重组标签蛋白在蛋白质纯化中的研究进展

重组蛋白的表达纯化是研究蛋白质结构与功能的重要环节之一,表达重组蛋白宿主细胞的监测筛选和重组蛋白低水平可溶性表达、低回收率以及不稳定易水解等问题一直是蛋白质高通量纯化工艺中的难题。近年来,将多肽或蛋白质作为标签,与目标蛋白共表达的方法已经很大程度地解决了这一问题。因此,针对不同特性的蛋白质选择合适的标签纯化策略在重组蛋白纯化研究中是相当关键的环节。本文回顾了几种传统标签蛋白的研究概况(His-tag,Arg-tag,Flag-tag,GST-tag,MBP-tag等),着重对近年来新开发的了几种标签蛋白(Si-tag,Halo-tag,Intein-CBD-tag,ELPs-tag等)进行了深入探讨,并对新标签蛋白在蛋白质纯化中的应用前景作以展望。     

萝卜硫素对急性低温暴露鼠骨骼肌Nrf2介导的抗氧化能力的影响

萝卜硫素(sulforaphane,SFN)是一种在十字花科植物中含量丰富,且具有抗氧化效应的天然物质。本文基于核因子E2相关因子2(nuclear factor E2-related factor 2,Nrf2)介导的抗氧化系统,探究不同时长低温暴露对骨骼肌抗氧化酶的影响及SFN对低温暴露骨骼肌抗氧化能力的作用。首先,30只雄性C57BL/6N小鼠随机分为常温对照组(0 h组)、低温暴露1 h组(1 h组)和低温暴露3 h组(3 h组)。其次,40只雄性C57BL/6N小鼠随机分为PBS常温对照组(PBS+Con),PBS低温暴露3 h组(PBS+Cold),SFN常温对照组(SFN+Con)和SFN低温暴露3 h组(SFN+Cold)。小鼠在急性温度干预前腹腔注射4次SFN或等体积PBS。急性低温暴露后,取小鼠骨骼肌,试剂盒检测活性氧(ROS)水平、总抗氧化能力(T-AOC)、还原型谷胱甘肽(GSH)和氧化型谷胱甘肽(GSSG)含量;荧光实时定量PCR检测Nrf2介导的抗氧化酶和参与生成谷胱甘肽相关酶的mRNA转录水平;Western blot检测Nrf2介导的抗氧化酶蛋白表达。结果显示,与0和1 h组相比,3 h组小鼠骨骼肌Nrf 2和抗氧化酶基因(Gpx 1、Hmox1、Cat、Sod 1和Nqo 1)的mRNA转录水平显著降低,ROS水平显著增加。与PBS+Con组相比,PBS+Cold组小鼠骨骼肌Nrf2和抗氧化酶(HMOX1和CAT)蛋白表达、GSH/GSSG比值及T-AOC水平显著降低,而GSSG含量和ROS水平增加。与PBS+Cold组相比,SFN+Cold组小鼠骨骼肌Nrf 2 mRNA及其蛋白表达、抗氧化酶(HMOX1和SOD1)蛋白表达、抗氧化酶基因(Gpx 1、Hmox 1、Cat、Sod 1和Nqo 1)mRNA转录水平、参与GSH生成的酶基因(Gclm和Gss)mRNA转录水平、GSH/GSSG比值以及T-AOC水平显著提高,而GSSG含量和ROS水平显著降低。综上,3 h急性低温暴露降低了Nrf2介导的抗氧化作用。而低温暴露前给予SFN补充,则激活了Nrf2介导的抗氧化酶和谷胱甘肽抗氧化系统,增强了骨骼肌抗氧化能力。     

硫氧还蛋白-1(Trx1)氧化还原状态的检测

硫氧还蛋白-1(thioredoxin-1,Trx1)是一种广泛存在于生物体内的氧化还原调节蛋白,其氧化还原状态的变化是细胞内发挥氧化还原调控作用的重要过程.本文建立了Trx1氧化还原状态的检测方法—氧化还原蛋白免疫印迹法(redox Western blot),即通过碘乙酸(IAA)标记Trx1,根据蛋白所带负电荷的不同,达到分离蛋白氧化与还原状态的目的,并根据能斯特方程计算出相应的氧化还原电势.本方法是在蛋白免疫印迹(Western blot)的基础上建立的,具有低成本、易操作的特点.实验中分别采用H2O2和DTT处理样本,利用此方法检测了细胞裂解液中、细胞内及过表达Trx1氧化还原电势的变化;并检测了HEK293细胞不同生长时期Trx1的氧化还原状态.     

亲和标签调节的 (S)-羰基还原酶2催化2-羟基苯乙酮的酶学性质

不同类型的亲和标签会影响酶的催化功能和酶学性质。近平滑假丝酵母Candida parapsilosis来源的(S)-羰基还原酶2 ((S)-carbonyl reductase 2,SCR2) 能催化2-羟基苯乙酮。文中在SCR2的N端添加不同类型的亲和标签,在大肠杆菌Escherichia coli中异源表达并纯化重组蛋白his6-SCR2、strep-SCR2和MBP-SCR2,研究了重组蛋白催化2-羟基苯乙酮的酶学性质。结果表明,不同类型的亲和标签对SCR2的酶学性质有一定的影响。其中,不同类型的亲和标签对重组蛋白稳定性影响较大：1) 在pH 6.0、30 ℃条件下保温13 h后,重组蛋白his6-SCR2和strep-SCR2的剩余酶活力是无融合标签SCR2的90.0%?95.2%,而MBP-SCR2的剩余酶活力是无融合标签SCR2的1.25倍。2) MBP-SCR2在50 ℃的半衰期比strep-SCR2、his6-SCR2和无融合标签SCR2长26.6%–48.8%。3) MBP-SCR2在?80 ℃存储60 d后,其酶活动力学参数kcat比his6-SCR2、strep-SCR2和无融合标签SCR2高1.25–1.45倍。根据三级结构分析推出重组蛋白MBP-SCR2中MBP的C末端的α螺旋具有稳定SCR2的N端无规则卷曲的作用,从而提高酶的稳定性。圆二色谱检测结果表明MBP标签对蛋白SCR2的二级结构有一定的影响,且解折叠温度 (Tm) 分析证明,MBP-SCR2的Tm比无融合标签SCR2提高近5 ℃。研究结果不仅为羰基还原酶家族增添了一种2-羟基苯乙酮的稳定高效催化剂MBP-SCR2,同时为其他短链醇脱氢酶的标签设计提供了借鉴和依据。     

NaCl胁迫下AM真菌对滨梅叶片中抗坏血酸-谷胱甘肽循环的影响

以接种Glomus mosseae的滨梅幼苗为试材,研究了AM真菌对2.0%NaCl胁迫下滨梅叶片抗坏血酸-谷胱甘肽循环系统的影响。结果显示,NaCl胁迫下滨梅幼苗叶片H2O2含量呈增加趋势,但接种菌幼苗叶片H2O2含量显著低于未接种苗的。NaCl胁迫下菌根苗叶片抗氧化酶(APX、DHAR和GR)活性、AsA和GSH含量、氧化还原力(AsA/DHA值和GSH/GSSG值)均显著高于未接种苗。这表明,NaCl胁迫下,AM真菌能促进滨梅叶AsA-GSH循环快速有效地运转,维持体内抗氧化物质较强的再生能力,从而提高抗氧化胁迫能力,菌根苗表现出较强的耐盐性。     

His-tag对胆固醇氧化酶基因在大肠杆菌中表达的影响

为考察组氨酸标签(His-tag)对Brevibacterium sp.DGCDC-82中胆固醇氧化酶基因(ChoAb)在大肠杆菌中表达的影响,将PCR扩增后得到的结构基因与pET28a(+)连接,构建重组质粒pETChoAb(不带His-tag),pETChoAbn(His-tag位于N端)和pETChoAbc(His-tag位于C端)并在大肠杆菌中进行表达.对重组酶进行酶活检测,结果表明His-tag位于ChoAb的C端和N端,COD单位体积酶活由未带标签时的1.72 U/mL分别提高到4.03 U/mL和11.36 U/mL.利用软件Quantity One对SDS-PAGE电泳条带进行灰度分析,结果显示与不带His-tag的COD相比,His-tag位于ChoAb的C端和N端,COD表达量由8.8％增加到16.4％与72.3％.同时菌体浓度分别提高了1.2倍和3.2倍.作为纯化标签,该研究结果对His-tag用于诊断用酶COD的分离纯化可以提供一定的理论指导.     

一种用于药物蛋白亲和纯化和跨膜转运的双功能标签的开发

目的:开发一种既能用于亲和纯化目标蛋白,又可介导不能自主进入细胞的药物蛋白跨膜转运到细胞内发挥活性的双功能标签。方法:从已有文献资料中挑选四种富含碱性氨基酸的钙调蛋白结合肽(calmodulin binding peptide,CBP),将其与绿色荧光蛋白(EGFP)融合表达,然后采用与钙调蛋白(calmodulin,CaM)亲和结合过程来筛选与CaM具有最高亲和力的CBP;随后采用荧光显微镜检测、激光共聚焦显微镜检测以及流式细胞术等技术来分析测定和比较候选CBP序列将EGFP重组蛋白自主转运进入细胞的能力。最后将筛选到的新型CBP双功能标签与凋亡蛋白融合表达,考察其与CaM亲和结合后纯化重组凋亡蛋白的能力,以MTT法分析此重组蛋白进入肿瘤细胞抑制生长的能力。结果:通过CaM-CBP亲和层析筛选出与CaM具高有亲和力的三种CBP序列;从重组蛋白胞内荧光检测结果得知,带有野生型骨骼肌肌球蛋白轻链激酶CBP序列(MLCK)的重组EGFP蛋白具有最佳跨膜转运效率,且显著高于来源于艾滋病毒的经典穿膜肽TAT的穿膜效率。以此MLCK新型双功能标签成功地通过CaM-CBP亲和结合纯化得到重组凋亡蛋白,并可将重组凋亡蛋白转运进入细胞内发挥抗肿瘤作用。重组凋亡蛋白对MGC-803、H460、HeLa三种肿瘤细胞生长的24h半抑制浓度(IC50)分别为:1. 18μmol/L、1. 23μmol/L、1. 23μmol/L。结论:筛选得到一种新型双功能标签MLCK,其可通过与CaM高亲和作用进行亲和纯化;同时标签本身还具有和典型穿膜肽一样的高效跨膜转运功能,可将药物蛋白自主转运进入细胞,发挥药物的生物活性。因此,新型双功能标签既可用于药物蛋白的亲和纯化,又兼具体内跨膜运输作用,可广泛用于各种新型药物的开发。     

摄入异烟肼对家蚕幼虫体内谷胱甘肽氧化还原循环和谷胱甘肽S-转移酶活性的影响

为了建立家蚕Bombyx mori的药物筛选和毒性评价模型, 以剂量为2 000 mg/kg的抗结核模药异烟肼饲喂家蚕5龄第3天幼虫后检测其中肠和脂肪体的抗氧化解毒相关代谢的变化。结果表明： 雌蚕中肠组织中, 总谷胱甘肽(GSH+2GSSG)、 还原型谷胱甘肽(reduced glutathione, GSH)和氧化型谷胱甘肽(oxidized glutathione, GSSG)含量均呈现迅速上升再缓慢下降趋势; 谷胱甘肽S 转移酶(glutathione S-transferase, GST)活性升高到较大值后逐渐降低; GSH/GSSG的比值下降表明, 在72 min后中肠组织向氧化态转移。脂肪体组织中, 总谷胱甘肽、 GSH和GSSG含量变化均呈现迅速下降再迅速上升的趋势; GST活性达到最大值后逐渐降低后趋于平稳; GSH/GSSG比值升高表明, 在72 min后脂肪体组织向还原态转移。无论雌蚕还是雄蚕, 总谷胱甘肽、 GSH和GSSG含量以及GST活性均是脂肪体高于中肠。雌蚕的总谷胱甘肽含量、 GSH和GSSG含量高于雄蚕, 但雄蚕的GST活性高于雌性。结果说明, 摄入异烟肼引起了家蚕幼虫体内谷胱甘肽氧化还原状态的改变和酶活性的变化, 在这个过程中脂肪体起主要解毒代谢作用。     

外源一氧化氮供体对NaCl胁迫下多裂骆驼蓬幼苗叶片ASA-GSH循环的影响

研究了外源一氧化氮(NO)供体硝普钠(SNP)对NaCl胁迫下多裂骆驼蓬幼苗抗坏血酸(ASA)-谷胱甘肽(GSH)循环抗氧化系统及H2O2和丙二醛(MDA)含量的影响。结果表明,0.15mmol.L-1SNP能提高300mmol.L-1NaCl胁迫下多裂骆驼蓬幼苗叶片抗坏血酸过氧化物酶(APX)、谷胱甘肽还原酶(GR)和谷胱甘肽转硫酶(GST)活性,增加还原型抗坏血酸(ASA)和谷胱甘肽(GSH)含量,降低脱氢抗坏血酸(DHA)和氧化型谷胱甘肽(GSSG)含量,提高ASA/DHA、GSH/GSSG比率,降低H2O2和MDA水平,对单脱氢抗坏血酸还原酶(MDAR)和脱氢抗坏血酸还原酶(DHAR)活性无显著影响。NO信号转导途径关键酶鸟苷酸环化酶(GC)抑制剂亚甲基蓝(MB)逆转了SNP对盐胁迫下APX、GR、GST活性和ASA、GSH、DHA,H2O2、MDA含量及ASA/DHA、GSH/GSSG比率的调节效应。由此表明,NO可能通过GC介导的cGMP信号转导参与ASA-GSH循环活性氧清除系统的调节,从而缓解盐胁迫诱导的氧化伤害。     

过表达Grx1抑制HEK293T细胞中H2O2诱导的p38MAPK信号通路

谷氧还蛋白1(glutaredoxin1, Grx1)是细胞内一种重要的巯基-二硫键氧化还原酶,在细胞内氧化还原状态的调控及抵抗氧化应激损伤过程中发挥重要作用.为进一步探讨Grx1的抗氧化机制,本实验将重组质粒pcDNA3.1(+)-hGrx1瞬时转染HEK293T细胞,经RT-PCR和Western印迹验证,细胞转染后实现了Grx1的过表达;以不同浓度H2O2为损伤因素,建立细胞氧化应激模型,检测过表达Grx1后细胞存活率,丙二醛(MDA)含量,超氧化物歧化酶(SOD)活力和乳酸脱氢酶(LDH)漏出率的变化,观察过表达Grx1后细胞的抗氧化能力;用终浓度100μmol/L H2O2作用于细胞,利用Western印迹检测120min内HEK293T细胞中p38MAPK磷酸化水平.实验结果表明,HEK293T细胞过表达Grx1后,缓解了细胞的氧化应激损伤;转染空载体组细胞p38MAPK磷酸化水平在H2O2刺激后5min开始升高,15min达到最高值,并可维持至120min左右;而过表达Grx1组细胞p38MAPK磷酸化水平在H2O2刺激后各时间段没有明显改变,提示Grx1通过抑制H2O2诱导的p38MAPK信号通路激活发挥其抗氧化作用.     

Production of biologically active human thioredoxin 1 protein in lettuce chloroplasts

The production of human therapeutic proteins in plants provides opportunities for low-cost production, and minimizes the risk of contamination from potential human pathogens. Chloroplast genetic engineering is a particularly promising strategy, because plant chloroplasts can produce large amounts of foreign target proteins. Oxidative stress is a key factor in various human diseases. Human thioredoxin 1 (hTrx1) is a stress-induced protein that functions as an antioxidant against oxidative stress, and overexpression of hTrx1 has been shown to suppress various diseases in mice. Therefore, hTrx1 is a prospective candidate as a new human therapeutic protein. We created transplastomic lettuce expressing hTrx1 under the control of the psbA promoter. Transplastomic plants grew normally and were fertile. The hTrx1 protein accumulated to approximately 1% of total soluble protein in mature leaves. The hTrx1 protein purified from lettuce leaves was functionally active, and reduced insulin disulfides. The purified protein protected mouse insulinoma line 6 cells from damage by hydrogen peroxide, as reported previously for a recombinant hTrx1 expressed in Escherichia coli. This is the first report of expression of the biologically active hTrx1 protein in plant chloroplasts. This research opens up possibilities for plant-based production of hTrx1. Considering that this expression host is an edible crop plant, this transplastomic lettuce may be suitable for oral delivery of hTrx1.     

根系温度对光核桃幼苗光合机构热稳定性的影响

以光核桃(Prunus mira)幼苗为材料, 通过控制根系温度研究了根系温度变化与叶片脱落酸(ABA)的关系及其对光合机构热稳定性的影响。结果表明: 1)环境高温(37和40 ℃)胁迫下保持根系温度适宜((25±2) ℃)时, 幼苗叶片相对含水量(Relative water content, RWC)下降较少, 但叶片ABA含量低, 超氧化物歧化酶(SOD)、抗坏血酸过氧化物酶(APX)、过氧化物酶(POD)和过氧化氢酶(CAT)活性低, 过氧化氢(H₂O₂)含量和膜质过氧化水平(丙二醛(Malondialdehyde, MDA)浓度)提高, 最大光化学效率(F_v/F_m)下降程度较大; 2)而同等环境高温(37和40 ℃)条件下根系温度逐步升高时, 幼苗叶片RWC降低, 叶片ABA含量增加, SOD、APX、POD、CAT活性高, H₂O₂含量高, MDA生成量低, F_v/F_m降低程度较小。与37 ℃相比, 40 ℃处理条件下各生理指标变化趋势相似, 但差异加大。因此认为: 高温胁迫条件下, 根系温度适宜时RWC高, 但导致光合机构伤害较重; 根系感受高温胁迫能够增加叶片ABA含量, 有助于保护光合机构、提高光合机构的抗热性。     

Thioredoxin and Thioredoxin Reductase Control Tissue Factor Activity by Thiol Redox-dependent Mechanism

Abnormally enhanced tissue factor (TF) activity is related to increased thrombosis risk in which oxidative stress plays a critical role. Human cytosolic thioredoxin (hTrx1) and thioredoxin reductase (TrxR), also secreted into circulation, have the power to protect against oxidative stress. However, the relationship between hTrx1/TrxR and TF remains unknown. Here we show reversible association of hTrx1 with TF in human serum and plasma samples. The association is dependent on hTrx1-Cys-73 that bridges TF-Cys-209 via a disulfide bond. hTrx1-Cys-73 is absolutely required for hTrx1 to interfere with FVIIa binding to purified and cell-surface TF, consequently suppressing TF-dependent procoagulant activity and proteinase-activated receptor-2 activation. Moreover, hTrx1/TrxR plays an important role in sensing the alterations of NADPH/NADP⁺ states and transducing this redox-sensitive signal into changes in TF activity. With NADPH, hTrx1/TrxR readily facilitates the reduction of TF, causing a decrease in TF activity, whereas with NADP⁺, hTrx1/TrxR promotes the oxidation of TF, leading to an increase in TF activity. By comparison, TF is more likely to favor the reduction by hTrx1-TrxR-NADPH. This reversible reduction-oxidation reaction occurs in the TF extracellular domain that contains partially opened Cys-49/-57 and Cys-186/-209 disulfide bonds. The cell-surface TF procoagulant activity is significantly increased after hTrx1-knockdown. The response of cell-surface TF procoagulant activity to H₂O₂ is efficiently suppressed through elevating cellular TrxR activity via selenium supplementation. Our data provide a novel mechanism for redox regulation of TF activity. By modifying Cys residues or regulating Cys redox states in TF extracellular domain, hTrx1/TrxR function as a safeguard against inappropriate TF activity.     

Generation of expressed sequence tags from low-CO2 and high-CO2 adapted cells of Chlamydomonas reinhardtii.

To characterize genes whose expression is induced in carbon-stress conditions, 12,969 and 13,450 5'-end expressed sequence tags (ESTs) were generated from cells grown in low-CO2 and high-CO2 conditions of the unicellular green alga, Chlamydomonas reinhardtii. These ESTs were clustered into 4436 and 3566 non-redundant EST groups, respectively. Comparison of their sequences with those of 3433 non-redundant ESTs previously generated from the cells under the standard growth condition indicated that 2665 and 1879 EST groups occurred only in the low-CO2 and high-CO2 populations, respectively. It was also noted that 96.2% and 96.0% of the cDNA species respectively obtained from the low-CO2 and high-CO2 conditions had no similar EST sequence deposited in the public databases. The EST species identified only in the low-CO2 treated cells included genes previously reported to be expressed specifically in low-CO2 acclimatized cells, suggesting that the ESTs generated in this study will be a useful source for analysis of genes related to carbon-stress acclimatization. The sequence information and search results of each clone will appear at the web site: http://www.kazusa.or.jp/en/plant/chlamy/EST/.     

Responses of rice cultivars with different nitrogen use efficiency to partial nitrate nutrition

BACKGROUND AND AIMS: There is increased evidence that partial nitrate (NO3-) nutrition (PNN) improves growth of rice (Oryza sativa), although the crop prefers ammonium (NH4+) to NO3- nutrition. It is not known whether the response to NO3- supply is related to nitrogen (N) use efficiency (NUE) in rice cultivars. Methods Solution culture experiments were carried out to study the response of two rice cultivars, Nanguang (High-NUE) and Elio (Low-NUE), to partial NO3- supply in terms of dry weight, N accumulation, grain yield, NH4+ uptake and ammonium transporter expression [real-time polymerase chain reaction (PCR)]. KEY RESULTS: A ratio of 75/25 NH4+ -N/NO3- -N increased dry weight, N accumulation and grain yield of 'Nanguang' by 30, 36 and 21 %, respectively, but no effect was found in 'Elio' when compared with those of 100/0 NH4+ -N/NO3- -N. Uptake experiments with 15N-NH4+ showed that NO3- increased NH4+ uptake efficiency in 'Nanguang' by increasing Vmax (14 %), but there was no effect on Km. This indicated that partial replacement of NH4+ by NO3- could increase the number of the ammonium transporters but did not affect the affinity of the transporters for NH4+. Real-time PCR showed that expression of OsAMT1s in 'Nanguang' was improved by PNN, while that in 'Elio' did not change, which is in accordance with the differing responses of these two cultivars to PNN. Conclusions Increased NUE by PNN can be attributed to improved N uptake. The rice cultivar with a higher NUE has a more positive response to PNN than that with a low NUE, suggesting that there might be a relationship between PNN and NUE.     

High-throughput Purification and Quality Assurance of Arabidopsis thaliana Proteins for Eukaryotic Structural Genomics

The Center for Eukaryotic Structural Genomics (CESG) has established procedures for the purification of Arabidopsis proteins in a high-throughput mode. Recombinant proteins were fused with (His)(6)-MBP tags at their N-terminus and expressed in Escherichia coli. Using an automated AKTApurifier system, fusion proteins were initially purified by immobilized metal affinity chromatography (IMAC). After cleavage of (His)(6)-MBP tags by TEV protease, (His)(6)-MBP tags were separated from target proteins by a subtractive 2nd IMAC. As a part of quality assurance, all purified proteins were subjected to MALDI-TOF and ESI mass spectrometry to confirm target identity and integrity, and determine incorporation of seleno-methionine (SeMet) and (15)N and (13)C isotopes. The protocols have been used successfully to provide high quality proteins that are suitable for structural studies by X-ray crystallography and NMR.     

Series of vectors to evaluate the position and the order of two different affinity tags for purification of protein complexes

A series of protein expression vectors with dual-affinity tags has been developed. With these constructed vectors, FLAG and hexahistidine tags were fused to a given protein at either the N- or the C-terminal ends or both, for a total of six combinations. Three auxotrophy markers were introduced into each construct, thus yielding 18 different vectors. These vectors allow evaluation of different positions and orders of two different tags. To confirm the efficacy of these vectors, we purified a histone acetyltransferase (Esa1p)-containing complex. First, an appropriate position of the tags was selected through small-scale purification. Next, large-scale purification was done for the selected construct, yielding an Esa1p-containing complex that was comparable to an Esa1p-containing complex (NuA4) obtained by a conventional activity-based purification. These vectors provide a convenient way to select the best position of tags for efficient purification of protein complexes also applicable in proteomics studies.     

A highly efficient multifunctional tandem affinity purification approach applicable to diverse organisms

Determining the localization, binding partners, and secondary modifications of individual proteins is crucial for understanding protein function. Several tags have been constructed for protein localization or purification under either native or denaturing conditions, but few tags permit all three simultaneously. Here, we describe a multifunctional tandem affinity purification (MAP) method that is both highly efficient and enables protein visualization. The MAP tag utilizes affinity tags inserted into an exposed surface loop of mVenus offering two advantages: (1) mVenus fluorescence can be used for protein localization or FACS-based selection of cell lines; and (2) spatial separation of the affinity tags from the protein results in high recovery and reduced variability between proteins. MAP purification was highly efficient in multiple organisms for all proteins tested. As a test case, MAP combined with liquid chromatography-tandem MS identified known and new candidate binding partners and modifications of the kinase Plk1. Thus the MAP tag is a new powerful tool for determining protein modification, localization, and interactions.     

The distribution of aquaporin subtypes (PIP1, PIP2 and gamma-TIP) is tissue dependent in soybean (Glycine max) root nodules

BACKGROUND AND AIMS The inner cortical cells (IC-cells) of legume root nodules have been previously shown to regulate the resistance to nodule O2 diffusion by a rapid contraction/expansion mechanism, which controls the volume of intercellular spaces and their occlusion by a liquid phase. The expression of aquaporins in IC-cells was also found to be involved in this nodule O2 diffusion mechanism. The aim of this study was to compare the expression of plasma membrane intrinsic proteins (PIP) aquaporin isoforms with tonoplast intrinsic protein (gamma-TIP) in both IC-cells and adjacent cell types. METHODS: Using immunogold labelling in ultra-thin sections of Glycine max nodules, the expression of two PIP isoforms was observed and compared with the gamma-TIP pattern. KEY RESULTS: The plasma membrane aquaporins PIP1 and PIP2 were expressed more in IC-cells and endodermis than in pericycle and infected cells. The tonoplast aquaporin gamma-TIP has shown a distribution pattern similar to that of the PIPs. CONCLUSIONS: PIPs and gamma-TIP aquaporins are highly expressed in both plasmalemma and tonoplast of nodule IC-cells. This distribution is consistent with the putative role of water fluxes associated with the regulation of nodule conductance to O2 diffusion and the subsequent ATP-dependent nitrogenase activity. In the endodermis, these aquaporins might also be involved in nutrient transport between the infected zone and vascular traces.     

Using sutures to attach miniature tracking tags to small bats for multimonth movement and behavioral studies

Determining the detailed movements of individual animals often requires them to carry tracking devices, but tracking broad-scale movement of small bats (<30 g) has been limited by transmitter technology and long-term attachment methods. This limitation inhibits our understanding of bat dispersal and migration, particularly in the context of emerging conservation issues such as fatalities at wind turbines and diseases. We tested a novel method of attaching lightweight global positioning system (GPS) tags and geolocating data loggers to small bats. We used monofilament, synthetic, absorbable sutures to secure GPS tags and data loggers to the skin of anesthetized big brown bats (Eptesicus fuscus) in Colorado and hoary bats (Lasiurus cinereus) in California. GPS tags and data loggers were sutured to 17 bats in this study. Three tagged bats were recaptured 7 months after initial deployment, with tags still attached; none of these bats showed ill effects from the tag. No severe injuries were apparent upon recapture of 6 additional bats that carried tags up to 26 days after attachment; however, one of the bats exhibited skin chafing. Use of absorbable sutures to affix small tracking devices seems to be a safe, effective method for studying movements of bats over multiple months, although additional testing is warranted. This new attachment method has the potential to quickly advance our understanding of small bats, particularly as more sophisticated miniature tracking devices (e.g., satellite tags) become available.