Mechanisms of adaptation in a predator-prey arms race: TTX-resistant sodium channels

Populations of the garter snake Thamnophis sirtalis have evolved geographically variable resistance to tetrodotoxin (TTX) in a coevolutionary arms race with their toxic prey, newts of the genus Taricha. Here, we identify a physiological mechanism, the expression of TTX-resistant sodium channels in skeletal muscle, responsible for adaptive diversification in whole-animal resistance. Both individual and population differences in the ability of skeletal muscle fibers to function in the presence of TTX correlate closely with whole-animal measures of TTX resistance. Demonstration of individual variation in an essential physiological function responsible for the adaptive differences among populations is a step toward linking the selective consequences of coevolutionary interactions to geographic and phylogenetic patterns of diversity.     

Single-channel and genetic analyses reveal two distinct A-type potassium channels in Drosophila

Whole-cell and single-channel voltage-clamp techniques were used to identify and characterize the channels underlying the fast transient potassium current (A current) in cultured myotubes and neurons of Drosophila. The myotube (A1) and neuronal (A2) channels are distinct, differing in conductance, voltage dependence, and gating kinetics. The myotube currents have a faster and more voltage-dependent macroscopic inactivation rate, a larger steady-state component, and a less negative steady-state inactivation curve than the neuronal currents. The myotube channels have a conductance of 12 to 16 picosiemens, whereas the neuronal channels have a conductance of 5 to 8 picosiemens. In addition, the myotube channel is affected by Shaker mutations, whereas the neuronal channel is not. Together, these data suggest that the two channels are separate molecular structures, the expression of which is controlled, at least in part, by different genes.     

牛fabp3和fabp4基因真核表达载体的构建 及在小鼠成肌细胞中的表达

【目的】分别构建牛fabp3和fabp4基因真核表达载体,并观察载体转染小鼠成肌细胞后基因的表达以及对内源fabp3和fabp4基因表达的影响,检测肉质性状形成相关基因在转基因细胞中的表达及对内源功能基因的影响。【方法】 以pDsRED质粒为骨架载体,分别将人肌红蛋白启动子与目的基因相连、CMV启动子与红色荧光蛋白报告基因相连构建肌肉特异性表达fabp3基因的载体pDsHF3和表达fabp4基因的载体pDsHF4;用脂质体瞬时转染小鼠成肌细胞,72 h后用2%孕马血清诱导成肌细胞向肌管的分化;利用real-time PCR技术检测转染前后成肌细胞中外源牛fabp3和fabp4基因和内源小鼠fabp3和fabp4基因的表达量。【结果】与对照组相比,小鼠成肌细胞分别转染pDsHF3和pDsHF4表达载体,24 h后外源牛fabp3和fabp4基因均获得高水平的表达,48 h后有所回落;而内源小鼠fabp3和fabp4基因在成肌细胞和诱导分化的肌管细胞中的表达均受到不同程度的影响。【结论】构建的真核表达载体能在成肌细胞中高效表达,外源牛fabp3或fabp4基因的转入能够影响小鼠成肌细胞及肌管中内源fabp3和fabp4基因的表达。     

Increased activity of calcium leak channels in myotubes of Duchenne human and mdx mouse origin

Elevated free Ca2+ concentrations found in adult dystrophic muscle fibers result in enhanced protein degradation. Since the difference in concentrations may reflect differences in entry, Ca2+ leak channels in cultures of normal and Duchenne human myotubes, and normal and mdx murine myotubes, have been identified and characterized. The open probability of leak channels is markedly increased in dystrophic myotubes. Other channel properties, such as mean open times, single channel conductance, ion selectivity, and behavior in the presence of pharmacological agents, were similar among myotube types. Compared to the Ca2+ concentrations in normal human and normal mouse myotubes, intracellular resting free Ca2+ concentrations ([Ca2+]i) in myotubes of Duchenne and mdx origin were significantly higher at a time when dystrophin is first expressed in normal tissue. Taken together, these findings suggest that the increased open probability of Ca2+ leak channels contributes to the elevated free intracellular Ca2+ concentration in Duchenne human and mdx mouse myotubes.     

Reconstitution and phosphorylation of chloride channels from airway epithelium membranes

Airway epithelial chloride secretion is controlled by the apical-membrane chloride permeability. Purified apical-membrane vesicles from bovine tracheal epithelium have now been shown to contain functional chloride channels by using the planar-bilayer technique. Three types of chloride channels were observed; a voltage-dependent, calcium-independent, 71-picoSiemen (in 150 mM NaCl) channel accounted for more than 80 percent of the vesicular chloride conductance and was under strict control of phosphorylation. The channel underwent a fast rundown in less than 2 to 3 minutes of recording, and reactivation required in situ exposure to a phosphorylating "cocktail" containing the catalytic subunit of the adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase. Mean open time and open probability were increased after phosphorylation, whereas slope conductance remained unchanged. Thus, metabolic control of tracheal chloride single channels can now be studied in vitro.     

Peptide neurotoxins from fish-hunting cone snails

To paralyze their more agile prey, the venomous fish-hunting cone snails (Conus) have developed a potent biochemical strategy. They produce several classes of toxic peptides (conotoxins) that attack a series of successive physiological targets in the neuromuscular system of the fish. The peptides include presynaptic omega-conotoxins that prevent the voltage-activated entry of calcium into the nerve terminal and release of acetylcholine, postsynaptic alpha-conotoxins that inhibit the acetylcholine receptor, and muscle sodium channel inhibitors, the mu-conotoxins, which directly abolish muscle action potentials. These distinct peptide toxins share several common features: they are relatively small (13 to 29 amino acids), are highly cross-linked by disulfide bonds, and strongly basic. The fact that they inhibit sequential steps in neuromuscular transmission suggests that their action is synergistic rather than additive. Five new omega-conotoxins that block presynaptic calcium channels are described. They vary in their activity against different vertebrate classes, and also in their actions against different synapses from the same animal. There are susceptible forms of the target molecule in peripheral synapses of fish and amphibians, but those of mice are resistant. However, the mammalian central nervous system is clearly affected, and these toxins are thus of potential significance for investigating the presynaptic calcium channels.     

钙钠离子对高粱和小麦乙烯释放的影响

本文阐述了钙、钠离子对小麦、高粱抗旱性不同的品种叶片与根乙烯释放的影响。钙和钠离子对小麦和高架根的乙烯释放有明显的抑制效应。钠离子胁迫下,小麦根乙烯释放量多于高粱根;钙离子胁迫下则以高粱根乙烯释放量为多。钙或钠离子胁迫下,高粱或小麦不抗旱品种根乙烯释放量多于抗早品种。就叶片乙烯释放而言,高粱乙烯释放量少于小麦。两离子对高粱抗旱品种叶片乙烯释有促进作用,对不抗旱品种影响甚微。小麦不抗旱品种叶片乙烯释放为钙钠处理所抑制。胁迫下两种作物抗旱品种叶片的乙烯释放量高于其不抗旱品种的。这些结果表明,植物的乙烯释放对盐分胁迫的反应随组织器官和基因型而异。     

Mechanism of the rapid effect of 17 beta-estradiol on medial amygdala neurons

The mechanism by which sex steroids rapidly modulate the excitability of neurons was investigated by intracellular recording of neurons in rat medial amygdala brain slices. Brief hyperpolarization and increased potassium conductance were produced by 17 beta-estradiol. This effect persisted after elimination of synaptic input and after suppression of protein synthesis. Thus, 17 beta-estradiol directly changes the ionic conductance of the postsynaptic membrane of medial amygdala neurons. In addition, a greater proportion of the neurons from females than from males responded to 17 beta-estradiol.     

Systemic delivery of recombinant proteins by genetically modified myoblasts

The ability to stably deliver recombinant proteins to the systemic circulation would facilitate the treatment of a variety of acquired and inherited diseases. To explore the feasibility of the use of genetically engineered myoblasts as a recombinant protein delivery system, stable transfectants of the murine C2C12 myoblast cell line were produced that synthesize and secrete high levels of human growth hormone (hGH) in vitro. Mice injected with hGH-transfected myoblasts had significant levels of hGH in both muscle and serum that were stable for at least 3 weeks after injection. Histological examination of muscles injected with beta-galactosidase-expressing C2C12 myoblasts demonstrated that many of the injected cells had fused to form multinucleated myotubes. Thus, genetically engineered myoblasts can be used for the stable delivery of recombinant proteins into the circulation.     

Calcium and sodium channels in spontaneously contracting vascular muscle cells

Electrophysiological recordings of inward currents from whole cells showed that vascular muscle cells have one type of sodium channel and two types of calcium channels. One of the calcium channels, the transient calcium channel, was activated by small depolarizations but then rapidly inactivated. It was equally permeable to calcium and barium and was blocked by cadmium, but not by tetrodotoxin. The other type, the sustained calcium channel, was activated by larger depolarizations, but inactivated very little; it was more permeable to barium than calcium. The sustained calcium channel was more sensitive to block by cadmium than the transient channel, but also was not blocked by tetrodotoxin. The sodium channel inactivated 15 times more rapidly than the transient calcium channel and at more negative voltages. This sodium channel, which is unusual because it is only blocked by a very high (60 microM) tetrodotoxin concentration but not by cadmium, is the first to be characterized in vascular muscle, and together with the two calcium channels, provides a basis for different patterns of excitation in vascular muscles.     

干旱胁迫对不同抗旱大豆品种质膜透性的影响

以抗旱性不同的12个大豆品种(系)为材料,对其电导率进行了测定。试验证明,不同大豆品种受到干旱胁迫时它们的相对电导率存在极显著差异,即抗旱性强的品种其相对电导率低,抗旱性弱的品种相对电导率高。联系产量性状进行比较,得出相对电导率与产量呈负相关性。     

绵羊Myostatin前肽对成肌细胞的分化作用

【目的】 研究绵羊Myostatin前肽对C2C12细胞分化的影响。【方法】 构建并包装Myostatin前肽基因过表达慢病毒质粒,包装慢病毒,分别用LV-CMV-MSTN-flag-GFP和LV-CMV -GFP慢病毒感染C2C12细胞,检测重组慢病毒对C2C12细胞的感染能力。【结果】 LV-CMV -GFP感染组可见明显的长条状肌管,LV-CMV-MSTN-flag-GFP组只见少量肌管的形成,肌管融合率的测定。未处理组,对照组,试验组肌管融合率分别为5.53%、6.62%、9.38%,试验组即转染Myostatin前肽的肌管融合率显著高于对照组和未处理组(P＜0.05)。【结论】 Myostatin前肽基因的过表达抑制了Myostatin基因的表达,影响肌管的形成,抑制了分化的进程。     

牛myf6基因真核表达载体的构建及在成肌细胞中的表达

【目的】构建牛myf6基因真核表达载体,并观察myf6真核表达载体转染鲁西黄牛成肌细胞后基因的表达和细胞形态的变化。【方法】在质粒pIRES2-EGFP的多克隆位点插入myf6基因构建真核表达载体pIRES2-EGFP-myf6,用脂质体技术转染鲁西黄牛成肌细胞,通过G418 筛选出稳定转染的细胞株。利用Western印记、Real-time PCR技术检测成肌细胞转染前后myf6基因、肌肉肌酸激酶基因和肌球蛋白轻链基因的表达量。【结果】与对照组相比,转染质粒的成肌细胞myf6蛋白和mRNA的表达量提高（P＜0.01）,肌肉肌酸激酶基因和肌球蛋白轻链基因的mRNA表达量提高（P＜0.01）。细胞形态观察显示成肌细胞融合为肌管。【结论】构建的真核表达载体pIRES2-EGFP-myf6能在成肌细胞中高效表达,myf6基因促进了成肌细胞向肌肉细胞分化。     

Diazepam inhibits myoblast fusion and expression of muscle specific protein synthesis

The presence of diazepam in culutres of chicken embryo myoblasts arrests normal muscle cell differentiation. High concentrations of the drug reversibly prevent myoblasts from fusing to form multinucleated myotubes. Lower concentrations of diazepam allow cell fusion to occur, but inhibit the synthesis and accumulation of myosin heavy chain, implying that cell fusion does not obligate myoblasts to synthesize and accumulate large quantities of muscle specific protein. The effect of diazepam on muscle cells in culture is direct and specific.     

Gating currents of the sodium channels: three ways to block them

Preceding the opening of the sodium channels of axon membrane there is a small outward current, gating current, that is probably associated with the molecular rearrangements that open the channels. Gating current is reversibly blocked by three procedures that block the sodium current: (i) internal perfusion with zinc ions, (ii) inactivation of sodium conductance by brief depolarization, and (iii) prolonged depolarization.     

Transfer of a protein encoded by a single nucleus to nearby nuclei in multinucleated myotubes

Specialized regions of muscle fibers may result from differential gene expression within a single fiber. In order to investigate the range of action of individual nuclei in multinucleated myotubes, C2 myoblasts were transfected to obtain stable cell lines that express a reporter protein that is targeted to the nucleus. Hybrid myotubes were then formed containing one or a few transfected nuclei as well as a large number of nuclei from the parental strain. In order to determine how far the products of a single nucleus extend, transfected nuclei were labeled with [3H]thymidine before fusion and the myotubes were stained to identify the reporter protein. In such myotubes the fusion protein was not confined to its nucleus of origin, but was restricted to nearby nuclei.     

Changes in sodium channel gating produced by point mutations in a cytoplasmic linker

Voltage-gated sodium channels are transmembrane proteins of approximately 2000 amino acids and consist of four homologous domains (I through IV). In current topographical models, domains III and IV are linked by a highly conserved cytoplasmic sequence of amino acids. Disruptions of the III-IV linker by cleavage or antibody binding slow inactivation, the depolarization-induced closed state characteristic of sodium channels. This linker might be the positively charged "ball" that is thought to cause inactivation by occluding the open channel. Therefore, groups of two or three contiguous lysines were neutralized or a glutamate was substituted for an arginine in the III-IV linker of type III rat brain sodium channels. In all cases, inactivation occurred more rapidly rather than more slowly, contrary to predictions. Furthermore, activation was delayed in the arginine to glutamate mutation. Hence, the III-IV linker does not simply act as a charged blocker of the channel but instead influences all aspects of sodium channel gating.     

干扰TP53INP2抑制牛成肌细胞分化

【背景】肌肉可以维持哺乳动物运动功能并调节机体代谢,它的数量和分布对肉质有重要影响,骨骼肌的生长发育及其遗传特性在很大程度上影响甚至决定家畜的产肉量和肉品质,研究骨骼肌的生长发育具有重要意义。TP53INP2对自噬的调控作用以及对前体脂肪细胞分化的调控机制已有研究,而对于牛成肌细胞分化的影响尚未报道。【目的】探究TP53INP2对秦川牛成肌细胞分化的影响,为肉牛肉用性状分子育种工作提供理论依据。【方法】利用实时荧光定量PCR（real-time fluorescence quantitative PCR,RT-qPCR）技术检测了TP53INP2在24月龄秦川牛不同组织中的表达特性,同时分析了其在体外培养的牛骨骼肌成肌细胞不同分化时期的表达规律;合成TP53INP2基因siRNA并转染秦川牛成肌细胞,转染后12h进行诱导分化,观察成肌细胞表型变化,并利用RT-qPCR技术和Western Blot技术分别检测其在诱导分化第四天时分化标志基因和蛋白的表达情况。【结果】1.RT-qPCR结果显示,TP53INP2在成年秦川牛背最长肌组织中表达量最高,在小肠组织中表达量最低。TP53INP2在成年牛心脏、肝脏、肾脏、网胃、瘤胃中表达量较高,在其余组织中表达量较低（与背最长肌相比）。2.随着成肌细胞的分化,该基因在第0—4天表达量呈上升趋势且在第4天达到峰值,随后表达量呈下降趋势。3.在成肌细胞中干扰TP53INP2后,试验组肌管的数量和长度均显著低于对照组。4.干扰TP53INP2并提取细胞总RNA,RT-qPCR结果显示,成肌细胞分化标志基因肌细胞生成素（MYOG）和肌球重链蛋白3（MYH3）相对于对照组表达量显著降低。5.干扰TP53INP2并提取细胞总蛋白,Western Blot结果显示,试验组MYOG、MYH3、MYOD的蛋白表达量均降低且与对照组相比差异极显著。【结论】干扰TP53INP2对牛成肌细胞的分化具有抑制作用,提示该基因可能对秦川牛肌肉组织的生长发育具有重要的调控作用,可对其进行深入的功能研究以期用于肉牛的分子育种实践中。     

马尾松广东种源与湖北种源的人工林生物量分配差异

采用解析木法与整株收获法分析已达轮伐期的马尾松广东种源和湖北当地种源的生长进程及地上、地下生物量分配格局,分析生长快速与缓慢种源人工林生长过程及生产力形成过程中生物量分配模式,探讨2个种源的长期适应性差异的机制。根据胸径（D_BH）大小将解析木划分为3个等级:12 cm≤D_BH < 18 cm,18 cm≤D_BH < 26 cm和26 cm≤D_BH < 34 cm。结果如下:①广东种源在25年生连年生长量达最大值0.015 m3·a-1,35年生平均生长量达最大值0.011 m3·a-1,湖北种源30年生时连年生长量达最大值0.011 m3·a-1,42年生时平均生长量达最大值0.007 1 m3·a-1。②广东种源分配较多比例的生物量给地下部分,2个种源的粗根生物量存在显著差异（P < 0.05）,随胸径增加,相对差异（R_E,GD/HB）分别为36.5%,62.9%和47.5%,而细根间生物量未达显著性差异。在胸径范围2 634 cm内,2个种源人工林在整株生物量、地上部分生物量、树干和针叶生物量差异显著（P < 0.05）。③湖北种源地下生物量分配比例在整个生长过程中均低于广东种源;在生长初期,湖北种源树干所占总生物量比例高于广东种源,而枝和叶的生物量分配比例低于广东种源;随着树龄增长,广东种源树干所占总生物量比例逐渐高于湖北种源,并趋于稳定;而枝、叶正好相反。因此,引种的广东种源生长性状及树干生物量均表现出快速增长的特点,数量成熟年龄较湖北种源提前,栽植快速成长的种源将增加人工林生物量。     

转录因子Sp1对成肌细胞中猪SKIP的表达调控作用

利用基因组PCR步移方法获得猪SKIP转录起始位点上游2 075 bp启动子序列。生物信息学分析发现该序列中存在4个Sp1结合位点和1个CpG岛。将启动子序列构建到pGL3-basic的双荧光素酶报告基因载体上,再利用RNA干扰技术分析转录因子Sp1对SKIP转录的影响。荧光素酶活性分析发现Sp1的表达抑制使成肌细胞中SKIP启动子活性显著下降,表明转录因子Sp1可能通过顺式作用元件GC-Box对成肌细胞分化过程中SKIP基因的转录激活起正向调控作用。