Harmonizing Labeling and Analytical Strategies to Obtain Protein Turnover Rates in Intact Adult Animals

Changes in the abundance of individual proteins in the proteome can be elicited by modulation of protein synthesis (the rate of input of newly synthesized proteins into the protein pool) or degradation (the rate of removal of protein molecules from the pool). A full understanding of proteome changes therefore requires a definition of the roles of these two processes in proteostasis, collectively known as protein turnover. Because protein turnover occurs even in the absence of overt changes in pool abundance, turnover measurements necessitate monitoring the flux of stable isotope–labeled precursors through the protein pool such as labeled amino acids or metabolic precursors such as ammonium chloride or heavy water. In cells in culture, the ability to manipulate precursor pools by rapid medium changes is simple, but for more complex systems such as intact animals, the approach becomes more convoluted. Individual methods bring specific complications, and the suitability of different methods has not been comprehensively explored. In this study, we compare the turnover rates of proteins across four mouse tissues, obtained from the same inbred mouse strain maintained under identical husbandry conditions, measured using either [¹³C₆]lysine or [²H₂]O as the labeling precursor. We show that for long-lived proteins, the two approaches yield essentially identical measures of the first-order rate constant for degradation. For short-lived proteins, there is a need to compensate for the slower equilibration of lysine through the precursor pools. We evaluate different approaches to provide that compensation. We conclude that both labels are suitable, but careful determination of precursor enrichment kinetics in amino acid labeling is critical and has a considerable influence on the numerical values of the derived protein turnover rates.     

Salinity Impact on Bacterial Community Composition in Five High-Altitude Lakes from the Tibetan Plateau,Western China

The influence of salinity and geographical distance on bacterial community composition (BCC) in five freshwater, oligosaline or polysaline lakes located at altitudes higher than 4400 m on the central and southern Tibetan Plateau were investigated using the 16S rRNA gene clone library approach together with multivariate analysis of environmental variables. A total of 10 clone libraries were constructed with two libraries in each lake, one in the epilimnion and the other in the hypolimnion. Geographical distance was not found to impact BCC significantly, but salinity, chl a and lake hydraulic retention time were significant factors influencing the BCC. Bacteria in lakes located on the central and southern Plateau owned the same community composition as that observed from the eastern Tibetan lakes. They were both predominated by Bacteroidetes and Cyanobacteria, had low taxon richness, and similar typical freshwater clusters and distributed characteristics. Supplemental materials are available for this article. Go to the publisher's online edition of Geomicrobiology Journal to view the supplemental file.     

Theophylline Steady-State Pharmacokinetics: Recent Concepts and Their Application in Chronotherapy of Reactive Airway Diseases

With the introduction of sustained-release theophyllíne formulations for once-daily dosing or for unequally divided twice-daily dosing, comparison with conventional equally divided twice-daily dosing has been focused on nocturnal serum theophylline concentrations (STCs), plateau properties and peak-trough fluctuation. The merits of various steady-state characteristics such as nocturnal excess, plateau time, residual concentration, peak-trough fluctuation, swing and AUC fluctuation are illustrated by 15 data sets from 7 multiple-dose studies, each including either 10–12 healthy volunteers or 12–20 COPD-patients. In all of the studies, STCs were determined at least every 2 hr over a 24-hr period in steady-state. Included in the studies were 7 sustained-release theophylline formulations which were administered either once daily (in the morning or in the evening), or twice daily (either equally divided, or unequally divided with one-third of the dose being given in the morning and two-thirds in the evening).     

青藏高原两种植被类型净初级生产力与气候因素的关系及周转值分析

基于2008—2016年青海海北站9年净初级生产力及气候因子监测数据,分析了青藏高原高寒小嵩草草甸和高寒金露梅灌丛两种植被净初级生产力年际动态,并探讨了气候因子对其影响及其不同土层深度根系周转值特征。结果表明:(1)年际尺度上,小嵩草草甸地上净初级生产力表现为显著增加趋势,增幅为7.02 g m~(-2) a~(-1),而金露梅灌丛地上净初级生产力相对较为稳定;对于其地下净初级生产力和总生产力,小嵩草草甸和金露梅灌丛均表现为增加趋势(P0.05),9年间小嵩草草甸地上、地下和总净初级生产力平均值分别为(217.55±9.95)、(1882.75±161.33) g m~(-2) a~(-1)和(2100.30±163.38) g m~(-2) a~(-1),金露梅灌丛地上、地下和总净初级生产力9年间平均值分别为(256.27±11.4)、(1614.31±173.03) g m~(-2) a~(-1)和(1870.58±177.93) g m~(-2) a~(-1)。(2)不同植被类型地上净初级生产力对气候因素响应不同,金露梅灌丛地上净初级生产力主要受温度影响,而温度对小嵩草草甸地上净初级生产力无显著影响。此外,降水不是限制高寒生态系统草地地上净初级生产力主要因子,相比于降水影响,高寒生态系统地上净初级生产力更受温度调控。(3)年均温和年降水对金露梅灌丛和小嵩草草甸地下净初级生产力均无显著影响(P0.05),表明高寒生态系统,其地下生产力受外界气候条件变化影响微弱,是一个稳定的碳库。(4)两种植被类型其根系周转值均随着土壤深度的增加呈逐渐增加趋势,且高寒灌丛根系周转值明显高于高寒草甸根系周转值。研究表明,在全球气候变暖背景下将会增加金露梅灌丛地上净初级生产力,而对小嵩草草甸地上净初级生产力无显著影响。     

青藏公路对高原鼠兔种内遗传分化的影响

研究采用7个微卫星标记分析在青海省西大滩区域,位于青藏公路两侧的4个高原鼠兔（Ochotona curzoniae）种群的遗传变异情况。分别采用软件TFPGA和GENE POP3．4计算各种群间的Nei’s标准遗传距离,基因分化系数（Fst）等参数,并对遗传距离进行UPGMA聚类分析。研究结果表明,公路同侧种群间平均遗传距离和基因分化系数分别为0．0808和0．0541;异侧种群间平均遗传距离和基因分化系数分别为0．1037和0．0705,公路东侧和西侧的两个种群分别聚为一类。青藏公路对分布于公路两侧的高原鼠兔种群间的基因交流产生了一定的阻隔效应,并导致种群间出现了一定程度的遗传分化。     

Akkermansia与高原牛肺水肿病相关性研究

【目的】本文通过对高原牛胃肠道菌群结构组成的分析,从微生物学角度探讨Akkermansia与高原牛肺水肿病的关系。【方法】本研究以沈阳地区健康娟姗牛为对照,以引进入拉萨半年的健康娟姗牛、拉萨本地健康黄牛以及引进入拉萨半年患肺水肿病的娟姗牛的粪便作为分析样本,采用Illumina MiSeq高通量测序技术测定样本中微生物16S rRNA基因V3–V4区序列,通过比较4种粪便样本菌群组成及丰度的差异,探讨Akkermansia与高原牛肺水肿病的相关性。【结果】Verrucomicrobia中Akkermansia在拉萨本地健康黄牛的胃肠道中的含量显著高于引进入拉萨半年的健康娟姗牛,在引进入拉萨半年患肺水肿病的娟姗牛胃肠道中的含量显著高于引进入拉萨半年的健康娟姗牛。在属水平上,沈阳地区健康娟姗牛胃肠道菌群中Akkermansia丰度占比为0.07%;引进入拉萨半年的健康娟姗牛胃肠道菌群中Akkermansia丰度占比为0.09%;拉萨本地黄牛胃肠道菌群中Akkermansia丰度占比为6.62%,是优势菌属;引进入拉萨半年的患肺水肿病的娟姗牛胃肠道菌群中Akkermansia丰度占比为11.85%,且是第一优势菌属。【结论】首次从微生物学角度探讨Akkermansia与高原牛肺水肿病的关系,为将Akkermansia丰度作为诊断肺水肿病的监测指标提供参考,但具体丰度值还有待进一步研究。     

青藏高原不同海拔高度下全缘叶绿绒蒿叶表皮特征研究

采用胶带粘取叶表皮法,利用光学显微镜观测不同海拔高度下全缘叶绿绒蒿叶片的表皮毛、气孔及表皮细胞结构特征,探讨全缘叶绿绒蒿叶表皮特征与海拔高度的关系。结果表明,随着海拔高度增加,全缘叶绿绒蒿叶片上、下表皮毛密度、气孔密度和表皮细胞密度逐渐增加;气孔器及表皮细胞的长度、宽度和面积逐渐减小;表皮细胞的形态随着海拔升高由无规则形向多边形变化,垂周壁由波状向弓形或平直变化。全缘叶绿绒蒿叶表皮结构在不同海拔高度下表现出的差异,可能是植物长期在高原生态环境下的综合反应,以结构上的变化来适应对高海拔地区的环境特点,为进一步研究高海拔地区植物的适应性提供一定依据。     

基于RAD测序技术的黄绿卷毛菇(Floccularia luteovirens)微卫星引物的开发

黄绿卷毛菇(Floccularia luteovirens)是广泛分布于青藏高原高寒草甸的一种重要的可食用的菌根真菌。先前的研究利用EST-SSR引物已经对该物种的遗传多样性及群落结构进行了研究,但研究表明EST-SSR位点多态性较低,因此想要进一步探讨形成现有遗传分布格局的机制(该物种小尺度空间范围内基株的密度及大小)需要重新开发具有更高多态性的基于核基因的微卫星位点。本研究利用RAD测序技术使用直接测序分析的方法重新开发了12对具有多态性的引物。测序结果去接头拼装后共获得46 036条重叠群。在这些序列当中共扫描到342条含有重复单元的序列,在这当中70.47%为三碱基重复(241),8.19%为二碱基重复(28)。随机选取48对检测,12对具有多态性且测序结果良好,基于3个居群63个个体的遗传多样性研究显示,所有样品中共获得60个单倍型,每个位点的单倍型数量介于2 (GSSR26L)至9 (GSSR7L,GSSR11L)之间。Gst值介于-0.03(GSSR46L)至0.28(GSSR6L)之间、Fst值介于-0.03(GSSR33L)至0.42(GSSR6L)之间。在引物GSSR47L扩增测序中发现,其扩增出的片段不仅具有重复单元数量的变化,重复单元本身也存在突变的现象,这是之前使用聚丙烯凝胶电泳所检测不到的。本研究获得的微卫星引物将为接下来小尺度空间下研究黄绿卷毛菇基株的密度、大小及动态变化提供有力的支持。     

纳木措可培养丝状真菌多样性及其与理化因子关系

微生物多样性在评估水体生态环境方面发挥着重要作用。本研究以青藏高原纳木措湖为研究对象, 开展水体可培养丝状真菌多样性及影响因子研究。通过膜过滤平置培养法、经典分类法和rRNA转录间隔区(ITS)序列分析对纳木措湖20个采样点的丝状真菌进行分离、纯化及鉴定, 测定水体理化指标, 综合分析丝状真菌空间分布格局与理化因子的相关性。菌种鉴定结果显示, 从纳木措水体样品中共分离纯化出1,412株丝状真菌, 隶属22属47种, 其中链格孢属(Alternaria)、青霉属(Penicillium)和毛霉属(Mucor)为优势属, 链格孢(Alternaria chlamydosporigena)和冻土毛霉(Mucor hiemalis)为优势种; Pearson相关性分析显示, 丝状真菌总丰度与温度、铵态氮、全磷呈显著正相关; 冗余分析显示, 铵态氮、温度、全磷、全氮、盐度及电导率是影响纳木措湖丝状真菌群落组成与分布的主要理化因子。综上所述, 纳木措水体可培养丝状真菌具有较高的物种多样性和空间异质性, 而且水体环境因子影响其分布。     

不同海拔高原鼠兔个性特征与SERT基因多态性关联

个性特征是指动物个体间稳定、可遗传的行为差异,与相关基因多态性有关,反映了动物对环境的适应模式,探究个性相关基因变异,将有助于更好地理解动物对环境的适应与进化机制。本文以高原鼠兔 (Ochotona curzoniae) 为对象,研究了5个不同海拔地区高原鼠兔的个性 (探究性、勇敢性和温顺性) 差异特征,并检测了个性相关基因5-羟色胺转运体 (SERT) 的单核苷酸多态性 (SNP) 和mRNA表达。结果发现,高海拔地区高原鼠兔的探究性和勇敢性显著高于低海拔地区,而高海拔地区高原鼠兔SERT基因mRNA表达量显著低于低海拔地区,提示不同海拔高原鼠兔个性特征差异可能与SERT基因mRNA表达有所关联。进一步检测不同海拔地区高原鼠兔SERT基因多态性及其分布差异情况,发现该基因存在6个SNP (其中5个位于外显子3,1个位于外显子5 );不同海拔地区鼠兔基因分布差异分析显示外显子5的c.A1063C 同义突变与海拔之间存在显著相关,高海拔地区该位点CC基因型分布频率显著高于低海拔地区;c.A1063C的基因型虽然与探究性、勇敢性无显著相关,但与温顺性显著相关。综上所述,随着海拔升高,高原鼠兔探究性和勇敢性增加,这有利于动物获取更多的食物资源,进而增加其生存机会。与此同时,SERT基因多态性显示与不同海拔地区的生存环境相适应的特征,且与温顺性有关,暗示不同海拔高原鼠兔个性差异可能与SERT基因SNP的差异有关。本研究从基因表达与突变的角度尝试阐述高原鼠兔适应不同海拔环境的行为差异,显示了不同海拔地区高原鼠兔行为生存策略潜在的分子机制。