三种麻醉方法在骨科动物实验中效果比较

目的比较3种麻醉方法在骨科动物实验中的效果,从而得出最佳方法。方法将80只实验动物随机分为3组,A组（速眠新II肌肉注射组）,B组（0.6%戊巴比妥钠静脉注射组）,C组（速眠新II肌肉注射＋0.6%戊巴比妥钠静脉注射联合用药组）,在骨科动物实验中分别进行麻醉、手术,比较3组的麻醉效果,观察其诱导期、麻醉期（麻醉维持时间）、苏醒期、麻醉效果、死亡率等。结果 A组麻醉的诱导期长,效果不好;B组麻醉的诱导期短,但麻醉效果不理想,死亡率高;C组麻醉的诱导期短,麻醉效果好,安全。结论 C组诱导期短,麻醉效果好,安全。无论在实验动物的伦理道德方面,还是在保证动物实验质量方面都是最佳选择。     

氯沙坦对机械通气大鼠肺组织TNF-α和MPO表达的影响

目的通过观察AngⅡ受体拮抗剂——氯沙坦对呼吸机所致肺组织TNF-α水平和髓过氧化物酶（MPO）活性的影响,探讨氯沙坦对呼吸机所致肺损伤的保护作用。方法 24只健康雄性Wister大鼠随机分为对照组、呼吸机所致肺损伤组（VILI）和氯沙坦干预组（LAP）,分别采用ELISA法测定大鼠肺组织中血管紧张素II（AngⅡ）含量,采用免疫组织化学染色法测定大鼠肺组织TNF-α蛋白表达水平,采用化学比色法测定肺组织匀浆中MPO活性。结果 VILI组大鼠肺组织AngⅡ含量、TNF-α蛋白表达水平以及肺组织匀浆中MPO活性均明显高于对照组（均P〈0.01）。氯沙坦干预组大鼠肺组织AngⅡ含量与VILI组比较无明显差异（P〉0.05）,但肺组织TNF-α蛋白表达水平、肺组织匀浆中MPO活性和肺W/D比值均较VILI组明显降低（均P〈0.01）,同时肺组织病理损伤程度也较VILI组明显减轻。结论 AngⅡ通过调控肺组织中TNF-α的表达在VILI发病中起重要作用,氯沙坦可阻断AngⅡ与AT1受体的结合,下调肺组织TNF-α表达,降低MPO的活性,对VILI具有一定防治作用。     

具有两个状态脉冲控制的害虫治理模型的阶一周期解的存在性和吸引性

在文献[10]的基础上,基于害虫治理问题,建立具有两个状态脉冲控制的HollingⅡ功能性反应的害虫治理模型,利用所定义的后继函数和分析的方法研究了系统阶一周期解的存在性和吸引性.     

凹耳蛙MHCII类B基因第二外显子多态性分析

两栖类正经历全球范围内的种群衰退,很多两栖动物集群灭绝事件与环境病原体(如壶菌(Batrachochytrium dendrobatidis)的侵扰有关。MHC基因的表达产物在有颌脊椎动物免疫应答过程中起关键作用,其多态性通常与动物对疾病的抗性或易感性密切相关,因而被认为是研究动物适应性进化的最佳候选基因之一。本文对中国特有的无尾两栖动物凹耳蛙(Odorrana tormota)MHC II类B基因多态性进行初步研究。首先,利用1对通用引物扩增出凹耳蛙MHC II类B基因exon2长约180bp的DNA片段。在此基础上,利用ligation-mediated PCR进一步获取侧翼未知序列,序列拼接后长2,030bp,包含exon2以及intron1和intron2的部分序列。基于上述序列设计出凹耳蛙B基因exon2特异性引物(IIQ1BU/IIQ1BD),对该物种黄山种群32个样品进行PCR扩增和克隆测序,共获得34个不同的等位基因,等位基因序列核苷酸和氨基酸变异位点的比例分别为16.17%(33/204)和26.87%(18/67),大多数氨基酸变异位点位于推测的抗原结合位点(antigen binding sites,ABS)。每个样品包含2-5个等位基因,结合等位基因序列特征以及cDNA表达分析结果,推测凹耳蛙至少拥有3个可表达的B基因座位。与文献报道的蛙科其他物种比较后发现,尽管凹耳蛙目前的分布区非常狭窄,但其MHC II类B基因多态性明显高于蛙科其他动物。等位基因碱基替换模式提示凹耳蛙MHC II类B基因曾经历过强烈的正选择作用,ABS区的dN值显著大于dS(P<0.05),PAML软件包CODEML程序中不同模型的似然比检测(likelihood rate test)结果同样支持上述推论,贝叶斯经验贝叶斯路径(Bayesian Em-pirical Bayes)共检测出5个显著受正选择作用的氨基酸位点。贝叶斯系统树的拓扑结构显示,无尾两栖类不同科的等位基因分别形成单系群,但蛙科不同属的等位基因未能形成单系群,蛙属绿池蛙(Rana clamitans)的1个等位基因与臭蛙属凹耳蛙的部分等位基因享有共同的谱系关系,提示蛙科不同属间的B基因存在跨种多态性。     

非诺贝特通过FoxO1 抑制心肌肥大

目的:探讨非诺贝特(fenofibrate)对血管紧张素Ⅱ(AngⅡ)诱导的肥大心肌细胞的抑制作用及对FoxO1表达的影响。方法:首先采用AngⅡ诱导心肌细胞肥大,将细胞分为三组:对照组:未给予任何干预;心肌细胞肥大组:AngⅡ(10-7mol/L)刺激细胞;治疗组:先给予fenofibrate(10-5mol/L),30min后AngⅡ(10-7mol/L)刺激细胞。应用蛋白免疫印迹法(western-blotting)和实时定量PCR法(real time PCR)检测各组细胞中转录因子FoxO1的蛋白质及mRNA含量,心肌细胞肥大的判断使用脑钠肽(brain natriuret icpepide BNP)。结果:心肌细胞肥大组的FoxO1表达较对照组明显降低,而治疗组的FoxO1表达较心肌肥大组明显升高。结论:非诺贝特可能通过上调FoxO1表达,从而抑制心肌细胞肥大。     

EGFR signaling modulates synaptic connectivity via Gurken

Synaptic target selection is critical for establishing functional neuronal circuits. The mechanisms regulating target selection remain incompletely understood. We describe a role for the EGF receptor and its ligand Gurken in target selection of octopaminergic Type II neurons in the Drosophila neuromuscular system. Mutants in happyhour, a regulator of EGFR signaling, form ectopic Type II neuromuscular junctions. These ectopic innervations are due to inappropriate target selection. We demonstrate that EGFR signaling is necessary and sufficient to inhibit synaptic target selection by these octopaminergic Type II neurons, and that the EGFR ligand Gurken is the postsynaptic, muscle-derived repulsive cue. These results identify a new pathway mediating cell-type and branch-specific synaptic repulsion, a novel role for EGFR signaling in synaptic target selection, and an unexpected role for Gurken as a muscle-secreted repulsive ligand.     

Artificial binding proteins (Affitins) as probes for conformational changes in secretin PulD

The DNA-binding protein Sac7d was previously modified to bind with high affinity to the N domain of the outer membrane secretin PulD from the bacterium Klebsiella oxytoca. Here, we show that binding of the Sac7d derivatives (affitins) to PulD is sensitive to conformational changes caused by denaturant and by the zwitterionic detergent Zwittergent 3-14 routinely used to extract secretins from outer membranes. This sensitivity to the conformational state of PulD allowed us to use the affitins as probes for the native structure of PulD and to devise protocols for examining in vitro synthesized protein in nonionic detergent and for the affinity purification of native PulD using affitins as ligands. When fused to periplasmic PhoA, three affitins inhibited PulD multimerization in vivo and caused loss of function. In two cases, this was likely to be due to dimerization of the affitin by the bound PhoA, as the effect was absent when the affitins were fused to monomeric MalE. In the third case, the MalE and PhoA moieties probably interfered sterically with PulD protomer interactions and, thereby, inhibited multimerization. None of the affitins tested interacted with PulD at sites of protomer interaction or blocked the secretin channel through which exoproteins cross the outer membrane in the Type II secretion pathway of which PulD is a key component.     

TRPM7 regulates myosin IIA filament stability and protein localization by heavy chain phosphorylation

Deregulation of myosin II-based contractility contributes to the pathogenesis of human diseases, such as cancer, which underscores the necessity for tight spatial and temporal control of myosin II activity. Recently, we demonstrated that activation of the mammalian α-kinase TRPM7 inhibits myosin II-based contractility in a Ca²⁺- and kinase-dependent manner. However, the molecular mechanism is poorly defined. Here, we demonstrate that TRPM7 phosphorylates the COOH-termini of both mouse and human myosin IIA heavy chains—the COOH-terminus being a region that is critical for filament stability. Phosphorylated residues were mapped to Thr1800, Ser1803 and Ser1808. Mutation of these residues to alanine and that to aspartic acid lead to an increase and a decrease, respectively, in myosin IIA incorporation into the actomyosin cytoskeleton and accordingly affect subcellular localization. In conclusion, our data demonstrate that TRPM7 regulates myosin IIA filament stability and localization by phosphorylating a short stretch of amino acids within the α-helical tail of the myosin IIA heavy chain.     

Extensive conformational transitions are required to turn on ATP hydrolysis in myosin

Conventional myosin is representative of biomolecular motors in which the hydrolysis of adenosine triphosphate (ATP) is coupled to large-scale structural transitions both in and remote from the active site. The mechanism that underlies such “mechanochemical coupling,” especially the causal relationship between hydrolysis and allosteric structural changes, has remained elusive despite extensive experimental and computational analyses. In this study, using combined quantum mechanical and molecular mechanical simulations and different conformations of the myosin motor domain, we provide evidence to support that regulation of ATP hydrolysis activity is not limited to residues in the immediate environment of the phosphate. Specifically, we illustrate that efficient hydrolysis of ATP depends not only on the proper orientation of the lytic water but also on the structural stability of several nearby residues, especially the Arg238-Glu459 salt bridge (the numbering of residues follows myosin II in Dictyostelium discoideum) and the water molecule that spans this salt bridge and the lytic water. More importantly, by comparing the hydrolysis activities in two motor conformations with very similar active-site (i.e., Switches I and II) configurations, which distinguished this work from our previous study, the results clearly indicate that the ability of these residues to perform crucial electrostatic stabilization relies on the configuration of residues in the nearby N-terminus of the relay helix and the “wedge loop.” Without the structural support from those motifs, residues in a closed active site in the post-rigor motor domain undergo subtle structural variations that lead to consistently higher calculated ATP hydrolysis barriers than in the pre-powerstroke state. In other words, starting from the post-rigor state, turning on the ATPase activity requires not only displacement of Switch II to close the active site but also structural transitions in the N-terminus of the relay helix and the “wedge loop,” which have been proposed previously to be ultimately coupled to the rotation of the converter subdomain 40 Å away.