P2X7 receptors regulate engulfing activity of non-stimulated resting astrocytes

We previously demonstrated that P2X7 receptors (P2X7Rs) expressed by cultured mouse astrocytes were activated without any exogenous stimuli, but its roles in non-stimulated resting astrocytes remained unknown. It has been reported that astrocytes exhibit engulfing activity, and that the basal activity of P2X7Rs regulates the phagocytic activity of macrophages. In this study, therefore, we investigated whether P2X7Rs regulate the engulfing activity of mouse astrocytes. Uptake of non-opsonized beads by resting astrocytes derived from ddY-mouse cortex time-dependently increased, and the uptaken beads were detected in the intracellular space. The bead uptake was inhibited by cytochalasin D (CytD), an F-actin polymerization inhibitor, and agonists and antagonists of P2X7Rs apparently decreased the uptake. Spontaneous YO-PRO-1 uptake by ddY-mouse astrocytes was reduced by the agonists and antagonists of P2X7Rs, but not by CytD. Down-regulation of P2X7Rs using siRNA decreased the bead uptake by ddY-mouse astrocytes. In addition, compared to in the case of ddY-mouse astrocytes, SJL-mouse astrocytes exhibited higher YO-PRO-1 uptake activity, and their bead uptake was significantly greater. These findings suggest that resting astrocytes exhibit engulfing activity and that the activity is regulated, at least in part, by their P2X7Rs.     

Rapid fluorescent visualization of multiple NAD(P)H oxidoreductases in homogenate,permeabilized cells,and tissue slices

Intracellular NAD(P)H oxidoreductases are a class of diverse enzymes that are the key players in a number of vital processes. The method we present and validate here is based on the ability of many NAD(P)H oxidoreductases to reduce the superoxide probe lucigenin, which is structurally similar to flavins, to its highly fluorescent water-insoluble derivative dimethylbiacridene. Two modifications of the method are proposed: (i) an express method for tissue homogenate and permeabilized cells in suspensions and (ii) a standard procedure for cells in culture and acute thin tissue slices. The method allows one to assess, visualize, and localize, using fluorescent markers of cellular compartments, multiple NADH and NADPH oxidoreductase activities. The application of selective inhibitors (e.g., VAS2870, a NOX2 inhibitor; plumbagin, a NOX4 inhibitor) allows one to distinguish and compare specific NAD(P)H oxidoreductase activities in cells and tissues and to attribute them to known enzymes. The method is simple, rapid, and flexible. It can be easily adapted to a variety of tasks. It will be useful for investigations of the role of various NAD(P)H oxidoreductases in a number of physiological and pathophysiological processes.     

Air and surface soil samples – two different pairs of shoes? Comparing the pollen spectrum on different days of the pollen season

The pollen spectra of air and surface soil samples from a rooftop (at 14 m) and from ground level (at 1.6 m) in the suburbs of Vienna (Austria) were compared. Two soil samples and two air samples were taken on four different days to account for possible differences: in winter when no pollination occurred (reference day), in spring during the main flowering of Betula (birch day), in spring/summer during the main flowering of Poaceae (grass day), and in autumn during the main flowering of Ambrosia (ragweed day). Thirty-five different pollen types were used to describe the pollen spectra. Frequencies of certain pollen types reflect a seasonal impact on both the surface soil and air samples and show a similarity between air and soil samples on most of the days. However, the seasonal impact is higher in the air samples and shows a high consistency for ground and rooftop level. Kendall’s tau correlation coefficients further substantiate the similarities of the samples especially for the pollen season days. Exceptions include the winter day when pollination was low and the air samples recorded nearly no pollen at all, and the ragweed day when Ambrosia pollen was abundant in three of four samples but not in the ground surface soil sample. Thus, (1) air and surface pollen samples record similar signals during the pollen season but not during the ragweed and winter season and (2) air and surface pollen samples show the impact of local vegetation also in pollen traps located at different heights.     

Polyethylenimine of various molecular weights as adjuvant for transfection mediated by cationic liposomes

Over the last years significant progress has been made in non-viral gene delivery mediated by cationic liposomes. However, the results obtained are still far from being satisfactory regarding transfection efficiency, particularly when compared to that achieved using viral vectors. We have previously demonstrated that association of transferrin with cationic liposomes significantly improves transfection in a large variety of cells, both in vitro and in vivo. In this work, several strategies have been explored in order to further improve transfection mediated by transferrin-associated lipoplexes. To this regard, the effect on transfection of pre-condensation of DNA with polyethylenimine of low MWs (2.7, 2.0 and 0.8 KDa) at various N/P ratios, lipid composition, cationic lipid/DNA (+/-) charge ratio and the presence of a surfactant in the lipoplexes was investigated. Two different modes for preparing the liposomes were tested and the extent of cell association of their complexes with DNA as well as their capacity to protect the carried DNA were evaluated. Our results show that complexes generated from cationic liposomes prepared by the ethanol injection method in which the carried DNA was pre-condensed with low MW polyethylenimine are highly efficient in mediating transfection. The differential modulating effect observed upon association of transferrin to various liposome formulations on transfection mediated by the polyethylenimine-complexes suggests that these complexes enter into the cells through different pathways (involving clathrin versus caveolin), most likely by taking advantage of their intrinsic biophysical properties to escape from the endosome to the cytosol.     

Sequence and hydropathy profile analysis of two classes of secondary transporters

A structural class in the MemGen classification of membrane proteins is a set of evolutionary related proteins sharing a similar global fold. A structural class contains both closely related pairs of proteins for which homology is clear from sequence comparison and very distantly related pairs, for which it is not possible to establish homology based on sequence similarity alone. In the latter case the evolutionary link is based on hydropathy profile analysis. Here, we use these evolutionary related sets of proteins to analyze the relationship between E-values in BLAST searches, sequence similarities in multiple sequence alignments and structural similarities in hydropathy profile analyses. Two structural classes of secondary transporters termed ST[3], which includes the Ion Transporter (IT) superfamily and ST[4], which includes the DAACS family (TC# 2.A.23) were extracted from the NCBI protein database. ST[3] contains 2051 unique sequences distributed over 32 families and 59 subfamilies. ST[4] is a smaller class containing 399 unique sequences distributed over 2 families and 7 subfamilies. One subfamily in ST[4] contains a new class of binding protein dependent secondary transporters. Comparison of the averaged hydropathy profiles of the subfamilies in ST[3] and ST[4] revealed that the two classes represent different folds. Divergence of the sequences in ST[4] is much smaller than observed in ST[3], suggesting different constraints on the proteins during evolution. Analysis of the correlation between the evolutionary relationship of pairs of proteins in a class and the BLAST E-value revealed that: (i) the BLAST algorithm is unable to pick up the majority of the links between proteins in structural class ST[3], (ii) ‘low complexity filtering’ and ‘composition based statistics’ improve the specificity, but strongly reduce the sensitivity of BLAST searches for distantly related proteins, indicating that these filters are too stringent for the proteins analyzed, and (iii) the E-value cut-off, which may be used to evaluate evolutionary significance of a hit in a BLAST search is very different for the two structural classes of membrane proteins.     

低温下抗氧化酶活性与冬油菜根细胞结冰关系的初步研究

以4个抗寒性不同的油菜品种的根为材料,于低温试验箱内连续降温处理(4℃/24 h,0℃/24 h,-3℃/24 h,-6℃/24 h,-9℃/24 h,-15℃/24 h,-21℃/24 h),检测其抗氧化酶(POD、CAT)活性,并结合POD同工酶电泳和冷冻前后幼苗的形态变化特征,探讨油菜根部抗氧化酶活性变化与细胞冰冻状态间的相关性以及油菜根的抗冻机理.结果显示:(1)弱抗寒性油菜品种(‘临油7号’和Vision)在低温冷冻后,叶面出现大量水渍,之后叶片萎缩直至焦枯,根部出现水渍后软化直至干枯死亡;强抗寒性品种(‘陇油6号’和‘陇油8号’)在冷冻后不产生水渍或水渍极少,其根在冻后损伤也较轻微.(2)在连续冷冻条件下,弱抗寒性油菜根部POD和CAT活性在降温初期有较大幅度的变化,之后就保持恒定,说明根部细胞已完全结冰;而强抗寒性油菜根部POD和CAT活性在降温初期变化较小,后期持续大幅增加,表明根部细胞仍保持溶液状态;POD同工酶分析也反映出类似的变化规律.研究表明,低温下冬油菜根部抗氧化酶活性变化和细胞冰冻状态之间存在相关性;细胞结冰通常会给植物造成致死性伤害,强抗寒油菜品种能安全越冬的关键就是能通过某种机制避免细胞内结冰,其结冰温度越低,抗寒性越强.     

酸雨背景下缙云山典型林分凋落物量和营养元素含量及其释放特征

选取缙云山针阔混交林、常绿阔叶林、楠竹林和灌木林作为研究对象,对4种林分的凋落物储量和营养元素释放量等进行观测,并应用室内模拟酸雨实验对4种林分凋落物进行淋溶模拟。结果表明:缙云山各林分现存凋落物厚度为1.4～4.5 cm,具有明显的分层结构;林分未分解U层、半分解S层和分解D层现存凋落物量分别为1.97～8.59、2.84～10.18和3.58～17.43 t·hm-2,林分年凋落物量为2.17～9.86 t·hm-2·a-1,凋落物总储量为14.21～32.42 t·hm-2,凋落物分解常数为0.15～0.31,林下凋落物分解95%时所需时间针阔混交林和楠竹林均在10年以上,凋落物分解速率比较缓慢;林下凋落物层营养元素含量以Ca、N为主,Fe、K、Mg次之;凋落物总的营养元素释放率表现为常绿阔叶林(0.80)>灌木林(0.72)>针阔混交林(0.50)>楠竹林(-0.17);与叶片相比,凋落物中N、K、Mn 3种营养元素含量明显降低;为探明酸雨影响营养元素循环的作用机理,对模拟酸雨的离子含量与凋落物淋滤液盐基离子含量进行了分析,其相关性大小表现为楠竹林(相关系数0.895)>针阔混交林(0.826)>灌木(0.700)>常绿阔叶林(0.699),楠竹林凋落物营养元素的淋滤受酸雨影响最大,常绿阔叶林受其影响最弱;推测在这一过程中以凋落物的吸附作用为主。     

濒危鱼类稀有白甲鱼沅江种群与西江种群形态度量学性状的差异性

分别以采自沅江水系142例和西江水系92例稀有白甲鱼标本为材料,采用差异系数法、方差分析法、判别分析法和主成分分析法,研究了稀有白甲鱼沅江种群、西江种群包括10个可数性状和31个比例性状等形态度量学性状的差异性.方差分析表明,2个种群之间共有6个可数性状和27个比例性状存在着显著性差异(P＜0.05).判别分析筛选出了9个对区分2个种群贡献较大的比例性状,并建立了2个种群的判别函数式.主成分分析显示,稀有白甲鱼种群鱼体沿背腹轴、头尾轴分别有16和6个比例性状表现出了明显的变化,在尾部有2个比例性状差异明显.可见2个种群之间形态度量学性状已显示了明显的分化.这可能是两个种群长期隔离,在不同水系生态环境中产生了适应性进化的结果.然而,差异系数的计算结果表明,两个种群之间形态度量学性状的差异尚未达到亚种分化的水平.     

小叶锦鸡儿灌丛下土壤水分对降雨的响应

以科尔沁沙地主要固沙灌木小叶锦鸡儿为研究对象,在其生长季次降雨21.5 mm后180 h内,利用TDR和微渗仪测量小叶锦鸡儿灌丛下不同部位的土壤含水量和土壤蒸发,并计算该灌丛下不同部位储水量和水量平衡.结果表明:降雨结束后初期,灌丛枝干的茎流作用使其根部的土壤含水量明显高于其他部位;灌丛根部水分的入渗速率大于灌丛中部和灌丛外缘.因冠幅的庇荫作用,灌丛下蒸发量小于灌丛外裸露沙地.水量平衡表明:小叶锦鸡儿灌丛下降雨后前期蒸散量明显高于灌丛外裸露沙地,与灌丛下根系的分布有直接关系.