and visceral processing area in the brainstem, to the ventral tegmental area (VTA) in the midbrain. The VTA contains a large population of dopaminergic neurons that have been shown to play a role in reward processing. Anterograde neural tracing methods were first used to confirm that a robust projection from the caudal PBN terminates in the dorsal VTA; this projection was larger on the contralateral side. In the next experiment, we combined dual retrograde tracing from the VTA and the gustatory ventral posteromedial thalamus (VPMpc) with taste-evoked Fos protein expression, which labels activated neurons. Mice were stimulated through an intraoral cannula with sucrose, quinine, or water, and PBN sections were processed for immunofluorescent detection of Fos and retrograde tracers. The distribution of tracer-labeled PBN neurons demonstrated that the populations of cells projecting to the VTA or VPMpc are largely independent. Quantification of cells double labeled for Fos and either tracer demonstrated that sucrose and quinine were effective in activating both pathways. These results indicate that information about both appetitive and aversive tastes is delivered to a key midbrain reward interface via direct projections from the PBN.

Hen protein-derived peptides as the blockers of human bitter taste receptors T2R4, T2R7 and T2R14.

Bitter sensation is mediated by various bitter taste receptors (T2Rs), thus T2R antagonists are actively explored. Our objective was to look for novel T2R blockers in hen protein hydrolysate (HPH). We screened the least bitter HPH fractions using electronic tongue, and analyzed their peptide sequences and calcium mobilization in HEK293T cells expressing T2Rs. The results showed that the HPH fractions with higher bitterness intensity had higher hydrophobicity, more hydrophobic amino acids, and more positively charged peptides, but fewer known umami peptides. The peptide fractions from the least bitter HPH fraction significantly inhibited quinine bitterness (P < 0.05), and also significantly inhibited quinine- or diphenhydramine-dependent calcium mobilization of HEK293T cells expressing human T2R4, T2R7, or T2R14 (P < 0.05). Among them, the first eluted (least bitter) peptide fraction showed the strongest bitter-inhibitory effect. In conclusion, HPH peptides are the blockers of T2R4, T2R7, and T2R14.

Endophyte composition and Cinchona alkaloid production abilities of Cinchona ledgeriana cultivated in Japan.

New eight endophytic filamentous fungi were isolated from the young stems of Cinchona ledgeriana (Rubiaceae) cultivated in Japan. They were classified into four genera based on phylogenetic analysis of the nucleotide sequences of the internal transcribed spacers (ITS1 and ITS2), including the 5.8S ribosomal DNA region. Of the eight fungi isolated, there were five genera Cladosporium, one Meira sp., one Diaporthe sp. and one Penicillium sp. Genus of Cladosporium and Meira were first isolated fungi from Cinchona plant. In a previous study, we applied the same process to the same plant cultivated in Indonesia. The endophyte compositions for the two cultivation regions were found to differ at the genera level. The ability of Cinchona endophytes cultivated in Japan to produce Cinchona alkaloids was also assessed. We found that three isolates have producing ability of Cinchona alkaloids. However, the amount produced was very small compared to that produced by the endophytes of Indonesian Cinchona ledgeriana. In addition, the total content amount of Cinchona alkaloids, especially quinine, produced by the extract of Cinchona cultivated in Japan was much smaller than that from Indonesia. These finding indicate that endophyte composition has an influence on the Cinchona alkaloid content amount in the Cinchona ledgeriana host.

Beef Protein-Derived Peptides as Bitter Taste Receptor T2R4 Blockers.

The aim of this work was to determine the T2R4 bitter taste receptor-blocking ability of enzymatic beef protein hydrolysates and identified peptide sequences. Beef protein was hydrolyzed with each of six commercial enzymes (alcalase, chymotrypsin, trypsin, pepsin, flavourzyme, and thermoase). Electronic tongue measurements showed that the hydrolysates had significantly ( p < 0.05) lower bitter scores than quinine. Addition of the hydrolysates to quinine led to reduced bitterness intensity of quinine with trypsin and pepsin hydrolysates being the most effective. Addition of the hydrolysates to HEK293T cells that heterologously express one of the bitter taste receptors (T2R4) showed alcalase, thermoase, pepsin, and trypsin hydrolysates as the most effective in reducing calcium mobilization. Eight peptides that were identified from the alcalase and chymotrypsin hydrolysates also suppressed quinine-dependent calcium release from T2R4 with AGDDAPRAVF and ETSARHL being the most effective. We conclude that short peptide lengths or the presence of multiple serine residues may not be desirable structural requirements for blocking quinine-dependent T2R4 activation.

Analysis of Species-Selectivity of Human, Mouse and Rat Cytochrome P450 1A and 2B Subfamily Enzymes using Molecular Modeling, Docking and Dynamics Simulations.

Cytochrome P450 (CYP) 1A and 2B subfamily enzymes are important drug metabolizing enzymes, and are highly conserved across species in terms of sequence homology. However, there are major to minor structural and macromolecular differences which provide for species-selectivity and substrate-selectivity. Therefore, species-selectivity of CYP1A and CYP2B subfamily proteins across human, mouse and rat was analyzed using molecular modeling, docking and dynamics simulations when the chiral molecules quinine and quinidine were used as ligands. The three-dimensional structures of 17 proteins belonging to CYP1A and CYP2B subfamilies of mouse and rat were predicted by adopting homology modeling using the available structures of human CYP1A and CYP2B proteins as templates. Molecular docking and dynamics simulations of quinine and quinidine with CYP1A subfamily proteins revealed the existence of species-selectivity across the three species. On the other hand, in the case of CYP2B subfamily proteins, no role for chirality of quinine and quinidine in forming complexes with CYP2B subfamily proteins of the three species was indicated. Our findings reveal the roles of active site amino acid residues of CYP1A and CYP2B subfamily proteins and provide insights into species-selectivity of these enzymes across human, mouse, and rat.

Optimizing Stem Length To Improve Ligand Selectivity in a Structure-Switching Cocaine-Binding Aptamer.

Understanding how aptamer structure and function are related is crucial in the design and development of aptamer-based biosensors. We have analyzed a series of cocaine-binding aptamers with different lengths of their stem 1 in order to understand the role that this stem plays in the ligand-induced structure-switching binding mechanism utilized in many of the sensor applications of this aptamer. In the cocaine-binding aptamer, the length of stem 1 controls whether the structure-switching binding mechanism for this aptamer occurs or not. We varied the length of stem 1 from being one to seven base pairs long and found that the structural transition from unfolded to folded in the unbound aptamer is when the aptamer elongates from 3 to 4 base pairs in stem 1. We then used this knowledge to achieve new binding selectivity of this aptamer for quinine over cocaine by using an aptamer with a stem 1 two base pairs long. This selectivity is achieved by means of the greater affinity quinine has for the aptamer compared with cocaine. Quinine provides enough free energy to both fold and bind the 2-base pair-long aptamer while cocaine does not. This tuning of binding selectivity of an aptamer by reducing its stability is likely a general mechanism that could be used to tune aptamer specificity for tighter binding ligands.

Comparison of the free and ligand-bound imino hydrogen exchange rates for the cocaine-binding aptamer.

Using NMR magnetization transfer experiments, the hydrogen exchange rate constants (k ex ) of the DNA imino protons in the cocaine-binding aptamer have been determined for the free, cocaine-bound, and quinine-bound states. The secondary structure of the cocaine-binding aptamer is composed of three stems built around a three-way junction. In the free aptamer the slowest exchanging imino protons are located in the middle of the stems. The highest k ex values were found for a nucleotide in the GAA loop of stem 3 and for nucleotides at the end of the stems that form the three-way junction structure and in the tandem GA mismatch. Upon ligand binding, the k ex values of nucleotides at the ligand binding site are reduced, indicating that these base pairs become more stable or less solvent accessible in the bound state. The imino proton k ex values of nucleotides located away from the binding site are only minimally affected by ligand binding.

Changes in sucrose and quinine taste reactivity patterns in infant rat pups after exposure to the other tastant.

The taste reactivity test is considered as an objective measure to assess the hedonic impact of tastes. Both the appetitive and aversive pattern of responses are plastic and can change based on previous experience. The present study assessed the repertoire of taste responses elicited by sucrose and quinine in preweanling rats, and described changes in these taste reactivity patterns after exposure to the other tastant. We exposed infant rats (17 days old at the start of training) to sweet (2% sucrose) or bitter (0.01% quinine) tastants during 4, 10-min trials in two different random sequences. The subjects were weighed before and after each trial to provide a measure of percent body weight gained. The following taste reactivity responses were registered: duration of mouthing and paw lick, frequency of chin rub, head shake and flailing of the forelimbs, frequency and duration of face washing, wall climbing and paw tread. The consummatory and affective taste responses changed depending on the order in which the solutions were administered. The order of exposure to the tastants did not affect the levels of sucrose intake. Conversely, rat pups showed more ingestive, and fewer aversive, responses to the sweet tastant when access to the solution followed the intraoral infusion of quinine. Likewise, intraoral delivery of quinine elicited a more aversive taste reactivity pattern when delivered after the access to sucrose than when presented to sucrose-naïve pups. This research contributes to the analysis of taste reactivity responses during the early ontogeny of the rat and highlights the importance of previous experiences on the subsequent assessment of rewards.