The role of 5‐HT2A receptor and 5‐HT2A/5‐HT1A receptor interaction in the suppression of catalepsy

In the present study, the 5‐HT_2A and 5‐HT_1A receptors functional activity and 5‐HT_2A receptor gene expression were examined in the brain of ASC/Icg and congenic AKR.CBAD13Mit76C mouse strains (genetically predisposed to catalepsy) in comparison with the parental catalepsy‐resistant AKR/J and catalepsy‐prone CBA/Lac mouse strains. The significantly reduced 5‐HT_2A receptor functional activity along with decreased 5‐HT_2A receptor gene expression in the frontal cortex was found in all mice predisposed to catalepsy compared with catalepsy‐resistant AKR/J. 5‐HT_2A agonist DOI (0.5 and 1 mg/kg, i.p.) significantly reduced catalepsy in ASC/Icg and CBA/Lac, but not in AKR.CBAD13Mit76C mice. Essential increase in 5‐HT_1A receptor functional activity was shown in catalepsy‐prone mouse strains in comparison with catalepsy‐resistant AKR/J mice. However, in AKR.CBAD13Mit76C mice it was lower than in ASC/Icg and CBA/Lac mice. The inter‐relation between 5‐HT_2A and 5‐HT_1A receptors in the regulation of catalepsy was suggested. This suggestion was confirmed by prevention of DOI anticataleptic effect in ASC/Icg and CBA/Lac mice by pretreatment with 5‐HT_1A receptor antagonist p‐MPPI (3 mg/kg, i.p.). At the same time, the activation of 5‐HT_2A receptor led to the essential suppression of 5‐HT_1A receptor functional activity, indicating the opposite effect of 5‐HT_2A receptor on pre‐ and postsynaptic 5‐HT_1A receptors. Thus, 5‐HT_2A/5‐HT_1A receptor interaction in the mechanism of catalepsy suppression in mice was shown.     

Subunit‐dependent inhibition and potentiation of 5‐HT3 receptor by the anticancer drug,topotecan

The 5‐hydroxytryptamine (serotonin, 5‐HT) type 3 (5‐HT3) receptor belongs to the superfamily of Cys‐loop ligand‐gated ion channels, and can be either homopentameric (5‐HT3A) or heteropentameric (5‐HT3AB) receptor. Several modulators are known, which either inhibit or potentiate this channel, but few have any appreciable selectivity between the two subtypes or can modulate one receptor differently to the other. In this study, we show that the anticancer drug, topotecan, bidirectionally modulates the 5‐HT3 receptor using a two‐electrode voltage clamp technique. Topotecan inhibited 5‐HT‐gated current through homomeric 5‐HT3A receptors. Interestingly, however, additional expression of the 5‐HT3B subunit changed the response to topotecan dramatically from an inhibitory to a potentiatory one. This effect was dependent on the level of 5‐HT3B subunit expression. Moreover, the effect was reduced in the receptors containing the 5‐HT3B(Y129S) polymorphic variant. These finding could explain individual differences in the sensitivity to topotecan‐induced nausea and vomiting.     

Transient expression of serotonin 5‐HT4 receptors in the mouse developing thalamocortical projections

The serotonin 5‐HT₄ receptor (5‐HT₄‐R) is an unusually complex G‐protein coupled receptor that is likely to play important roles in brain development and that may underlie the comorbidity of central and peripheral abnormalities in some developmental disorders. We studied the expression of 5‐HT₄‐Rs in the developing mouse forebrain at embryonic days 13, 15, 17, and at postnatal days 3 and 14 by using immunohistochemistry, tract tracing, and quantitative RT‐PCR. The developing thalamocortical projections transiently expressed 5‐HT₄‐Rs in the embryonic brain and the 5‐HT₄‐R expression in the forebrain changed from axonal to somatic around birth. From embryonic days 13–17, the forebrain mRNA levels of the 5‐HT_4(a)‐R and 5‐HT_4(b)‐R splice variants increased nine‐ and fivefold, respectively, whereas the levels of the 5‐HT_4(e)‐R and 5‐HT_4(f)‐R variants remained relatively low throughout the studied period of embryonic development. These results suggest that during development 5‐HT₄‐R expression undergoes a dynamic regulation and that this regulation may be important for the normal development of sensory and limbic processing. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2010.     

β‐arrestin1 phosphorylation by GRK5 regulates G protein‐independent 5‐HT4 receptor signalling

G protein‐coupled receptors (GPCRs) have been found to trigger G protein‐independent signalling. However, the regulation of G protein‐independent pathways, especially their desensitization, is poorly characterized. Here, we show that the G protein‐independent 5‐HT₄ receptor (5‐HT₄R)‐operated Src/ERK (extracellular signal‐regulated kinase) pathway, but not the G_s pathway, is inhibited by GPCR kinase 5 (GRK5), physically associated with the proximal region of receptor’ C‐terminus in both human embryonic kidney (HEK)‐293 cells and colliculi neurons. This inhibition required two sequences of events: the association of β–arrestin1 to a phosphorylated serine/threonine cluster located within the receptor C‐t domain and the phosphorylation, by GRK5, of β–arrestin1 (at Ser⁴¹²) bound to the receptor. Phosphorylated β‐arrestin1 in turn prevented activation of Src constitutively bound to 5‐HT₄Rs, a necessary step in receptor‐stimulated ERK signalling. This is the first demonstration that β‐arrestin1 phosphorylation by GRK5 regulates G protein‐independent signalling.     

The role of 5‐HT2c receptor on corticosterone‐mediated food intake

Corticosterone plays an important role in feeding behavior. However, its mechanism remains unclear. Therefore, the present study aimed to investigate the effect of corticosterone on feeding behavior. In this study, cumulative food intake was increased by acute corticosterone administration in a dose‐dependent manner. Administration of the 5‐HT_2c receptor agonist m‐chlorophenylpiperazin (mCPP) reversed the effect of corticosterone on food intake. The anorectic effects of mCPP were also blocked by the 5‐HT_2c receptor antagonist RS102221 in corticosterone‐treated mice. Both corticosterone and mCPP increased c‐Fos expression in hypothalamic nuclei, but not the nucleus of the solitary tract. RS102221 inhibited c‐Fos expression induced by mCPP, but not corticosterone. In addition, mCPP had little effect on TH and POMC levels in the hypothalamus. Furthermore, mCPP antagonized decreasing effect of the leptin produced by corticosterone. Taken together, our findings suggest that 5‐HT_2c receptors and leptin may be involved in the effects of corticosterone‐induced hyperphagia.     

Anxiety and increased 5‐HT1A receptor response in NCAM null mutant mice

Mice deficient in the neural cell adhesion molecule (NCAM) show behavioral abnormalities as adults, including altered exploratory behavior, deficits in spatial learning, and increased intermale aggression. Here, we report increased anxiety‐like behavior of homozygous (NCAM^−/−) and heterozygous (NCAM^+/−) mutant mice in a light/dark avoidance test, independent of genetic background and gender. Anxiety‐like behavior was reduced in both NCAM^+/+ and NCAM^−/− mice by systemic administration of the benzodiazepine agonist diazepam and the 5‐HT_1A receptor agonists buspirone and 8‐OH‐DPAT. However, NCAM^−/− mice showed anxiolytic‐like effects at lower doses of buspirone and 8‐OH‐DPAT than NCAM^+/+ mice. Such increased response to 5‐HT_1A receptor stimulation suggests a functional change in the serotonergic system of NCAM^−/− mice, likely involved in the control of anxiety and aggression. However, 5‐HT_1A receptor binding and tissue content of serotonin and its metabolite 5‐hydroxyindolacetic acid were found unaltered in every brain area of NCAM^−/− mice investigated, indicating that expression of 5‐HT_1A receptors as well as synthesis and release of serotonin are largely unchanged in NCAM^−/− mice. We hypothesize a critical involvement of endogenous NCAM in serotonergic transmission via 5‐HT_1A receptors and inwardly rectifying K⁺ channels as the respective effector systems. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 343–355, 1999     

Chronic voluntary ethanol intake hypersensitizes 5‐HT1A autoreceptors in C57BL/6J mice

Alcoholism is a complex disorder involving, among others, the serotoninergic (5‐HT) system, mainly regulated by 5‐HT_1A autoreceptors in the dorsal raphe nucleus. 5‐HT_1A autoreceptor desensitization induced by chronic 5‐HT reuptake inactivation has been associated with a decrease in ethanol intake in mice. We investigated here whether, conversely, chronic ethanol intake could induce 5‐HT_1A autoreceptor supersensitivity, thereby contributing to the maintenance of high ethanol consumption. C57BL/6J mice were subjected to a progressive ethanol intake procedure in a free‐choice paradigm (3–10% ethanol versus tap water; 21 days) and 5‐HT_1A autoreceptor functional state was assessed using different approaches. Acute administration of the 5‐HT_1A receptor agonist ipsapirone decreased the rate of tryptophan hydroxylation in striatum, and this effect was significantly larger (+75%) in mice that drank ethanol than in those drinking water. Furthermore, ethanol intake produced both an increased potency (+45%) of ipsapirone to inhibit the firing of 5‐HT neurons, and a raise (+35%) in 5‐HT_1A autoreceptor‐mediated stimulation of [³⁵S]GTP‐γ‐S binding in the dorsal raphe nucleus. These data showed that chronic voluntary ethanol intake in C57BL/6J mice induced 5‐HT_1A autoreceptor supersensitivity, at the origin of a 5‐HT neurotransmission deficit, which might be causally related to the addictive effects of ethanol intake.     

Improved fruit α‐tocopherol,carotenoid, squalene and phytosterol contents through manipulation of Brassica juncea 3‐HYDROXY‐3‐METHYLGLUTARYL‐COA SYNTHASE1 in transgenic tomato

3‐Hydroxy‐3‐methylglutaryl‐coenzyme A synthase (HMGS) in the mevalonate (MVA) pathway generates isoprenoids including phytosterols. Dietary phytosterols are important because they can lower blood cholesterol levels. Previously, the overexpression of Brassica juncea wild‐type (wt) and mutant (S359A) BjHMGS1 in Arabidopsis up‐regulated several genes in sterol biosynthesis and increased sterol content. Recombinant S359A had earlier displayed a 10‐fold higher in vitro enzyme activity. Furthermore, tobacco HMGS overexpressors (OEs) exhibited improved sterol content, plant growth and seed yield. Increased growth and seed yield in tobacco OE‐S359A over OE‐wtBjHMGS1 coincided with elevations in NtSQS expression and sterol content. Herein, the overexpression of wt and mutant (S359A) BjHMGS1 in a crop plant, tomato (Solanum lycopersicum), caused an accumulation of MVA‐derived squalene and phytosterols, as well as methylerythritol phosphate (MEP)‐derived α‐tocopherol (vitamin E) and carotenoids, which are important to human health as antioxidants. In tomato HMGS‐OE seedlings, genes associated with the biosyntheses of C10, C15 and C20 universal precursors of isoprenoids, phytosterols, brassinosteroids, dolichols, methylerythritol phosphate, carotenoid and vitamin E were up‐regulated. In OE‐S359A tomato fruits, increased squalene and phytosterol contents over OE‐wtBjHMGS1 were attributed to heightened SlHMGR2, SlFPS1, SlSQS and SlCYP710A11 expression. In both tomato OE‐wtBjHMGS1 and OE‐S359A fruits, the up‐regulation of SlGPS and SlGGPPS1 in the MEP pathway that led to α‐tocopherol and carotenoid accumulation indicated cross‐talk between the MVA and MEP pathways. Taken together, the manipulation of BjHMGS1 represents a promising strategy to simultaneously elevate health‐promoting squalene, phytosterols, α‐tocopherol and carotenoids in tomato, an edible fruit.     

Preparative isolation and purification of four flavonoids from the petals of nelumbo nucifera by high‐speed counter‐current chromatography

Introduction – Flavonoids, the primary constituents of the petals of Nelumbo nucifera, are known to have antioxidant properties and antibacterial bioactivities. However, efficient methods for the preparative isolation and purification of flavonoids from this plant are not currently available. Objective – To develop an efficient method for the preparative isolation and purification of flavonoids from the petals of N. nucifera by high‐speed counter‐current chromatography (HSCCC). Methodology – Following an initial clean‐up step on a polyamide column, HSCCC was utilised to separate and purify flavonoids. Purities and identities of the isolated compounds were established by HPLC‐PAD, ESI‐MS, ¹H‐NMR and ¹³C‐NMR. Results – The separation was performed using a two‐phase solvent system composed of ethyl acetate–methanol–water–acetic acid (4 : 1 : 5 : 0.1, by volume), in which the upper phase was used as the stationary phase and the lower phase was used as the mobile phase at a flow‐rate of 1.0 mL/min in the head‐to‐tail elution mode. Ultimately, 5.0 mg syringetin‐3‐O‐β‐d‐glucoside, 6.5 mg quercetin‐3‐O‐β‐d‐glucoside, 12.8 mg isorhamnetin‐3‐O‐β‐d‐glucoside and 32.5 mg kaempferol‐3‐O‐β‐d‐glucoside were obtained from 125 mg crude sample. Conclusion – The combination of HSCCC with a polyamide column is an efficient method for the preparative separation and purification of flavonoids from the petals of N. nucifera. Copyright © 2009 John Wiley & Sons, Ltd.     

Large‐scale synthesis of tert‐butyl (3R,5S)‐6‐chloro‐3,5‐dihydroxyhexanoate by a stereoselective carbonyl reductase with high substrate concentration and product yield

To biosynthesize the (3R,5S)‐CDHH in an industrial scale, a newly synthesized stereoselective short chain carbonyl reductase (SCR) was successfully cloned and expressed in Escherichia coli. The fermentation of recombinant E. coli harboring SCR was carried out in 500 L and 5000 L fermenters, with biomass and specific activity of 9.7 g DCW/L, 15749.95 U/g DCW, and 10.97 g DCW/L, 19210.12 U/g DCW, respectively. The recombinant SCR was successfully applied for efficient production of (3R,5S)‐CDHH. The scale‐up synthesis of (3R,5S)‐CDHH was performed in 5000 L bioreactor with 400 g/L of (S)‐CHOH at 30°C, resulting in a space‐time yield of 13.7 mM/h/g DCW, which was the highest ever reported. After isolation and purification, the yield and d.e. of (3R,5S)‐CDHH reached 97.5% and 99.5%, respectively. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:612–620, 2017     

A single in vivo cocaine administration impairs 5‐HT1B receptor‐induced long‐term depression in the nucleus accumbens

The nucleus accumbens (NAc) is a crucial forebrain nucleus implicated in reward‐based decision‐making. While NAc neurons are richly innervated by serotonergic fibers, information on the functional role of serotonin 5‐hydroxytryptamine (5‐HT) in the NAc is still sparse. Here, we demonstrate that brief application of 5‐HT or 5‐HT_1B receptor agonist CP 93129 induced a long‐term depression (LTD) of glutamatergic transmission in NAc neurons. This LTD was presynaptically mediated and inducible by endogenous 5‐HT. Remarkably, a single cocaine exposure impaired the induction of LTD by 5‐HT or CP 93129. The inhibition was blocked when a selective dopamine D₁ receptor antagonist SCH23390 was coadministered with cocaine. Cocaine treatment resulted in increased phosphorylation of presynaptic proteins, rabphilin 3A and synapsin 1, and significantly attenuated CP 93129‐induced decrease in rabphilin 3A and synapsin 1 phosphorylation. Application of cAMP‐dependent protein kinase inhibitor KT5720 caused a prominent synaptic depression in NAc neurons of mice with a history of cocaine exposure. Our results reveal a novel 5‐HT_1B receptor‐mediated LTD in the NAc and suggest that cocaine exposure may result in elevated phosphorylation of presynaptic proteins involved in regulating glutamate release, which counteracts the presynaptic depressant effects of 5‐HT_1B receptors and thereby impairs the induction of LTD by 5‐HT.     

GC Separation of Enantiomers of Alkyl Esters of 2‐Bromo Substituted Carboxylic Acids Enantiomers on 6‐TBDMS‐2,3‐di‐alkyl‐ β‐ and γ‐Cyclodextrin Stationary Phases

The gas chromatographic separation of enantiomers of 2‐Br carboxylic acid derivatives was studied on four different 6‐TBDMS‐2,3‐di‐O‐alkyl‐ β‐ and ‐γ‐CD stationary phases. The differences in thermodynamic data {ΔH and –ΔS} for the 15 structurally related racemates were evaluated. The influence of structure differences in the alkyl substituents covalently attached to the stereogenic carbon atom, as well as in the ester group of the homologous analytes, and the selectivity of modified β‐ and γ‐ cyclodextrin derivatives was studied in detail. The cyclodextrin cavity size, as well as elongation of alkyl substituents in positions 2 and 3 of 6‐TBDMS‐β‐CD, also affected their selectivity. The quality of enantiomeric separations is influenced mainly by alkyl chains of the ester group of the molecule and this appears to be independent of the CD stationary phase used. In some cases the separations occur as the result of external adsorption rather than inclusion complexations with the chiral selector. It was found that the temperature dependencies of the selectivity factor were nonlinear. Chirality 26:279–285, 2014. © 2014 Wiley Periodicals, Inc.