禾谷缢管蚜阳离子通道基因RSC1的克隆、分子特性及诱导表达分析

【目的】SC1通道(sodium channel 1)是昆虫体内一种重要的离子通道,被认为是一种开发新型杀虫剂的神经靶标。本研究拟克隆禾谷缢管蚜Rhopalosiphum padi的SC1通道基因,并初步分析其生理功能及其与SC1类通道、电压门控钠离子通道、电压门控钙离子通道的进化关系。【方法】采用RT-PCR技术,克隆了禾谷缢管蚜SC1基因完整的开放阅读框;利用实时荧光定量PCR技术,分析禾谷缢管蚜成蚜在不同浓度的高效氯氟氰菊酯诱导下SC1基因表达变化。【结果】获得了禾谷缢管蚜SC1基因(命名为RSC1)完整的开放阅读框(Gen Bank登录号为KU640190),其长度6 687bp,编码2 228个氨基酸。RSC1具有SC1通道的结构特征,有一个不同于电压门控钠离子通道和电压门控钙离子通道的特殊DEEA模体(motif)。系统进化分析结果显示,RSC1与电压门控钠离子通道组成一个进化枝,电压门控钙离子通道组成另外一个进化枝,SC1与电压门控钠离子通道在进化上有更近的起源关系。实时荧光定量PCR分析结果表明,LC15,LC35和LC503种剂量的高效氯氟氰菊酯处理6 h后,禾谷缢管蚜RSC1基因表达量相对于清水对照显著下调,表达量分别为对照的0.57,0.82和0.78倍;3种剂量的高效氯氟氰菊酯处理24 h后,禾谷缢管蚜RSC1基因表达量分别为对照的2.19,1.33和1.19倍,其中LC15(0.1484 mg/L)胁迫下RSC1基因的表达量显著上调。【结论】SC1类通道与电压门控钠离子通道在进化起源上有更近的关系。RSC1通道可能是高效氯氟氰菊酯的次级靶标。由于RSC1和其同源基因只存在于节肢动物中,脊椎动物尚未发现该类基因,因此这类通道可能作为开发新型杀虫剂的神经靶标。     

Blockade of L-type voltage-gated Ca channel inhibits ischemia-induced neurogenesis by down-regulating iNOS expression in adult mouse

Neurogenesis in the adult mammalian hippocampus may contribute to repairing the brain after injury. The signals that regulate neurogenesis in the dentate gyrus following ischemic stroke insult are not well known. We have previously reported that inducible nitric oxide synthase (iNOS) expression is necessary for ischemia-stimulated neurogenesis in the adult dentate gyrus. Here, we show that mice subjected to 90 min of middle cerebral artery occlusion (MCAO) significantly increased the number of new neurons and up-regulated iNOS expression in the dentate gyrus. Blockade of the L-type voltage-gated Ca(2+) channel (L-VGCC) prevented neurogenesis in the dentate gyrus and subventricular zone (SVZ), and down-regulated iNOS expression in the dentate gyrus after cerebral ischemia. This study suggests that Ca(2+) influx through L-VGCC is involved in ischemia-induced neurogenesis by up-regulating iNOS expression.     

钙激活钾通道在黎芦碱致神经细胞损伤中的作用

目的:观察新生SD大鼠原代培养皮层神经元的钙激活钾通道(Kca)在黎芦碱致神经元损伤模型上的激活、抑制效应.方法:采用细胞贴附和内面向外两种膜片钳单通道记录方法记录新生SD大鼠原代培养皮层神经元的Kca电生理活动.结果:黎芦碱在胞外可激活Kca.在有钙浴液内,细胞贴附式,钳制膜电位 30 mV,加入不同浓度黎芦碱(μmol/L:15、25、50、75),通道开放概率由0.005分别增加为0.014±0.003、0.085±0.010、0.132±0.016、0.059±0.006(P＜0.01),在50μmol/L以内表现出浓度依赖性.无钙浴液内,细胞贴附式膜片上,钳制膜电位 50 mV,随药物浓度(μmol/L)增加为15、40、60、100时,通道开放概率由0.005分别增加为0.014±0.010、0.113±0.006、0.141±0.004、0 295±0.009(P＜0.05).6例内面向外式膜片上,钳制膜电位 40 mV,分别加入黎芦碱25 μmol/L、50μmol/L 3 min后,通道开放概率由0.011±0.008分别增加为0.010±0.010、0.012±0.007(P＞0.05).黎芦碱在胞内Kca开放概率,平均开放/关闭时间,电流幅值均无明显变化.结论:黎芦碱通过影响胞内游离钙水平间接调节Kca,在缺血缺氧早期,胞内游离钙增高激活Kca开放.     

BACE1 modulates filopodia-like protrusions induced by sodium channel beta4 subunit

Processing of APP by BACE1 plays a crucial role in the pathogenesis of Alzheimer disease (AD). Recently, the voltage-gated sodium channel (Na_v) β4 subunit (β4), an auxiliary subunit of Na_v that is supposed to serve as a cell adhesion molecule, has been identified as a substrate for BACE1. However, the biological consequence of BACE1 processing of β4 remains illusive. Here, we report the biological effects of β4 processing by BACE1. Overexpression of β4 in Neuro2a cells promoted neurite extension and increased the number of F-actin rich filopodia-like protrusions. While coexpression of BACE1 together with β4 further accelerated neurite extension, the number of filopodia-like protrusions was reduced. Overexpression of C-terminal fragment of β4 that was generated by BACE1 (β4-CTF) partially recapitulated the results obtained with BACE1 overexpression. These results suggest that the processing of β4 by BACE1 regulates neurite length and filopodia-like protrusion density in neurons.     

Hypotonic shocks activate rat TRPV4 in yeast in the absence of polyunsaturated fatty acids

Transient-receptor-potential channels (TRPs) underlie the sensing of chemicals, heat, and mechanical force. We expressed the rat TRPV1 and TRPV4 subtypes in yeast and monitored their activities in vivo as Ca²⁺ rise using transgenic aequorin. Heat and capsaicin activate TRPV1 but not TRPV4 in yeast. Hypotonic shocks activate TRPV4 but not TRPV1. Osmotic swelling is modeled to activate enzyme(s), producing polyunsaturated fatty acids (PUFAs) to open TRPV4 in mammalian cells. This model relegates mechanosensitivity to the enzyme and not the channel. Yeast has only a single Δ9 fatty-acid monodesaturase and cannot make PUFAs suggesting an alternative mechanism for TRPV4 activation. We discuss possible explanations of this difference.     

Effect of the exogenous soyabean phyto-oestrogen genistein on sperm quality, ATP content and fertilization rates in channel catfish Ictalurus punctatus (Rafinesque) and walleye Sander vitreus (Mitchill)

This study examined whether the soya phyto-oestrogen genistein would have an effect on spermatozoa quality and in vitro fertilization capacity in mature channel catfish Ictalurus punctatus or walleye Sander vitreus . For both species, motility time and motility rank were significantly different among treatment groups and control, with higher genistein concentrations producing significantly lower motility time and motility rank ( P ≤ 0·01). Walleye and channel catfish ATP content was significantly lower compared to control treatments at several incubation concentrations and was significantly related to fertilization rate. Fertilization rate was significantly dependant on genistein incubation concentrations ( P ≤ 0·01). Additionally, logistic regression showed a significant negative relation between genistein concentration and fertilization in channel catfish ( P ≤ 0·01). These results establish that genistein could play a role in reproductive performance within aquaculture species. In addition, these findings warrant further examination of the impact of phyto-oestrogens in broodstock feeds.     

NaV1.2 EFL domain allosterically enhances Ca2+ binding to sites I and II of WT and pathogenic calmodulin mutants bound to the channel CTD

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Linkage of hyperkalaemic periodic paralysis in Quarter horses to the horse adult skeletal muscle sodium channel gene

A genetic disease observed in certain Quarter horses is hyperkalaemic periodic paralysis (HYPP). This disease causes attacks of paralysis which can be induced by ingestion of potassium. Recent studies have shown that HYPP in humans is due to single base changes within the adult skeletal muscle sodium channel gene. A large Quarter horse pedigree segregating dominant HYPP was studied to determine if mutations of the sodium channel gene are similarly responsible for HYPP in horses. We used cross-species, PCR-mediated, cDNA cloning and sequencing of the horse adult skeletal muscle sodium channel alpha-subunit gene to identify a polymorphism, and then used this polymorphism to see if the horse sodium channel gene was genetically linked to HYPP in horses. The sodium channel gene was indeed found to be tightly linked to HYPP (LOD = 2.7, theta = 0). Our results suggest that HYPP in horses involves the same gene as the clinically similar human disease, and indicates that these are homologous disorders. The future identification of the specific sodium channel mutation causing HYPP in Quarter horses will permit the development of accurate molecular diagnostics of this condition, as has been recently shown for humans.