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.     

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.     

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.     

Ca2+ influx into identified leech neurons induced by 5‐hydroxytryptamine

The role of 5‐hydroxytryptamine (5‐HT, serotonin) in the control of leech behavior is well established and has been analyzed extensively on the cellular level; however, hitherto little is known about the effect of 5‐HT on the cytosolic free calcium concentration ([Ca²⁺]_i) in leech neurons. As [Ca²⁺]_i plays a pivotal role in numerous cellular processes, we investigated the effect of 5‐HT on [Ca²⁺]_i (measured by Fura‐2) in identified leech neurons under different experimental conditions, such as changed extracellular ion composition and blockade of excitatory synaptic transmission. In pressure (P), lateral nociceptive (N1), and Leydig neurons, 5‐HT induced a [Ca²⁺]_i increase which was predominantly due to Ca²⁺ influx since it was abolished in Ca²⁺‐free solution. The 5‐HT‐induced Ca²⁺ influx occurred only if the cells depolarized sufficiently, indicating that it was mediated by voltage‐dependent Ca²⁺ channels. In P and N1 neurons, the membrane depolarization was due to Na⁺ influx through cation channels coupled to 5‐HT receptors, whereby the dose‐dependency suggests an involvement in excitatory synaptic transmission. In Leydig neurons, 5‐HT receptor‐coupled cation channels seem to be absent. In these cells, the membrane depolarization activating the voltage‐dependent Ca²⁺ channels was evoked by 5‐HT‐triggered excitatory glutamatergic input. In Retzius, anterior pagoda (AP), annulus erector (AE), and median nociceptive (N2) neurons, 5‐HT had no effect on [Ca²⁺]_i. © 2004 Wiley Periodicals, Inc. J Neurobiol, 2005     

Regulation of the 5‐HT3A Receptor‐Mediated Current by Alkyl 4‐Hydroxybenzoates Isolated from the Seeds of Nelumbo nucifera

Four known alkyl 4‐hydroxybenzoates, i.e., methyl 4‐hydroxybenzoate ( 1 ), ethyl 4‐hydroxybenzoate ( 2 ), propyl 4‐hydroxybenzoate ( 3 ), and butyl 4‐hydroxybenzoate ( 4 ), were isolated from the seeds of Nelumbo nucifera Gaertner (Nymphaeaceae) for the first time. The structures of the isolates were identified by 1D‐ and 2D‐NMR spectroscopy and comparison with published values. The compounds were evaluated for their effects on the 5‐HT‐stimulated inward current (I_5‐HT) mediated by the human 5‐HT₃A receptors expressed in Xenopus oocytes. Compounds 1 and 2 enhanced the I_5‐HT, but 4 reduced it. These results indicate that 4 is an inhibitor of the 5‐HT₃A receptors expressed in Xenopus oocytes.     

Enantioselective synthesis and antioxidant activity of 3‐(3,4‐dihydroxyphenyl)‐glyceric acid—Basic monomeric moiety of a biologically active polyether from Symphytum asperum and S. caucasicum

The racemic and enantioselective synthesis of a novel glyceric acid derivative, namely, 2,3‐dihydroxy‐3‐(3,4‐dihydroxyphenyl)‐propionic acid as well as the antioxidant activities is described. The virtually pure enantiomers, (+)‐(2R,3S)‐2,3‐dihydroxy‐3‐(3,4‐dihydroxyphenyl)‐propionic acid and (?)‐(2S,3R)‐2,3‐dihydroxy‐3‐(3,4‐dihydroxyphenyl)‐propionic acid were synthesized for the first time via Sharpless asymmetric dihydroxylation of trans‐caffeic acid derivatives using the enantiocomplementary catalysts, (DHQD)₂‐PHAL and (DHQ)₂‐PHAL. The determination of enantiomeric purity of the novel chiral glyceric acid derivatives was performed by high‐performance liquid chromatographic techniques on the stage of their alkylated precursors. The novel glyceric acid derivatives show strong antioxidant activity against hypochlorite and N,N‐diphenyl‐N‐picryl‐hydrazyl free radical. Their antioxidant activity is about 40‐fold higher than that of the corresponding natural polyether and three‐fold higher of trans‐caffeic acid itself. Chirality, 2010. © 2010 Wiley‐Liss, Inc.     

Conformation of poly(γ‐glutamic acid) in aqueous solution

Local conformation and overall conformation of poly(γ‐DL‐glutamic acid) (PγDLGA) and poly(γ‐L‐glutamic acid) (PγLGA) in aqueous solution was studied as a function of degree of ionization ε by ¹H‐NMR, circular dichroism, and potentiometric titration. It was clarified that their local conformation is represented by random coil over an entire ε range and their overall conformation is represented by expanded random‐coil in a range of ε > ε^*, where ε^* is about 0.3, 0.35, 0.45, and 0.5 for added‐salt concentration of 0.02M, 0.05M, 0.1M, and 0.2M, respectively. In a range of ε < ε^*, however, ε dependence of their overall conformation is significantly differentiated from each other. PγDLGA tends to aggregate intramolecularly and/or intermolecularly with decreasing ε, but PγLGA still behaves as expanded random‐coil. It is speculated that spatial arrangement of adjacent carboxyl groups along the backbone chain essentially affects the overall conformation of PγGA in acidic media. © 2015 Wiley Periodicals, Inc. Biopolymers 105: 191–198, 2016.     

LncRNA Gm2044 promotes 17β‐estradiol synthesis in mpGCs by acting as miR‐138‐5p sponge

Long noncoding RNAs (lncRNAs) have been demonstrated to play vital roles in mammalian reproduction. Our previous research revealed that lncRNA Gm2044 is highly expressed in mouse spermatocytes and regulates male germ cell function. The gene annotation database BioGPS shows that Gm2044 is not only highly expressed in testicular tissue but also in ovarian tissue, which suggests that Gm2044 may be involved in female reproductive development. In this study, we confirmed that lncRNA Gm2044 promotes 17β‐estradiol synthesis in mouse pre‐antral follicular granulosa cells (mpGCs). Furthermore, bioinformatics methods, western blot, and the luciferase assay proved that Gm2044 functions as a miR‐138‐5p sponge to inhibit the direct target of miR‐138‐5p, Nr5a1, which enhances 17β‐estradiol synthesis through cyp19a1 activation. Taken together, our results provide an insight into the mechanistic roles of lncRNA Gm2044 for 17β‐estradiol synthesis by acting as competing‐endogenous RNAs to modulate the function of mpGCs. Studying the potential lncRNAs, which regulate estradiol release, will be beneficial for the diagnosis and treatment of steroid hormone‐related disease.     

Lewis acid catalysed methylation of N‐(9H‐fluoren‐9‐yl)methanesulfonyl (Fms) protected lipophilic α‐amino acid methyl esters

This work reports an efficient Lewis acid catalysed N‐methylation procedure of lipophilic α‐amino acid methyl esters in solution phase. The developed methodology involves the use of the reagent system AlCl₃/diazomethane as methylating agent and α‐amino acid methyl esters protected on the amino function with the (9H‐fluoren‐9‐yl)methanesulfonyl (Fms) group. The removal of Fms protecting group is achieved under the same conditions to those used for Fmoc removal. Thus the Fms group can be interchangeable with the Fmoc group in the synthesis of N‐methylated peptides using standard Fmoc‐based strategies. Finally, the absence of racemization during the methylation reaction and the removal of Fms group were demonstrated by synthesising a pair of diastereomeric dipeptides. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

High yield and cost‐effective production of poly(γ‐glutamic acid) with Bacillus subtilis

Poly(γ‐glutamic acid) (γ‐PGA) is a promising biopolymer with many potential industrial and pharmaceutical applications. To reduce the production costs, the effects of yeast extract and L ‐glutamate in the substrate for γ‐PGA production were investigated systematically at shake flask scale. The results showed that lower concentrations of yeast extract (40 g/L) and L ‐glutamate (30 g/L) were beneficial for the cost‐effective production of γ‐PGA in the formulated medium. By maintaining the glucose concentration in the range of 3–10 g/L via a fed‐batch strategy in a 10‐L fermentor, the production of γ‐PGA was greatly improved with the highest γ‐PGA concentration of 101.1 g/L, a productivity of 2.19 g/L·h and a yield of 0.57 g/g total substrate, which is about 1.4‐ to 3.2‐fold higher than those in the batch fermentation. Finally, this high‐density fermentation process was successfully scaled up in a 100‐L fermentor. The present work provides a powerful approach to produce this biopolymer as a bulk chemical in large scale.     

Nitric oxide promotes MPK6‐mediated caspase‐3‐like activation in cadmium‐induced Arabidopsis thaliana programmed cell death

Nitric oxide (NO), a vital cell‐signalling molecule, has been reported to regulate toxic metal responses in plants. This work investigated the effects of NO and the relationship between NO and mitogen‐activated protein kinase (MAPK) in Arabidopsis (Arabidopsis thaliana) programmed cell death (PCD) induced by cadmium (Cd²⁺) exposure. With fluorescence resonance energy transfer (FRET) analysis, caspase‐3‐like protease activation was detected after Cd²⁺ treatment. This was further confirmed with a caspase‐3 substrate assay. Cd²⁺‐induced caspase‐3‐like activity was inhibited in the presence of the NO‐specific scavenger 2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide (cPTIO), suggesting that NO mediated caspase‐3‐like protease activation under Cd²⁺ stress conditions. Pretreatment with cPTIO effectively inhibited Cd²⁺‐induced MAPK activation, indicating that NO also affected the MAPK pathway. Interestingly, Cd²⁺‐induced caspase‐3‐like activity was significantly suppressed in the mpk6 mutant, suggesting that MPK6 was required for caspase‐3‐like protease activation. To our knowledge, this is the first demonstration that NO promotes Cd²⁺‐induced Arabidopsis PCD by promoting MPK6‐mediated caspase‐3‐like activation.     

Metabolic precursors enhance the production of poly‐ε‐lysine by Streptomyces noursei NRRL 5126

Poly‐ε‐lysine produced by streptomyces species is a promising biopolymer owing to its antimicrobial activity and safety for humans. A number of nutritional factors influencing poly‐ε‐lysine production by Streptomyces noursei NRRL 5126 were studied. Various metabolic precursors such as amino acids, tricarboxylic acid cycle intermediates and cofactors were investigated for improved production of poly‐ε‐lysine. Results indicated L ‐aspartate (2 mM) and citric acid (5 mM) to substantially increase the poly‐ε‐lysine production from 97.08 to 409.94 mg/L. Addition of citric acid after 24 h and L ‐aspartate after 36 h of fermentation medium further enhanced poly‐ε‐lysine production to 497.67 mg/L after a total fermentation time of 108 h. However, the use of cofactors of enzymes involved in the biosynthesis of poly‐ε‐lysine inhibited its production which is believed to be due to diversion of the flux to other metabolites.     

Interaction between abscisic acid and nitric oxide in PB90‐induced catharanthine biosynthesis of catharanthus roseus cell suspension cultures

Elicitations are considered to be an important strategy to improve production of secondary metabolites of plant cell cultures. However, mechanisms responsible for the elicitor‐induced production of secondary metabolites of plant cells have not yet been fully elucidated. Here, we report that treatment of Catharanthus roseus cell suspension cultures with PB90, a protein elicitor from Phytophthora boehmeriae, induced rapid increases of abscisic acid (ABA) and nitric oxide (NO), subsequently followed by the enhancement of catharanthine production and up‐regulation of Str and Tdc, two important genes in catharanthine biosynthesis. PB90‐induced catharanthine production and the gene expression were suppressed by the ABA inhibitor and NO scavenger respectively, showing that ABA and NO are essential for the elicitor‐induced catharanthine biosynthesis. The relationship between ABA and NO in mediating catharanthine biosynthesis was further investigated. Treatment of the cells with ABA triggered NO accumulation and induced catharanthine production and up‐regulation of Str and Tdc. ABA‐induced catharanthine production and gene expressions were suppressed by the NO scavenger. Conversely, exogenous application of NO did not stimulate ABA generation and treatment with ABA inhibitor did not suppress NO‐induced catharanthine production and gene expressions. Together, the results showed that both NO and ABA were involved in PB90‐induced catharanthine biosynthesis of C. roseus cells. Furthermore, our data demonstrated that ABA acted upstream of NO in the signaling cascade leading to PB90‐induced catharanthine biosynthesis of C. roseus cells. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:994–1001, 2013     

The role of calcium in apoptosis induced by 7β‐hydroxycholesterol and cholesterol‐5β,6β‐epoxide

Oxysterols, such as 7β‐hydroxy‐cholesterol (7β‐OH) and cholesterol‐5β,6β‐epoxide (β‐epoxide), may have a central role in promoting atherogenesis. This is thought to be predominantly due to their ability to induce apoptosis in cells of the vascular wall and in monocytes/macrophages. Although there has been extensive research regarding the mechanisms through which oxysterols induce apoptosis, much remains to be clarified. Given that experimental evidence has long associated alterations of calcium (Ca²⁺) homeostasis to apoptotic cell death, the aim of the present study was to determine the influence of intracellular Ca²⁺ changes on apoptosis induced by 7β‐OH and β‐epoxide. Ca²⁺ responses in differentiated U937 cells were assessed by epifluorescence video microscopy, using the ratiometric dye fura‐2. Over 15‐min exposure of differentiated U937 cells to 30 μM of 7β‐OH induced a slow but significant rise in fura‐2 ratio. The Ca²⁺ channel blocker nifedipine and the chelating agent EGTA blocked the increase in cytoplasmic Ca²⁺. Moreover, dihydropyridine (DHP) binding sites identified with BODIPY‐FLX‐DHP were blocked following pretreatment with nifedipine, indicating that the influx of Ca²⁺ occurred through L‐type channels. However, following long‐term incubation with 7β‐OH, elevated levels of cytoplasmic Ca²⁺ were not maintained and nifedipine did not provide protection against apoptotic cell death. Our results indicate that the increase in Ca²⁺ may be an initial trigger of 7β‐OH–induced apoptosis, but following chronic exposure to the oxysterol, the influence of Ca²⁺ on apoptotic cell death appears to be less significant. In contrast, Ca²⁺ did not appear to be involved in β‐epoxide–induced apoptosis. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:324–332, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20295     

Genetic manipulation of the γ‐aminobutyric acid (GABA) shunt in rice: overexpression of truncated glutamate decarboxylase (GAD2) and knockdown of γ‐aminobutyric acid transaminase (GABA‐T) lead to sustained and high levels of GABA accumulation in rice kernels

Gamma‐aminobutyric acid (GABA) is a non‐protein amino acid commonly present in all organisms. Because cellular levels of GABA in plants are mainly regulated by synthesis (glutamate decarboxylase, GAD) and catabolism (GABA‐transaminase, GABA‐T), we attempted seed‐specific manipulation of the GABA shunt to achieve stable GABA accumulation in rice. A truncated GAD2 sequence, one of five GAD genes, controlled by the glutelin (GluB‐1) or rice embryo globulin promoters (REG) and GABA‐T‐based trigger sequences in RNA interference (RNAi) cassettes controlled by one of these promoters as well, was introduced into rice (cv. Koshihikari) to establish stable transgenic lines under herbicide selection using pyriminobac. T₁ and T₂ generations of rice lines displayed high GABA concentrations (2–100 mg/100 g grain). In analyses of two selected lines from the T₃ generation, there was a strong correlation between GABA level and the expression of truncated GAD2, whereas the inhibitory effect of GABA‐T expression was relatively weak. In these two lines both with two T‐DNA copies, their starch, amylose, and protein levels were slightly lower than non‐transformed cv. Koshihikari. Free amino acid analysis of mature kernels of these lines demonstrated elevated levels of GABA (75–350 mg/100 g polished rice) and also high levels of several amino acids, such as Ala, Ser, and Val. Because these lines of seeds could sustain their GABA content after harvest (up to 6 months), the strategy in this study could lead to the accumulation GABA and for these to be sustained in the edible parts.     

Vinpocetine and α‐tocopherol prevent the increase in DA and oxidative stress induced by 3‐NPA in striatum isolated nerve endings

Vinpocetine is a neuroprotective drug that exerts beneficial effects on neurological symptoms and cerebrovascular disease. 3‐nitropropionic acid (3‐NPA) is a toxin that irreversibly inhibits succinate dehydrogenase, the mitochondrial enzyme that acts in the electron transport chain at complex II. In previous studies in striatum‐isolated nerve endings (synaptosomes), we found that vinpocetine decreased dopamine (DA) at expense of its main metabolite 3,4‐dihydroxyphenylacetic acid (DOPAC), and that 3‐NPA increased DA, reactive oxygen species (ROS), DA‐quinone products formation, and decreased DOPAC. Therefore, in this study, the possible effect of vinpocetine on 3‐NPA‐induced increase in DA, ROS, lipid peroxidation, and DA‐quinone products formation in striatum synaptosomes were investigated, and compared with the effects of the antioxidant α‐tocopherol. Results show that the increase in DA induced by 3‐NPA was inhibited by both 25 μM vinpocetine and 50 μM α‐tocopherol. Vinpocetine, as α‐tocopherol, also inhibited 3‐NPA‐induced increase in ROS (as judged by DCF fluorescence), lipid peroxidation (as judged by TBA‐RS formation), and DA‐quinone products formation (as judged by the nitroblue tetrazolium reduction method). As in addition to the inhibition of complex II exerted by 3‐NPA, 3‐NPA increases DA‐oxidation products that in turn can inhibit other sites of the respiratory chain, the drop in DA produced by vinpocetine and α‐tocopherol may importantly contribute to their protective action from oxidative damage, particularly in DA‐rich structures.