咖啡酸苯乙酯通过AMPK/FOXO3a信号通路缓解猪精液常温保存氧化应激的作用机制

【目的】探究咖啡酸苯乙酯（caffeic acid phenethyl ester,CAPE）通过AMPK/FOXO3a信号通路缓解氧化应激对猪精液的影响及其分子机制。【方法】选取8头成年（1—2岁）、健康状况良好长白种公猪进行鲜精采集,将质检合格的样品混池待用。试验设计如下：首先,在常温基础稀释液中分别添加0、0.02、0.04、0.06、0.08和0.10 g·L^-1浓度的CAPE,将精液样品与各个浓度组依次混合后放置室温平衡,随即转入17 ℃电子恒温冰箱进行常温保存。全自动精子分析仪（CASA）测定1—5 d精子运动学参数（总活率与渐进性活力）,筛选出最佳适宜浓度为0.06 g·L^-1。其次,将0.06 g·L^-1 CAPE与400 μmol·L^-1的H₂O₂建立氧化胁迫模型,在第1、3、5天分别采用CASA测定精子总活率与渐进性活力,荧光探针技术评估质膜完整性与顶体完整性,抗氧化试剂盒检测过氧化氢酶（catalase,CAT）与超氧化物歧化酶（superoxide dismutase,SOD）活性,在第5 天利用实时荧光定量 PCR技术测定AMPK/FOXO3a信号通路下游靶向基因（AMPK、FOXO3a、SOD1、SOD2、CAT）及凋亡基因BAX的mRNA表达水平,蛋白免疫印记技术测定AMPK、p-AMPK蛋白表达。【结果】（1）浓度筛选试验中,0.06 g·L^-1的CAPE在保存第3 天显著提高精子的总活率并维持至第5天（P<0.05）,不仅如此,精子的渐进性活力在第1 天显著高于其他处理组（P<0.05）。（2）氧化胁迫试验中,H₂O₂处理组的精子质膜完整性、顶体完整性、总活率、渐进性活力、SOD与CAT活性在保存的第5天均极限显著低于空白组与CAPE+H₂O₂混合组（P<0.001）,而空白组与CAPE+H₂O₂混合组的总活率、顶体完整性、CAT与SOD活性不存在显著差异（P>0.05）。与CAPE+H₂O₂混合组相比,H₂O₂处理组AMPK、FOXO3a、SOD1、SOD2、CAT的mRNA表达水平显著降低（P<0.05）,BAX的表达水平显著升高（P<0.05）,而空白组与CAPE+H₂O₂混合组的AMPK、SOD1、CAT与BAX不存在显著差异（P>0.05）。在蛋白表达水平中,CAPE+H₂O₂混合组与H₂O₂组相比,AMPK、p-AMPK 与p-AMPK/AMPK比率显著提升（P <0.05）。【结论】在常温基础稀释液中添加0.06 g·L^-1CAPE可通过促进磷酸化AMPK的表达,诱导AMPK/FOXO3a信号下游抗氧化分子的转录,继而缓解H₂O₂介导的氧化危害对精液品质产生的影响,延长精液的保存时间,但CAPE保护精子氧化损伤更为深入的分子机制仍需作进一步探究。     

Role of Akt signal pathway in inhibition of neuronal apoptosis by Chinese medicine Zuogui pill in adult rats treated with monosodium glutamate

AIM: To explore the role of Akt signal pathway in apoptosis of neural cells in adult rats treated with Zuogui Pill, a Chinese medicine. METHODS: Flowcytometry and Western blotting methods were used to investigate the changes of cellular apoptosis rate and Akt signal pathway. RESULTS: Monosodium glutamate (MSG) could increase cellular apoptosis rate and significantly restrained the phosphorylation of Akt (Ser473) and Akt (Thr308), and markedly increased the levels of phospho-FKHR (ser256), GSK-3β (Ser9) and PTEN. Zuogui Pill partly inhibited the above effects of MSG. CONCLUSION: Zuogui pill effectively inhibits the neural apoptosis induced by MSG, and Akt pathway is involved in the neuronal protection of Zuogui pill.     

鸭维甲酸诱导基因I克隆及其结构域功能分析

【目的】克隆鸭维甲酸诱导基因I（retinoic acid inducible gene I,RIG-I）,分析其不同结构域的功能。【方法】根据GenBank上公布的鸭RIG-I序列设计引物,利用RT-PCR克隆鸭RIG-I基因CDS（coding sequence）区,根据保守结构域预测结果,构建携带6*his组氨酸标签的不同结构域缺失突变体的真核表载体（RIG-I-Full、RIG-I-N和RIG-I-C）,转染鸡胚成纤维细胞DF1,经RT-PCR、间接免疫荧光方法鉴定重组质粒在细胞中转录与表达;同时,利用RT-qPCR检测RLR抗病毒信号通路中的IFN-β、Mx1和PKR等下游基因的表达变化。【结果】鸭RIG-I基因CDS区全长2 802 bp,共编码933个氨基酸;不同结构域缺失突变体的真核表载体转染DF1细胞后,重组蛋白均在DF1细胞中表达;RT-qPCR结果显示,N端能显著激活RLR通路上IFN-β、Mx1及PKR基因的表达上调。【结论】 duRIG-I及不同区段均能在DF1细胞中表达,其中N端在调节RLR抗病毒信号通路下游基因的表达过程中发挥了重要作用。     

芒果炭疽病菌环境pH信号调控基因PalF的克隆与分析

通过本地查找胶孢炭疽病菌(Colletotrichum gloeosporioides)橡胶菌株全基因组测序数据,获得PalF基因的序列信息。利用同源克隆的方法扩增胶孢炭疽病菌芒果菌株PalF基因,获得片段大小为2 369 bp的目的基因片段。对该基因序列进行分析发现,该片段含有完整的开放式阅读框,与橡胶炭疽菌PalF基因的序列同源性达100%。用RT-PCR的方法获得PalF基因的cDNA序列,序列进行分析发现该基因全长为2 310 bp,其中含有1个大小为59 bp的内含子,推测编码769个氨基酸。对PalF基因的氨基酸序列进行推测保守结构域分析可知PalF蛋白中含有Arrestin-C保守结构域,推测该基因参与环境pH感应信号转导途径(Ambient pH-sensing signal transduetion pathway)的调控,为进一步研究该基因在环境pH感应信号转导途径中的作用奠定了基础。     

脂肪细胞膜免疫降脂的研究

本文综述了利用脂肪细胞膜免疫技术进行畜禽脂肪代谢研究的成果，其技术路线主要包括试验途径和方法、免疫机理、降脂效果及特异性脂肪膜蛋白的研究启示，从而为脂肪细胞膜免疫降脂试验提供成功的范例和科学的依据，并开辟降低脂肪、提高瘦肉率研究的新领域。     

Mechanical Signalling in Plant: Perception and Transduction Pathway

The metabolism and development of plants are controlled through interplay between genetic and environmental cues.For optimal growth plants require the ability to coordinate complex developmental processes and at the same time sense and respond to environmental stimuli.The responses can be divided into three main events: stimulus perception,generation and transmission of a signal,and subsequent changes in downstream biochemical processes.Because of space limitation this review does not provide an exhaustive account of plant signal transduction.Rather,the authors intention is to cover new and important results in this field,including the mechanochemical transform mediated by integrin and integrin-like protein,the change of membrane fluidity respond to stimulus,and the wide accepted transduction pathway of calcium signaling system.The research emphasis and tendency are prospected.     

Testing assumptions of cost surface analysis—a tool for invasive species management

Applied ecology could benefit from new tools that identify potential movement pathways of invasive species, particularly where data are sparse. Cost surface analysis (CSA) estimates the permeability (friction) across a landscape and can be applied to dispersal modelling. Increasingly used in a diversity of applications, several fundamental assumptions that might influence the outputs of CSA (cost surfaces and least-cost pathways) have yet to be systematically examined. Thus, we explore two issues: the presumed relationship between habitat preferences and dispersal behaviour as well as the degree of landscape fragmentation through which an organism moves by modelling a total of 18 sensitivity and dispersal scenarios. We explored the effect of fragmentation by altering the friction values (generally assigned using expert opinion) associated with patch and linear features. We compared these sensitivity scenarios in two sites that differed in fragmentation. We also used eastern grey squirrels (Sciurus carolinensis) as an example invading species and compared diffusion models and two contrasting cost surface dispersal scenarios. The diffusion model underestimated spread because squirrels did not move randomly through the landscape. Despite contrasting assumptions regarding dispersal behaviour, the two cost surfaces were strikingly similar while the least-cost paths differed. Furthermore, while the cost surfaces were insensitive to changes in friction values for linear features, they were sensitive to assumptions made for patch features. Our results suggest that movement in fragmented landscapes may be more sensitive to assumptions regarding friction values than contiguous landscapes. Thus, the reliability of CSA may depend not only on the range of friction values used for patches but also the degree of contiguity in the landscape.     

Comparative analysis of MAPK and PI3K/AKT pathway activation and inhibition in human and canine melanoma

The lack of advanced animal models of human cancers is considered a barrier to developing effective therapeutics. Canine and human melanomas are histologically disparate but show similar disease progression and response to therapies. The purpose of these studies was to compare human and canine melanoma tumours and cell lines regarding MAPK and PI3K/AKT signalling dysregulation, and response to select molecularly targeted agents. Pathway activation was investigated via microarray and mutational analysis. Growth inhibition and cell cycle effects were assessed for pathway inhibitors AZD6244 (MAPK) and rapamycin (PI3K/AKT) in human and canine melanoma cells. Human and canine melanoma share similar differential gene expression patterns within the MAPK and PI3K/AKT pathways. Constitutive pathway activation and similar sensitivity to AZD6244 and rapamycin was observed in human and canine cells. These results show that human and canine melanoma share activation and sensitivity to inhibition of cancer‐related signalling pathways despite differences in activating mutations.     

When, why, and how to perform cardiac radiofrequency catheter ablation

Radiofrequency catheter ablative techniques provide a unique opportunity to cure dogs of certain supraventricular tachyarrhythmias, rather than simply controlling the rhythm with drug therapy. Accessory pathways, which can participate as one limb of a rapid, narrow complex tachyarrhythmia circuit, have a particularly high cure rate with radiofrequency catheter ablation. Focal atrial tachycardias also provide a substrate that can be amenable to ablation. Tachycardia-induced myocardial dysfunction resulting from frequent tachyarrhythmic episodes will resolve after a successful ablation. This report outlines the indications, equipment, and techniques used in dogs for performing catheter ablation with temperature-controlled radiofrequency energy delivery.     

降糖益肾方干预胰岛素信号通路改善转基因 2型糖尿病1VIKR鼠肾损伤的研究

目的观察降糖益肾方对高脂饮食转基因2型糖尿病MKR鼠血糖、血胰岛素及肾皮质中胰岛素受体、磷脂酞肌醇-3一激酶蛋白表达的影响、方法选取MKR鼠40只经鉴定后,随机分为MKR鼠组、MKR鼠高脂组、降糖益肾方组和糖适贝那组〔MKR鼠组用基础饲料喂养,MKR鼠高脂组、降糖益肾方组和糖适贝那组用高脂饲料喂养,连续喂养8周后,降糖益肾方组给予降糖益肾方,糖适贝那组给予糖适平加贝那普利,同时MKR鼠组和MKR鼠高脂组分别给予蒸馏水灌胃4周〔4周后处死小鼠收集标本,免疫组织化学SABC法检测肾小球中胰岛素受体1(IRS-1),磷脂酞肌醇-3一激}(PI-3K)的蛋白表达〔结果降糖益肾方明显降低高脂饮食MKR鼠肾小球中IRS-1和PI-3 K的蛋白表达水平,与MKR鼠高脂组比较,差异有统计学意义(P<0.01)、结论降糖益肾方改善糖尿病肾病系膜细胞增殖的机制可能与千预胰岛素信号通路有关、