A transcription assay for EWS oncoproteins in Xenopus oocytes

Aberrant chromosomal fusion of the Ewing's sarcoma oncogene(EWS)to several different cellular partners produces the Ewing's family of oncoproteins(EWSfusion-proteins,EFPs)and associated tumors(EFTs).EFPs are potent transcriptional activators,dependent on the N-terminal region of EWS(the EWS-activationdomain,EAD)and this function is thought to be central to EFT oncogenesis and maintenance.Thus EFPs are promising therapeutic targets,but detailed molecular studies will be pivotal for exploring this potential.Such studies have so far largely been restricted to intact mammalian cells while recent evidence has indicated that a mammalian cell-free transcription system may not support bona fide EAD function.Therefore,the lack of manipulatable assays for the EAD presents a significant barrier to progress.Using Xenopus laevis oocytes we describe a plasmid-based micro-injection assay that supports efficient,bona fide EAD transcriptional activity and hence provides a new vehicle for molecular dissection of the EAD.

Identification of transient receptor potential channel genes and functional characterization of TRPA1 in Spodoptera frugiperda

Spodoptera frugiperda is a highly destructive pest that has become a global problem due to its robust reproductive and migratory capabilities.Transient receptor potential(TRP)channels,which constitute a vast ion channel family,play pivotal roles in sensing the external environment and maintaining internal homeostasis in insects.TRP channels have been widely investigated for their critical roles in regulating various insect behaviors in recent years.In this study,we identified 15 TRP gene loci encoding 26 transcripts in the genome of S.frugiperda and analyzed their expression profiles at different developmental stages.The results revealed that S.frugiperda possesses four TRPC genes,six TRPA genes,one TRPM gene,two TRPV genes,one TRPN gene,and one TRPML gene,while a canonical TRPP is absent.Moreover,the SfruTRPA1 was functionally characterized using the Xenopus oocyte expression system.The results showed that SfruTRPA1 is activated by temperature increases from 20 to 45℃,and there is no significant desensitization after repeated stimuli within the same temperature range.Additionally,SfruTRPA1 is activated by certain natural chemicals,including allyl isothiocyanate(AITC)and cinnamaldehyde(CA).These findings provide valuable insights to the TRP genes in S.frugiperda.

An Accurate Model for the Expression of the Antisense RNA Block Gene:the Firefly Luciferase Gene-Xenopus Oocyte System

The plasmid p^(SV-Luc20)or the mRNA of luciferase gene transcribed from p^(SP64-Luc12)was introduced intothe nucleus or cytoplasm of Xenopus oocytes at stages 5-6 by microinjection.Then the injectedoocytes were incubated in MB medium at 18℃ for definite periods,and the crude enzyme ofluciferase was prepared.Results indicated that the luciferase gent and its mRNA could be transcribedand translated into the enzymatic protein of luciferase with high biological activity,and could alsocatalyze the substrates to emit light.If different ratios of firefly lucifcrasc gene and its antisense RNA were introduced together into thenucleus or cytoplasm of Xenopus oocytes,then the expression of firefly luciferase gone was severelyblocked.Since the lucifcrasc activity can be measured rapidly and quantitatively and the Xenopusoocytes obtained easily,the firefly luciferase gene-Xenopus oocyte system is an excellent model forrevealing quantitatively how the antisense RNA can block gene expression.

Effects of Glutamic Acid on C-type Inactivation of Kv1.4ΔN Channel

Objectives Acidosis has an inhibitory effect on the inactivation of Kv1.4 ΔN channel through the position H508. So in order to show the effects of glutamic acid on the mutant Kv 1.4 channel that lacks N-type inactivation （Kv1.4 Δ2-146）, we studied in the expression system of the Xenopus oocytes. Methods The two-electrode voltage-clamp technique （TEV） was used to record the currents. Results Acidosis increased fKv1.4 Δ2-146 C-type inactivation. After application of glutamic acid （1 mmol/L） to Kv1.4 Δ2-146 increased C-type inactivation further, changed inactivation time constants from （2.02 ± 0.39 s ） to （1.71 ± 0.23 s） （P〈 0.05） at ＋50mv, and shifted the steady-state inactivation curves of Kv1.4 ΔN to positive potential, which was from （-44.30 ± 0.59 mV） to （-39.88 ± 0.29 mV）（P〈0.05）. and slowed the rate of recovery from inactivation, which was from （1.64 ± 0.19 s） to （1.91 ± 0.23 s）（P〈 0.05）. Conclusions Together, these results suggest that 1 mmol/L glutamic acid accelerates the C-type inactivation of Kv1.4 ΔN in pH 6.8.

Deglycosylation altered the gating properties of rNav1.3:glycosylation/deglycosylation homeostasis probably complicates the functional regulation of voltage-gated sodium channel

Objective To examine the effect of deglycosylation on gating properties of rNav1.3. Methods rNav1.3 was expressed in Xenopus oocyte, with glycosylation inhibition by using tunicamycin. Two-electrode voltage clamp was employed to record the whole-cell sodium current and data were analyzed by Origin software. Those of glycosylated rNav1.3 were kept as control. Results Compared with glycosylated ones, the steady-state activation curve of deglycosylated rNav1.3 was positively shifted by about 10 mV, while inactivation curve was negatively shifted by about 8 mV. Conclusion Glycosylation altered the gating properties of rNav 1.3 and contributed to the functional diversity.

Preliminary Study on γ-Aminobutyric Acid (GABA_A) Like Receptor in Rat Testis

For making it clear whether GABAA-like receptor is in cells or rat testis and how itsgene expresses in tissue cells, the mRNA of rat testis was microinjected into Xenopusoocyte. The membrane electrobiological results showed that an inward 30 nA micro-currentwas elicited, under two-electrode voltageclamped configuration, by extracellular GABA(500 μmol/L), and both Bicuculline and Picrotoxin could inhibit this effect. It is an indication that the mRNA of rat testis can express GABA receptor in the membrane of Xenonpus oocyte. Using Muscimol as a excitant of GABA receptor type A, the micro--currentwas also elicited to generate; however, using Baclofen as a excitant of GABA receptortype B, the micro-current could not be induced. These results indicate that GABA receptorexpressed in Xenopus oocyte membrane is not type B but for type A. On the basis of the research result above, 24-mer oligonucleotides to be complementary to the high conservativeregion of the cDNAs of neur-GABA receptors were synthesized as probes to hybridize respectively with the RNAs Of brain and testis of adult rat, and of rat testes of the variousages. The dot hybridizations showed that the hybrid signal of rat testis was stronger thanthat of rat brain, meaning that, compared with rat brain, the RNA of rat testis has morehomologous regions with probes, and also implying that the GABA receptor from theRNA Of rat testis may be similar to neural-GABA receptor. This receptor therefore iscalled as GABA_A-like receptor. The dot blot analysis above also showed the testis GABA_Alike receptor. The dot blot analysis above also showed testis GABA_A like receptor mRNAcontent changed with the develOPmental age of rats. There is a very low level of gene expression in the rat testis on day 5 postnatal; there is the highest expression of GABA_A-likereceptor gene in rat testes on day 30to 100 after birth; the expression begins to drop on day200 after birth.

Characterization of the pheromone receptors in Mythimna loreyi reveals the differentiation of sex pheromone recognition in Mythimna species

Pheromone receptors(PRs)are key proteins in the molecular mechanism of pheromone recognition,and exploring the functional differentiation of PRs between closely related species helps to understand the evolution of moth mating systems.Pheromone components of the agricultural pest Mythimna loreyi have turned into(Z)-9-tetradecen-1-yl acetate(Z9-14:OAc),(Z)-7-dodecen-1-yl acetate(Z7-12:OAc),and(Z)-11-hexadecen-1-yl acetate,while the composition differs from that of M.separata in the genus Mythimna.To understand the molecular mechanism of pheromone recognition,we sequenced and analyzed antennal transcriptomes to identify 62 odorant receptor(OR)genes.The expression levels of all putative ORs were analyzed using differentially expressed gene analysis.Six candidate PRs were quantified and functionally characterized in the Xenopus oocytes system.MlorPR6 and MlorPR3 were determined to be the receptors of major and minor components Z9-14:OAc and Z7-12:OAc.MlorPR1 and female antennae(FA)-biased MlorPR5 both possessed the ability to detect pheromones of sympatric species,including(Z,E)-9,12-tetradecadien-1-ol,(Z)-9-tetradecen-1-ol,and(Z)-9-tetradecenal.Based on the comparison of PR functions between M.loreyi and M.separata,we analyzed the differentiation of pheromone recognition mechanisms during the evolution of the mating systems of 2 Mythimna species.

Functional characterization of sex pheromone receptors in Spodoptera frugiperda, S. exigua, and S. litura moths

Moths possess an extremely sensitive and diverse sex pheromone processing system,in which pheromone receptors(PRs)are essential to ensure communication between mating partners.Functional properties of some PRs are conserved among species,which is important for reproduction.However,functional differentiation has occurred in some homologous PR genes,which may drive species divergence.Here,using genome analysis,17 PR genes were identified from Spodoptera frugiperda,S.exigua,and S.litura,which belong to 6 homologous groups(odorant receptor[OR]6,11,13,16,56,and 62);of which 6 PR genes(OR6,OR11,OR13,OR16,OR56,and OR62)were identified in S.frugiperda and S.exigua,and 5 PR genes were identified in S.litura,excluding OR62.Using heterologous expression in Xenopus oocytes,we characterized the functions of PR orthologs including OR6,OR56,and OR62,which have not been clarified in previous studies.OR6 orthologs were specifically tuned to(Z,E)-9,12-tetradecadienyl acetate(Z9,E12-14:OAc),and OR62 orthologs were robustly tuned to Z7-12:OAc in S.frugiperda and S.exigua.The optimal ligand for OR56 was Z7-12:OAc in S.frugiperda,but responses were minimal in S.exigua and S.litura.In addition,SfruOR6 was male antennae-specific,whereas SfruOR56 and SfruOR62 were male antennae-biased.Our study further clarified the functional properties of PRs in 3 Spodoptera moth species,providing a comprehensive understanding of the mechanisms of intraspecific communication and interspecific isolation in Spodoptera.

Influence of Y151 F mutation in loop B on the agonist potency in insect nicotinic acetylcholine receptor

Nicotinic acetylcholine receptors （nAChRs） are ligand-gated ion channels, which mediate fast cholinergic synaptic transmission in insect and vertebrate nervous systems. The nAChR agonist-binding site is present at the interface of adjacent subunits and is formed by loops A-C present in α subunits together with loops D-F present in either non-α subunits or homomer-forming α subunits. Although Y151 in loop B has been identified as important in agonist binding, various residues at the 151-site are found among vertebrate and invertebrate nAChR α subunits, such as F 151. In Xenopus oocytes expressing Nlα1 or Nlα1^Y151F plus rat β2, Y151F mutation was found to significantly change the rate of receptor desensitization and altered the pharmacological properties of acetylcholine, but not imidacloprid, including the decrease Of Imax, the increase of EC50 （the concentration causing 50% of the maximum response） and the fast time-constant of decay （τf）. By comparisons of residue structure, the hydroxyl group in the side chain of Y151 was thought to be important in the interaction between Nlα1/β2 nAChRs and acetylcholine, and the phenyl group to be important between Nlα1/β2 nAChRs and imidacloprid.

δ-opioid Receptor Induced Inhibition of Sodium Channel Function

Objective： To study the precise role of DOR in the regulation of sodium channels at present. Methods： With Xenopus oocytes co-expressing sodium channel subtype 2 （Nav1.2） and DOR. Results： 1） Nav1.2 expression induced tetrodotoxin-sensitive inward currents; 2） DOR expression reduced the inward currents; 3） activation of DOR reduced the amplitude of the current and rightly shifted the activation curve of the current in the oocytes with both Nav1.2 and DOR, but not in ones with Nav1.2 alone; 4） the DOR agonist-induced inhibition of Nay 1.2 currents was in a dose-dependent manner and saturable; 5） the DOR agonist had no effect on naive oocytes. Conclusion： These data represent the first demonstration that activation of DOR inhibits Na^＋ channel function by decreasing the amplitude of sodium currents and increasing its threshold of activation. This novel finding has far-reaching impacts on novel solutions of certain neurological disorders such as hypoxic/ischemic injury, epilepsy and pain. Also, our data may improve the understanding of the mechanisms underlying acupuncture since acupuncture is known to activate the brain opioid system.

The actions of neonicotinoid insecticides on nicotinic acetylcholine subunits Ldα1 and Ldα8 of Leptinotarsa decemlineata and assembled receptors

The insect nicotinic acetylcholine receptor(nAChR)is a pentameric channel protein and also a target for neonicotinoids.There are few reported studies on the molecular interactions of Leptinotarsa decemlineata nAChRs with neonicotinoids.In this study,we analyzed the response of acetylcholine and neonicotinoids(thiamethoxam[TMX],imidacloprid[IMI],and clothianidin[CLO])on hybrid receptors constructed by nAChRα1 andα8 subunits of L.decemlineata(Ldα1 and Ldα8)co-expressed with ratβ2 subunit(rβ2)at different capped RNA(cRNA)ratios in Xenopus oocytes.In addition,we evaluated the expression changes of Ldα1 and Ldα8 after median lethal dose of TMX treatment for 72 h by quantitative polymerase chain reaction(qPCR).The resulting functional nAChRs Ldα1/rβ2 and Ldα1/Ldα8/rβ2 showed different pharmacological characteristics.The neonicotinoids tested showed lower agonist affinity on Ldα1/Ldα8/rβ2 compared to Ldα1/rβ2 at same ratios of subunit cRNAs.The sensitivities of neonicotinoids tested for Ldα1/rβ2 and Ldα1/Ldα8/rβ2 at cRNA ratios of 5:1,1:1 and 5:5:1,1:1:1,respectively,were lower than those for nAChRs at ratios of 1:5 and 1:1:5,respectively,whereas the values of maximum response(Imax)varied.For Ldα1/Ldα8/rβ2,a reduction of Lda8 cRNA resulted in increased sensitivity to IMI and decreased sensitivity to TMX.The expression of Ldα1 and Ldα8 significantly decreased in adults by 82.12%and 47.02%,respectively,while Ldα8 was significantly upregulated by 2.44 times in 4th instar larvae after exposure to TMX.We infer that Ldα1 and Ldα8 together play an important role in the sensitivity of L.decemlineata to neonicotinoids.