外源γ-氨基丁酸对低氧胁迫下甜瓜幼苗活性氧代谢的影响

以甜瓜品种‘西域一号’幼苗为材料,采用营养液水培方法,设置正常通气(对照)、正常通气+GABA(5mmol.L-1)、低氧胁迫、低氧胁迫+GABA(5mmol.L-1)4个处理,研究了外源γ-氨基丁酸(GABA)对正常通气和低氧胁迫下甜瓜幼苗活性氧代谢的影响。结果表明:与正常通气处理相比,低氧胁迫处理导致甜瓜幼苗体内O2.-产生速率和H2O2、MDA含量显著增加,同时SOD、POD、CAT、APX、GR等抗氧化酶活性和抗氧化物质AsA、GSH含量显著提高。低氧胁迫下外源GABA能显著提高甜瓜幼苗叶片SOD、CAT、APX、GR等酶活性和AsA、GSH含量,降低了植株体内O2.-产生速率和H2O2、MDA含量;而正常通气条件下添加外源GABA处理对甜瓜幼苗活性氧代谢的影响较小,仅CAT、GR活性和AsA、GSH含量显著提高,而H2O2、MDA含量显著降低。结果证明,添加外源GABA可以通过显著提高低氧胁迫下抗氧化酶活性和抗氧化物质含量来降低甜瓜幼苗活性氧积累,维持其细胞膜结构稳定性,从而有效减轻低氧胁迫对甜瓜幼苗的伤害。     

一氧化氮参与外源谷胱甘肽对盐胁迫下番茄幼苗抗氧化损伤的调控

采用营养液水培,研究外源谷胱甘肽(GSH)对NaCl和NaCl+Hb(牛血红蛋白,一种一氧化氮清除剂)处理下番茄(Lycopersicon esculentum)幼苗叶片中一氧化氮合酶(NOS)活性及其一氧化氮(NO)水平、膜脂过氧化程度、活性氧(ROS)积累和ROS清除能力的影响。结果表明:施用Hb使盐胁迫下番茄幼苗叶片的氧化损伤程度加剧,NO含量下调,但对GSH含量无显著影响。外源GSH的施用显著提高了盐胁迫下番茄幼苗叶中内源GSH与NO水平,单脱氢抗坏血酸还原酶(MDHAR)和硝酸还原酶(NR)活性,超氧化物歧化酶(SOD)活性(除72 h),过氧化氢酶(CAT)活性(除48 h),处理48和72 h的谷胱甘肽还原酶(GR)、抗坏血酸过氧化物酶(APX)和脱氢抗坏血酸还原酶(DHAR)活性;降低电解质渗透率、硫代巴比妥酸(TBARS)含量以及处理48和72 h的过氧化氢(H_2O_2)和超氧阴离子(O_2ˉ·)含量。外源喷施GSH亦显著提高NaCl+Hb处理下番茄叶中抗坏血酸(AsA)、GSH和NO水平,CAT和APX活性,处理24和48 h的NR活性以及处理48和72 h的NOS活性;降低电解质渗透率及H_2O_2和TBARS含量。表明外源GSH通过介导内源GSH和NO水平上调,提高抗氧化酶活性和ROS的清除能力来缓解NaCl和NaCl+Hb处理导致的氧化损伤。因此,NO参与了外源GSH对盐胁迫下番茄幼苗抗氧化损伤的调控。     

不同耐铝型杉木幼苗叶片抗氧化系统对铝胁迫的响应特征

为探讨铝胁迫对杉木抗坏血酸-谷胱甘肽(AsA-GSH)循环的影响,明确AsA-GSH循环在杉木耐铝性中的作用,以耐铝(YX26)和铝敏感型(YX5)杉木家系为材料,分析了铝胁迫对不同耐铝型杉木叶片氧化损伤、抗氧化酶活性和AsA-GSH循环系统的影响。结果表明:(1)铝胁迫显著增加杉木叶片丙二醛(MDA)含量,而且YX5叶片中MDA含量增幅显著大于YX26。(2)铝胁迫不同程度增加了2个杉木家系叶片过氧化物酶(POD)、抗坏血酸过氧化物酶(APX)和单脱氢抗坏血酸还原酶(MDAR)活性以及抗坏血酸(AsA)、脱氢抗坏血酸(DHA)、还原型谷胱甘肽(GSH)和氧化型谷胱甘肽(GSSG)含量,而且除AsA含量外,铝胁迫下YX26中上述酶活性和非酶性抗氧化剂含量的增幅均大于YX5。(3)铝胁迫下YX5叶片中过氧化氢酶(CAT)和脱氢抗坏血酸还原酶(DHAR)的活性受到显著抑制,而YX26中这两个酶的活性却有所增加,且YX26中的DHAR活性显著高于对照。(4)铝胁迫抑制了2个杉木家系超氧化物歧化酶(SOD)活性,但YX26中SOD活性的降幅小于YX5。研究认为,铝胁迫下通过维持AsA-GSH循环酶活性和非酶性抗氧化系统的高效运转,增强自身活性氧清除能力是耐铝型杉木家系具有较强铝耐能力的生理基础。     

H_2S对干旱胁迫下白三叶幼苗抗氧化防御能力的影响

以‘拉丁诺’白三叶(Trifolium repens cv.‘Ladino’)为试验材料,研究外源H2S处理对PEG6 000(聚乙二醇)模拟干旱胁迫下白三叶叶片相对含水量(RWC)、膜脂过氧化、活性氧成分、抗氧化酶、抗坏血酸-谷胱甘肽循环代谢和非酶抗氧化物质的影响,以揭示H_2S调控白三叶抗旱性的生理机制。结果显示:(1)0.2 mmol/L的外源NaHS(H_2S供体)能显著提高干旱胁迫下白三叶的叶片相对含水量,维持显著较低的电解质渗透率(EL)和丙二醛(MDA)含量。(2)与直接干旱胁迫相比,干旱胁迫下外源添加NaHS处理的白三叶叶片内超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)活性显著增强,抗坏血酸-谷胱甘肽循环代谢中关键酶抗坏血酸过氧化物酶(APX)、脱氢抗坏血酸还原酶(DHAR)、单脱水抗坏血酸还原酶(MDHAR)和谷胱甘肽还原酶(GR)活性及其抗氧化中间产物抗坏血酸(AsA)、谷胱甘肽(GSH)含量也显著提高。(3)叶片类黄酮、总酚和原花青素的含量在一定的胁迫时间范围内亦显著增加,并伴随着活性氧成分O_2~(-·)产生速率和H_2O_2水平降低。研究认为,外源H2S能通过促进干旱胁迫下白三叶体内的多重抗氧化防御能力来提高其幼苗的抗旱性。     

外源ABA对大蒜试管苗玻璃化发生和抗氧化系统的影响

以大蒜品种‘二水早’试管苗为材料,从活性氧代谢的角度研究了外源ABA、H_2O_2和H_2O_2+ABA处理下的试管苗玻璃化率、活性氧积累与组织定位和抗氧化系统的响应特征,探讨ABA缓解试管苗玻璃化过程的机理。结果表明:(1)外源H_2O_2处理可诱导大蒜试管苗玻璃化发生,外源ABA处理下玻璃化率最低,可以缓解H_2O_2诱导的玻璃化的发生。(2)试管苗O_2~产生速率和H_2O_2含量在H_2O_2处理下最高,在ABA处理下最低;在添加H_2O_2的培养基中同时添加ABA能显著减少因外源H_2O_2处理引起的O_2~产生和H_2O_2积累。(3)试管苗CAT、POD和APX活性在外源H_2O_2处理前期(0~8d)均上升并显著高于对照,但其CAT、APX活性在处理后期(8~16d)下降,其同期POD活性也增加缓慢;各抗氧化酶的活性在外源ABA与H_2O_2+ABA处理前期(0~8d)均呈直线上升趋势,而它们在H_2O_2+ABA处理后期(8~16d)均显著高于H_2O_2处理。(4)各处理试管苗抗坏血酸和谷胱甘肽含量随处理时间先升高后降低,并以外源ABA处理下最高,外源H_2O_2处理下最低。(5)试管苗O_2~和H_2O_2产生部位主要在基部和叶尖,且外源ABA处理下组织中ROS的积累最少。(6)在ABA+H_2O_2处理下,大蒜试管苗内丙二醛含量和膜相对透性显著低于对照和H_2O_2处理。研究发现,外源ABA处理可有效降低大蒜试管苗的内源O_2~产生速率和H_2O_2含量,提高抗氧化酶活性和抗氧化物质含量,抑制活性氧在试管苗内的产生和运输,显著降低试管苗玻璃化率;外源ABA可通过增强大蒜试管苗抗氧化能力来抑制玻璃化发生。     

24-表油菜素内酯预处理对干旱胁迫下葡萄幼苗抗氧化系统和渗透调节物质的影响

以酿酒葡萄‘雷司令’(Riesling)一年生营养袋扦插苗为材料,采用人工气候室水培试验,考察在聚乙二醇6000(PEG)模拟干旱条件下,不同浓度(0.05、0.10和0.20mg/L)24-表油菜素内酯(EBR)预处理对‘雷司令’幼苗活性氧、抗氧化物质、渗透调节物质含量和抗氧化酶活性的影响,以揭示EBR预处理对干旱胁迫下葡萄幼苗的抗旱机理。结果显示:(1)与正常生长(对照)相比,干旱胁迫显著提高葡萄幼苗叶片中超氧阴离子自由基(■)、过氧化氢(H_2O_2)和丙二醛(MDA)含量;与干旱胁迫处理(PEG)相比,不同浓度EBR预处理均可降低叶片中■、H_2O_2和MDA的含量。(2)与对照相比,PEG处理显著降低葡萄幼苗叶片的抗坏血酸(AsA)和还原型谷胱甘肽(GSH)含量;与PEG处理相比,各浓度EBR预处理均可显著提高葡萄叶片AsA与GSH的含量,且以0.10mg/LEBR处理效果最好。(3)随着干旱胁迫时间的延长,葡萄幼苗叶片中的超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、过氧化物酶(POD)与抗坏血酸过氧化物酶(APX)活性均呈先上升后下降的变化趋势,而在正常生长条件下酶活性基本保持不变;EBR预处理的葡萄叶片SOD、CAT、POD和APX活性均始终高于同期PEG处理。(4)PEG处理条件下,渗透调节物质脯氨酸和可溶性蛋白的含量整体高于对照;与PEG处理相比,不同浓度EBR预处理在干旱胁迫中后期均能显著提高葡萄叶片中脯氨酸和可溶性蛋白含量。研究表明,在干旱胁迫下,外源EBR预处理能够提高葡萄叶片抗氧化系统酶活性和渗透调节物质含量,有效降低干旱胁迫诱导的活性氧过度积累及膜脂过氧化程度,提高葡萄幼苗的抗旱能力,且以0.10mg/L EBR处理效果最佳。     

外源H_2S对NO_3~-胁迫下番茄幼苗生理生化特性的影响

采用营养液水培方法,通过外源施加H2S供体NaHS(100μmol/L),研究了信号分子H2S对100mmol/L NO3-胁迫下番茄幼苗生理生化特性的影响。结果表明:(1)NO3-胁迫下,随着处理时间的延长,番茄幼苗的株高、根长、鲜重和干重显著降低,叶绿素(a、b)含量、净光合速率、气孔导度、蒸腾速率均显著降低,而胞间CO2浓度以及丙二醛(MDA)、H2O2含量增加,超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)和抗坏血酸过氧化物酶(APX)活性显著降低,抗坏血酸(AsA)和还原性谷胱甘肽(GSH)含量显著降低。(2)与NO3-胁迫处理相比,外源NaHS处理1、3、5d后,番茄幼苗的株高、根长、鲜重和干重显著增加,叶绿素(a、b)含量、净光合速率、气孔导度、蒸腾速率均显著升高,而胞间CO2浓度显著降低;MDA和H2O2含量降低,SOD、POD、CAT和APX活性显著增强,AsA和GSH含量显著增加,而且幼苗的硝酸还原酶、谷氨酰胺合成酶、谷氨酸合酶的活性显著增强;L-半胱氨酸脱巯基酶活性和内源H2S含量增加。研究认为,外源H2S可能通过提高抗氧化物酶的活性和增加抗氧化物质含量来缓解NO3-对番茄幼苗造成的伤害,从而增强其对NO3-胁迫耐性。     

根区亚低温胁迫下外源GABA对山定子根系抗氧化功能的影响

以当年生山定子实生苗为试材,采用温室盆栽土培试验,模拟根区亚低温(5℃)条件,设置亚低温(L)、亚低温+外源γ氨基丁酸(LG)和亚低温+氨己烯酸(LV)处理,分别于处理后0、12、24、48、96和144h后剪取白色幼嫩根系,测定山定子根系内源γ-氨基丁酸(GABA)含量、渗透调节物质含量、活性氧含量和抗氧化酶活性等指标,研究根区亚低温下GABA对山定子根系抗氧化系统的调节效应。结果显示:(1)根区亚低温下,山定子根系内源GABA含量略高于对照,渗透调节物质也不同程度积累,超氧阴离子(O_2~-·)含量、过氧化氢(H_2O_2)含量显著升高,膜脂过氧化程度加深。(2)外源施加GABA进一步促进了内源GABA的积累,明显增加根系中可溶性糖、可溶性蛋白和脯氨酸(Pro)等渗透调节物质含量,显著提高根系的超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、抗坏血酸过氧化物酶(APX)等酶活性,最终使得山定子根系中H_2O_2等的含量降低,膜脂过氧化程度缓解。(3)GABA专一性抑制剂VGB处理后的效果与外源GABA处理结果相反。研究表明,GABA代谢对山定子根系抵御亚低温胁迫引发的氧化胁迫有积极作用,外源GABA具有通过诱导内源GABA代谢提高根系抗氧化能力的生理效应。     

外源亚精胺对高温下黄瓜幼苗叶片抗氧化系统的影响

以黄瓜热敏感品种‘长春密刺'和耐热品种‘津春4号'为试材,在人工气候箱内采用营养液栽培法研究了外源亚精胺(Spd)预处理对短期高温胁迫(42℃)下黄瓜幼苗叶片抗氧化系统的影响.结果显示:(1)随高温胁迫时间的延长,2个黄瓜品种幼苗叶片相对电导率升高、MDA含量增加,热敏感品种的膜脂过氧化程度大于耐热品种;高温胁迫4h,SOD活性降低,POD、CAT、APX活性升高,抗坏血酸(AsA)含量升高,类胡萝卜素(Car)含量降低,且随胁迫时间反映出耐热品种抗氧化系统的自我调节能力大于热敏感品种.(2)外源Spd预处理能有效抑制高温胁迫引起的膜脂过氧化伤害,增强SOD、POD、APX活性以及AsA和Car含量,增强植株抗氧化能力,且在热敏感品种上的应用效果优于耐热品种.研究表明,外源Spd预处理能有效提高黄瓜幼苗叶片处于高温胁迫时的抗氧化能力,对缓解高温胁迫有重要作用.     

外源H_2S通过减轻低温光抑制增强黄瓜幼苗耐冷性

以‘津优3号’黄瓜(Cucumis sativus)为试材,叶面喷施硫化氢(H_2S)供体硫氢化钠(NaHS)、H_2S合成抑制剂氨氧基乙酸(AOA)、清除剂次牛磺酸(HT)或去离子水(对照),研究H_2S对低温下黄瓜幼苗光合作用和抗氧化系统的影响。结果表明:低温胁迫初期,黄瓜幼苗叶片的内源H_2S与L-/D-半胱氨酸脱巯基酶(L/DCD)活性快速升高,4或6 h后降低。随着低温胁迫时间的延长,黄瓜幼苗的丙二醛(MDA)含量、电解质渗漏率(EL)和冷害指数逐渐增加,NaHS处理的增加幅度明显小于对照,而AOA和HT处理的与对照差异不显著。低温下黄瓜幼苗的净光合速率(P_n)、气孔导度(G_s)、蒸腾速率(T_r)、光下光系统Ⅱ(PSⅡ)实际光化学效率(Φ_(PSⅡ))和暗下PSⅡ最大光化学效率(F_v/F_m),以及核酮糖-1,5-二磷酸羧化酶(RuBPCase)和果糖-1,6-二磷酸酯酶(FBPase)活性逐渐降低,胞间CO_2浓度(C_i)和初始荧光(F_o)趋于升高。与对照相比,NaHS处理的P_n、G_s、T_r、RuBPCase和FBPase活性,以及Φ_(PSⅡ)和F_v/F_m均较高,C_i和F_o较低,而AOA和HT处理的气体交换参数、光合酶活性及荧光参数多与对照差异不显著。随着低温胁迫时间的延长,黄瓜幼苗的过氧化物酶(POD)活性逐渐增加,超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、抗坏血酸过氧化物酶(APX)和谷胱甘肽还原酶(GR)活性,以及还原型谷胱甘肽(GSH)和抗坏血酸(AsA)含量先升高,后降低。NaHS处理的SOD、POD、CAT、APX和GR活性及GSH和AsA含量明显高于对照,AOA和HT处理的低于对照或与对照差异不显著。由此可见,H_2S受低温胁迫诱导,外源H_2S可通过减轻低温光抑制增强黄瓜幼苗耐冷性。     

Role and Cost Effectiveness of PET/CT in Management of Patients with Cancer

PET/CT is a relatively new imaging technology, whose undoubted advantages are valuable in clinical oncology as well as in all fields of diagnosis, staging, and treatment. The hardware combination of anatomy and function has been the true evolution in imaging. PET using 18F-fluorodeoxyglucose (FDG) is increasingly used for the staging of solid malignancies, including colon, lung, etc., but anatomic information is limited. Integrated PET/CT enables optimal anatomic delineation of PET findings and identification of FDG-negative lesions on computed tomography (CT) images and might improve preoperative staging. However, controversy still exists in relation to the application of PET/CT in clinical practice, mainly because of its high cost. It is evident that apart from additional costs, potential savings also are associated with PET/CT as a result of avoiding additional imaging examinations or invasive procedures and by helping clinicians make the optimum treatment decisions. The authors review the literature on the role of PET/CT in management of various tumors and discuss the medicoeconomic usefulness.     

Ouabain-induced Internalization and Lysosomal Degradation of the Na+/K+-ATPase

Internalization of the Na⁺/K⁺-ATPase (the Na⁺ pump) has been studied in the human lung carcinoma cell line H1299 that expresses YFP-tagged α1 from its normal genomic localization. Both real-time imaging and surface biotinylation have demonstrated internalization of α1 induced by ≥100 nm ouabain which occurs in a time scale of hours. Unlike previous studies in other systems, the ouabain-induced internalization was insensitive to Src or PI3K inhibitors. Accumulation of α1 in the cells could be augmented by inhibition of lysosomal degradation but not by proteosomal inhibitors. In agreement, the internalized α1 could be colocalized with the lysosomal marker LAMP1 but not with Golgi or nuclear markers. In principle, internalization could be triggered by a conformational change of the ouabain-bound Na⁺/K⁺-ATPase molecule or more generally by the disruption of cation homeostasis (Na⁺, K⁺, Ca²⁺) due to the partial inhibition of active Na⁺ and K⁺ transport. Overexpression of ouabain-insensitive rat α1 failed to inhibit internalization of human α1 expressed in the same cells. In addition, incubating cells in a K⁺-free medium did not induce internalization of the pump or affect the response to ouabain. Thus, internalization is not the result of changes in the cellular cation balance but is likely to be triggered by a conformational change of the protein itself. In physiological conditions, internalization may serve to eliminate pumps that have been blocked by endogenous ouabain or other cardiac glycosides. This mechanism may be required due to the very slow dissociation of the ouabain·Na⁺/K⁺-ATPase complex.     

NHERF2/NHERF3 Protein Heterodimerization and Macrocomplex Formation Are Required for the Inhibition of NHE3 Activity by Carbachol

NHERF1, NHERF2, and NHERF3 belong to the NHERF (Na⁺/H⁺ exchanger regulatory factor) family of PSD-95/Discs-large/ZO-1 (PDZ) scaffolding proteins. Individually, each NHERF protein has been shown to be involved in the regulation of multiple receptors or transporters including Na⁺/H⁺ exchanger 3 (NHE3). Although NHERF dimerizations have been reported, results have been inconsistent, and the physiological function of NHERF dimerizations is still unknown. The current study semiquantitatively compared the interaction strength among all possible homodimerizations and heterodimerizations of these three NHERF proteins by pulldown and co-immunoprecipitation assays. Both methods showed that NHERF2 and NHERF3 heterodimerize as the strongest interaction among all NHERF dimerizations. In vivo NHERF2/NHERF3 heterodimerization was confirmed by FRET and FRAP (fluorescence recovery after photobleach). NHERF2/NHERF3 heterodimerization is mediated by PDZ domains of NHERF2 and the C-terminal PDZ domain recognition motif of NHERF3. The NHERF3-4A mutant is defective in heterodimerization with NHERF2 and does not support the inhibition of NHE3 by carbachol. This suggests a role for NHERF2/NHERF3 heterodimerization in the regulation of NHE3 activity. In addition, both PDZ domains of NHERF2 could be simultaneously occupied by NHERF3 and another ligand such as NHE3, α-actinin-4, and PKCα, promoting formation of NHE3 macrocomplexes. This study suggests that NHERF2/NHERF3 heterodimerization mediates the formation of NHE3 macrocomplexes, which are required for the inhibition of NHE3 activity by carbachol.     

A Dual Tracer PET-MRI Protocol for the Quantitative Measure of Regional Brain Energy Substrates Uptake in the Rat

We present a method for comparing the uptake of the brain''s two key energy substrates: glucose and ketones (acetoacetate [AcAc] in this case) in the rat. The developed method is a small-animal positron emission tomography (PET) protocol, in which ¹¹C-AcAc and ¹⁸F-fluorodeoxyglucose (¹⁸F-FDG) are injected sequentially in each animal. This dual tracer PET acquisition is possible because of the short half-life of ¹¹C (20.4 min). The rats also undergo a magnetic resonance imaging (MRI) acquisition seven days before the PET protocol. Prior to image analysis, PET and MRI images are coregistered to allow the measurement of regional cerebral uptake (cortex, hippocampus, striatum, and cerebellum). A quantitative measure of ¹¹C-AcAc and ¹⁸F-FDG brain uptake (cerebral metabolic rate; μmol/100 g/min) is determined by kinetic modeling using the image-derived input function (IDIF) method. Our new dual tracer PET protocol is robust and flexible; the two tracers used can be replaced by different radiotracers to evaluate other processes in the brain. Moreover, our protocol is applicable to the study of brain fuel supply in multiple conditions such as normal aging and neurodegenerative pathologies such as Alzheimer''s and Parkinson''s diseases.     

Plasticity and diversity of tRNA anticodon determinants of substrate recognition by eukaryotic A37 isopentenyltransferases

The N(6)-(isopentenyl)adenosine (i(6)A) modification of some tRNAs at position A37 is found in all kingdoms and facilitates codon-specific mRNA decoding, but occurs in different subsets of tRNAs in different species. Here we examine yeasts' tRNA isopentenyltransferases (i.e., dimethylallyltransferase, DMATase, members of the Δ(2)-isopentenylpyrophosphate transferase, IPPT superfamily) encoded by tit1(+) in Schizosaccharomyces pombe and MOD5 in Saccharomyces cerevisiae, whose homologs are Escherichia coli miaA, the human tumor suppressor TRIT1, and the Caenorhabditis elegans life-span gene product GRO-1. A major determinant of miaA activity is known to be the single-stranded tRNA sequence, A36A37A38, in a stem-loop. tRNA(Trp)(CCA) from either yeast is a Tit1p substrate, but neither is a Mod5p substrate despite the presence of A36A37A38. We show that Tit1p accommodates a broader range of substrates than Mod5p. tRNA(Trp)(CCA) is distinct from Mod5p substrates, which we sort into two classes based on the presence of G at position 34 and other elements. A single substitution of C34 to G converts tRNA(Trp)(CCA) to a Mod5p substrate in vitro and in vivo, consistent with amino acid contacts to G34 in existing Mod5p-tRNA(Cys)(GCA) crystal structures. Mutation of Mod5p in its G34 recognition loop region debilitates it differentially for its G34 (class I) substrates. Multiple alignments reveal that the G34 recognition loop sequence of Mod5p differs significantly from Tit1p, which more resembles human TRIT1 and other DMATases. We show that TRIT1 can also modify tRNA(Trp)(CCA) consistent with broad recognition similar to Tit1p. This study illustrates previously unappreciated molecular plasticity and biological diversity of the tRNA-isopentenyltransferase system of eukaryotes.     

Is polyphenol induction simply a result of altered carbon and nitrogen accumulation?

Carbon translocation in plants is shaped by phyllotaxis and regulated by source/sink interactions that respond to the demands of growth and defense. We have studied this extensively in poplar saplings, and recently showed that unlike carbon import, nitrogen is not translocated to sink leaves in response to application of jasmonic acid. Here we report that this is also true for young trees in the field. We discuss the importance of transport processes in establishing local C:N ratios, and suggest that the JA-induced flow of C but not N to sink tissues, and their corresponding increases in C-based defenses, may simply reflect a plant adaptation to handle excess reduced carbon and energy.     

Inhibition of vacuolar-type (H+)-ATPase by the cytostatic macrolide apicularen A and its role in apicularen A-induced apoptosis in RAW 264.7 cells

Apicularen A and the known vacuolar-type (H(+))-ATPase (V-ATPase) inhibitor bafilomycin A(1) induced apoptosis of RAW 264.7 cells, while apicularen B, an N-acetyl-glucosamine glycoside of apicularen A, was far less effective. Apicularen A inhibited vital staining with acridine orange of the intracellular organelles of RAW 264.7 cells, inhibited the ATP-dependent proton transport into inside-out microsome vesicles, and inhibited the bafilomycin A(1)-sensitive ATP hydrolysis. The IC(50) values of the proton transport were 0.58 nM for apicularen A, 13 nM for apicularen B, and 0.95 nM for bafilomycin A(1). Furthermore, apicularen A inhibited the bafilomycin A(1)-sensitive ATP hydrolysis more potently than apicularen B. F-ATPase and P-ATPase were not inhibited by apicularen A. We concluded that apicularen A inhibits V-ATPase, and thus induces apoptosis in RAW 264.7 cells.     

Endosomal Na+ (K+)/H+ exchanger Nhx1/Vps44 functions independently and downstream of multivesicular body formation

The multivesicular body (MVB) is an endosomal intermediate containing intralumenal vesicles destined for membrane protein degradation in the lysosome. In Saccharomyces cerevisiae, the MVB pathway is composed of 17 evolutionarily conserved ESCRT (endosomal sorting complex required for transport) genes grouped by their vacuole protein sorting Class E mutant phenotypes. Only one integral membrane protein, the endosomal Na+ (K+)/H+ exchanger Nhx1/Vps44, has been assigned to this class, but its role in the MVB pathway has not been directly tested. Herein, we first evaluated the link between Nhx1 and the ESCRT proteins and then used an unbiased phenomics approach to probe the cellular role of Nhx1. Select ESCRT mutants (vps36Δ, vps20Δ, snf7Δ, and bro1Δ) with defects in cargo packaging and intralumenal vesicle formation shared multiple growth phenotypes with nhx1Δ. However, analysis of cellular trafficking and ultrastructural examination by electron microscopy revealed that nhx1Δ cells retain the ability to sort cargo into intralumenal vesicles. In addition, we excluded a role for Nhx1 in Snf7/Bro1-mediated cargo deubiquitylation and Rim101 response to pH stress. Genetic epistasis experiments provided evidence that NHX1 and ESCRT genes function in parallel. A genome-wide screen for single gene deletion mutants that phenocopy nhx1Δ yielded a limited gene set enriched for endosome fusion function, including Rab signaling and actin cytoskeleton reorganization. In light of these findings and the absence of the so-called Class E compartment in nhx1Δ, we eliminated a requirement for Nhx1 in MVB formation and suggest an alternative post-ESCRT role in endosomal membrane fusion.     

The twins K and Na in plants

In the earth's crust and in seawater, K⁺ and Na⁺ are by far the most available monovalent inorganic cations. Physico-chemically, K⁺ and Na⁺ are very similar, but K⁺ is widely used by plants whereas Na⁺ can easily reach toxic levels. Indeed, salinity is one of the major and growing threats to agricultural production. In this article, we outline the fundamental bases for the differences between Na⁺ and K⁺. We present the foundation of transporter selectivity and summarize findings on transporters of the HKT type, which are reported to transport Na⁺ and/or Na⁺ and K⁺, and may play a central role in Na⁺ utilization and detoxification in plants. Based on the structural differences in the hydration shells of K⁺ and Na⁺, and by comparison with sodium channels, we present an ad hoc mechanistic model that can account for ion permeation through HKTs.     

Solution structure of recombinant somatomedin B domain from vitronectin produced in Pichia pastoris

The cysteine-rich somatomedin B domain (SMB) of the matrix protein vitronectin is involved in several important biological processes. First, it stabilizes the active conformation of the plasminogen activator inhibitor (PAI-1); second, it provides the recognition motif for cell adhesion via the cognate integrins (alpha(v)beta(3), alpha(v)beta(5), and alpha(IIb)beta(3)); and third, it binds the complex between urokinase-type plasminogen activator (uPA) and its glycolipid-anchored receptor (uPAR). Previous structural studies on SMB have used recombinant protein expressed in Escherichia coli or SMB released from plasma-derived vitronectin by CNBr cleavage. However, different disulfide patterns and three-dimensional structures for SMB were reported. In the present study, we have expressed recombinant human SMB by two different eukaryotic expression systems, Pichia pastoris and Drosophila melanogaster S2-cells, both yielding structurally and functionally homogeneous protein preparations. Importantly, the entire population of our purified, recombinant SMB has a solvent exposure, both as a free domain and in complex with PAI-1, which is indistinguishable from that of plasma-derived SMB as assessed by amide hydrogen ((1)H/(2)H) exchange. This solvent exposure was only reproduced by one of three synthetic SMB products with predefined disulfide connectivities corresponding to those published previously. Furthermore, this connectivity was also the only one to yield a folded and functional domain. The NMR structure was determined for free SMB produced by Pichia and is largely consistent with that solved by X-ray crystallography for SMB in complex with PAI-1.