人白细胞抗原-G1对NK92效靶识别过程的影响

人白细胞抗原G(human leukocyte antigen-G,HLA-G)属非经典的主要组织相容性复合体(major histocompatibility complex, MHC)Ⅰ类分子,为自然杀伤细胞（natural killer,NK）抑制性受体的识别配体,可以向NK细胞传递抑制性信号,从而抑制NK细胞的细胞毒作用.为了研究HLA-G对NK与靶细胞识别作用的影响机制,采用激光扫描共聚焦显微镜和流式细胞术对全长HLA-G1的表达和功能进行了探讨,并对效靶识别过程中靶细胞［Ca²⁺］_i（胞内自由钙离子浓度）进行了实时检测.结果发现,全长的HLA-G1表达于K562、JAR和CHO细胞的胞浆和胞膜上,它能够部分地抑制NK92的细胞毒作用. NK92与CHO和GFP-CHO细胞作用后,靶细胞［Ca²⁺］_i出现了明显的升高,HLA-G1的表达则抑制了靶细胞［Ca²⁺］_i的升高.结果提示：靶细胞［Ca²⁺］_i的升高是有效的细胞杀伤作用的必要条件,HLA-G的免疫抑制功能可能与靶细胞胞内钙离子浓度升高的抑制作用有密切关系.     

共聚焦显微术测定大鼠和黄鼠心肌细胞游离Ca2+浓度与温度的关系

运用共聚焦激光扫描显微成像术对比研究非冬眠动物大鼠和冬眠动物黄鼠心肌细胞胞内Ca²⁺浓度 ( [Ca²⁺]_i)随温度的变化 .首先标定了不同温度下Ca²⁺探针indo 1的解离常数 ,提出并证明按α定态设定标定溶液pH值的必要性 .细胞荧光分析显示 ,大鼠心肌细胞 [Ca²⁺ ]_i 随温度降低显著上升 ,低温下频繁出现自发钙波 ,胞内发生钙超载 ;相比较冬眠动物黄鼠心肌细胞[Ca²⁺ ]_i  在相同条件下保持稳定 ,避免发生钙超载 .认识其中的钙稳态机制可能对有关医学问题有潜在的指导意义 .     

硒对NO诱导的内皮细胞内游离钙离子浓度变化的影响

用Fura-2显微荧光测钙技术,研究了用外源性一氧化氮（NO）供体S-亚硝基谷胱甘肽（GSNO）诱导的,人脐静脉内皮细胞系ECV-304细胞胞内游离钙离子浓度（［Ca²⁺］_i ）升高以及硒的抑制效应.结果表明,GSNO作用于ECV-304细胞,短时间内即可导致其胞内游离钙离子浓度升高.胞外液换为无钙液或向胞外液中加入CdCl₂（1 mmol/L）对GSNO引起的［Ca²⁺］_i升高无影响.提示,GSNO刺激主要引起胞内钙库释放.而且,一氧化氮清除剂血红蛋白（Hb）对这一过程有抑制作用,说明GSNO引起的胞内钙库释放由NO介导.经亚硒酸钠（1 μmol/L）处理的细胞,其NO引起的［Ca²⁺］_i升高幅度明显被抑制,说明NO的这种作用可能与细胞的氧化还原状态有关.     

用Fura－2双波长荧光法测定神经细胞内游离钙

采用新型Ca²⁺荧光指示剂Fura-2建立双波长荧光法测定分离的大鼠神经细胞内游离钙浓度([Ca²⁺]_i).结果显示,在静息状态下,其[Ca²⁺]_i为109±12nmol/L.30mmol/L KCl可显著增加[Ca²⁺]_i,并且KCl的这种效应呈一定的剂量依赖关系,提示该法灵敏、可靠.     

脉冲电场对粒细胞内Ca2+浓度影响的动态变化

利用激光共聚焦扫描显微镜和装有CCD系统的荧光显微镜 ,研究在单脉冲电场作用下经fluo 3/AM标记的鸡胚小脑粒细胞内自由Ca²⁺浓度 ( [Ca ²⁺]_i)的动态变化过程 .结果表明 :在单个电脉冲作用下 ,细胞内Ca²⁺浓度立刻升高并达到其最大值 .Ca²⁺浓度升高的幅度以及升高的速率具有电场强度的依赖性 .当细胞外Ca²⁺被过量的EGTA络合或细胞膜上的Ca²⁺通道被La ³⁺堵塞后 ,细胞内的Ca²⁺浓度仍然升高 .细胞内不同区域的Ca²⁺浓度同时升高 ;两极内的Ca²⁺浓度早于胞体的Ca²⁺浓度达到最大值并迅速恢复 .     

单细胞内Ca2+时空变化的激光共聚焦显微测定

应用激光扫描共聚焦显微系统(LSCM)和Fluo-3/AM荧光探剂标记技术, 测定了单个活细胞胞内游离Ca²⁺的动态变化与立体分布影像. 结果显示, 在37℃, Fluo-3/AM终浓度为6μmol/L的条件下, C57BL/6J小鼠巨噬细胞负载1h左右即可获得良好的标记效果. 相反, 若探剂浓度太高或负载时间太长, 胞内荧光强度太强, 影响在共聚焦显微镜镜下分辨细胞内结构. 因此用LSCM研究细胞内游离Ca²⁺变化时, 荧光探剂的负载应以获得最适荧光信号而不是以最大荧光强度为标准. 上述方法在其他如平滑肌细胞、卵母细胞中的测定亦获得满意的结果, 这对进一步研究各种生理和病理条件下细胞内Ca²⁺信号的动态变化、与跨膜Ca²⁺梯差的关系及对活细胞功能活动的调节提供了一种可行的、直观的研究手段.     

Peculiarities of phospholipase C-dependent release of CA2+ from intracellular stores upon activation of choline and purine receptors in myocytes of the guinea-pig small intestine

Using a two-wave fluorescence probe, Fura-2, we studied changes in the intracellular concentration of calcium ions ([Ca²⁺]_i) resulting from activation of muscarinic and purine receptors in single myocytes of the guinea-pig small intestine. Applications of the respective agonists added to the normal Krebs solution (1.0, 10.0, and 100.0 μM carbachol, CCh, as well as 10.0 and 100.0 μM ATP) induced a rise in the [Ca²⁺]_i. Carbachol evoked an increase in the [Ca²⁺]_i, including two components (a rapid and a plateaulike), while ATP under analogous conditions led only to a short-lasting rise in the [Ca²⁺]_i. Transients induced by CCh or ATP applied in different concentrations, which exceeded a certain level, did not significantly differ from each other in their amplitudes, i.e., they were generated according to an all-or-none principle. In the nominally Ca-and Mg-free solution, CCh and ATP induced only rapid increases in the [Ca²⁺]_i in myocytes. The absence of the slow component in the [Ca²⁺]_i elevation upon the action of CCh under such conditions indicates that the effect of ATP, as compared with that of CCh, is not related to activation of the entry of Ca²⁺ ions into cells through voltage-operated calcium channels. After the addition of CCh, repeated application of CCh or ATP induced no effect, while application of CCh after the addition of ATP initiated a rise in the [Ca²⁺]_i. These data show that intracellular calcium stores are depleted completely upon the action of CCh, while they are depleted only partially after the action of ATP. An inhibitor of phospholipase C (PLC), U-73122 (5.0 μM), completely blocked rises in the [Ca²⁺]_i induced by both CCh and ATP; therefore, the release of Ca²⁺ ions from the intracellular calcium stores after application of these agonists is mediated by PLC. We hypothesize that the difference in the release of Ca²⁺ ions from the intracellular stores observed in our experiments upon activation of choline and purine receptors (partial and complete depletion of the stores upon the action of ATP and CCh, respectively) is responsible for the opposite functional effects of the above-mentioned neurotransmitters on smooth muscles. Neirofiziologiya/Neurophysiology, Vol. 38, No. 1, pp. 3–10, January–February, 2006.     

多形核白细胞上嘌呤受体的初步研究

ATP和ADP能激活多型核白细胞引起细胞内［Ca²⁺］_i的明显升高,AMP则无此作用.多型核白细胞对ATP和ADP具有不同的浓度依赖性.当细胞外的钙离子被螯合后,ATP和ADP仍能引起细胞内游离钙浓度的升高.结果表明多形核白细胞存在着对ATP和ADP敏感的P2型嘌呤受体,并且属于P2型受体中的P2Y亚类.     

酿酒酵母细胞增殖对Ca2+需求的新证据*

本文研究了酿酒酵母细胞增殖对Ca²⁺需求的证据。结果表明:SD-Ca培养基中外加1mmol/L的Ca²⁺明显促进酿酒酵母细胞增殖,外源Ca²⁺浓度在0~20mmol/L范围内变动时,随Ca²⁺浓度增加,细胞生长到达稳定期的终浓度也越大;5、10mmol/L的EGTA可明显延缓细胞生长的延滞期,但是最终不能抑制细胞增殖;酿酒酵母在SD-Ca培养基中继代培养4次,随增殖代数增加,细胞总钙含量没有明显变化,说明酵母能够在低钙介质中生长可能是因为具有捕捉和富集钙的功能;以Fluo-3作为胞质Ca²⁺指示剂,通过激光扫描共聚焦显微镜观察,发现随胞外Ca²⁺浓度增加,胞质中游离Ca²⁺浓度也相应增加。这些证据都揭示了Ca²⁺在酿酒酵母细胞增殖过程中是必需的。     

亚硒酸钠对巨噬细胞内游离Ca2+和Mg2+的影响

用单细胞阳离子测定系统研究了SeO^2-₃对巨噬细胞内游离Ca²⁺和Mg²⁺的影响.实验结果表明:SeO^2-₃高于10^-4mol/L时,有显著的细胞毒性.SeO^2-₃对细胞的毒性作用使细胞内游离Ca²⁺和Mg²⁺的浓度升高但Ca²⁺浓度的升高速率比Mg²⁺快.还有,高于10^-4mol/L的SeO^2-₃对红细胞膜上的Ca²⁺-ATP酶活性有明显抑制作用.     

Simultaneous multi-parameter observation of Harringtonine-treating HL-60 cells with both two-photon and confocal laser scanning microscopy

Harringtonine (HT), a kind of anticancer drug isolated from Chinese herb-Cephalotaxus hainanensis Li, can induce apoptosis in promyelocytic leukemia HL-60 cells. With both two-photon laser scanning microscopy and confocal laser scanning microscopy in combination with the fluorescent probe Hoechst 33342, tetramethyrhodamine ethyl ester (TMRE) and Fluo 3-AM, we simultaneously observed HT-induced changes in nuclear morphology, mitochondrial membrane potential and intracellular calcium concentration ([Ca2+]i) in HL-60 cells, and developed a real-time, sensitive and invasive method for simultaneous multi-parameter observation of drug- treating living cells at the level of single cell.     

The musculature of Squatinella rostrum (Milne, 1886) (Rotifera: Lepadellidae) as revealed by confocal laser scanning microscopy with additional new data on its trophi and overall morphology

Wilts, E.F., Wulfken, D., Ahlrichs, W.H. and Martínez Arbizu, P. 2012. The musculature of Squatinella rostrum (Milne, 1886) (Rotifera: Lepadellidae) as revealed by confocal laser scanning microscopy with additional new data on its trophi and overall morphology.—Acta Zoologica (Stockholm) 93 : 14–27. The monogonont rotifer Squatinella rostrum was investigated with light, scanning electron and confocal laser scanning microscopy to reveal new morphological data on its inner and outer anatomy. In total, the visualized somatic musculature displays five paired longitudinal muscles (musculi longitudinales I–V) and nine circular muscles (musculi circulares I–IX). Compared to other species, S. rostrum is characterized by the absence of several longitudinal and circular muscles (e.g. musculus longitudinalis capitis, corona sphincter and pars coronalis). A reconstruction of the mastax musculature revealed a total number of seven paired and two unpaired mastax muscles. Possibly homologous somatic and mastax muscles in other, thus far investigated rotifers are discussed. Moreover, we provide a phylogenetic evaluation of the revealed morphological characters and suggest possible autapomorphic characters supporting Squatinella and Lepadellidae. Finally, we refer to some striking similarities in the morphology, ecology and way of movement of Squatinella and Bryceella that may indicate a closer relationship of both taxa.     

Body musculature of Beauchampiella eudactylota (Gosse, 1886) (Rotifera: Euchlanidae) with additional new data on its trophi and overall morphology

This study presents the results of confocal laser scanning microscopy and fluorescence‐labelled phalloidin used to visualize the system of body musculature in Beauchampiella eudactylota. Moreover, the poorly known trophi of B. eudactylota are described based on scanning electron microscopy. In total, four paired longitudinal muscles (musculi longitudinales I–IV) and three circular muscles (musculi circulares I–III) were identified. Among these are the musculus longitudinalis ventralis, the musculus longitudinalis dorsalis and the musculus circumpedalis as documented in previous studies for other rotifer species. Compared to other species, B. eudactylota is characterized by the low number of lateral longitudinal muscles and the absence of some longitudinal muscles (musculi longitudinales capitum) and circular muscles (corona sphincter, musculus pars coronalis). Moreover, scanning electron microscopic data on the trophi of B. eudactylota reveal a number of striking similarities to the trophi in some species of Epiphanidae. This suggests that either (1) these similarities represent plesiomorphic characters present both in Epiphanidae and B. eudactylota or (2) they are synapomorphic features of B. eudactylota and some species of Epiphanidae, which would question the monophyly of Euchlanidae.     

The metareticulate pollen morphology of Alternanthera Forssk. (Gomphrenoideae,Amaranthaceae) and its taxonomic implications

Alternanthera (Amaranthaceae) is a diverse genus largely restricted to the American Tropics that belongs to the alternantheroid clade containing C₄ and C₃–C₄ intermediate species. This research focuses on the study of pollen characters by studying 13 species, representatives of the two major clades and subclades of Alternanthera. General palynological comparisons were conducted with light microscopy (LM), scanning electron microscopy (SEM) and with confocal laser scanning microscopy (CLSM) for exine ultrastructure. Twenty-five characters were measured and described for Alternanthera and among these, 14 pollen characters were used to discriminate pollen groups using cluster analysis and canonical analysis of principal coordinates (CAP). Pollen form and ornamentation, pores number, spines length, number of ektexinous bodies and nanospines on the ektexinous bodies on pore membranes, arrangement of nanopores and spines on structural elements, and metareticula form were taxonomically important and therefore used to construct the first palynological key to the alternantheroid clade species. Our study indicates that the seemingly subtle morphological variation of pollen is useful for recognising three main pollen types within Alternanthera. The much needed palynological terminology for describing the mesoporium in the metareticulate pollen of Amaranthaceae is provided.     

Use of confocal laser scanning microscopy on soil thin-sections for improved characterization of microbial growth in unconsolidated soils and aquifer materials

Confocal laser scanning microscopy (CLSM) was utilized to examine samples from an aquifer microcosm that was used to investigate microbially mediated losses in hydraulic conductivity. Samples were fixed, dehydrated and dried to prepare the biological material in a fashion similar to that used previously for viewing under the scanning electron microscope. Then, samples were prepared as thin-sections by employing soil micromorphological techniques. Serial images generated by the CLSM technique were visualized using computer three-dimensional rendering software. Results from the CLSM technique were compared with simple fluorescence microscopy of thin-sections and scanning electron microscopy (SEM) of samples from the microcosm. Computer visualization of serial sections with the CLSM technique provided images on a submicron scale in three dimensions. SEM has a much higher resolution, on a nanometer scale, but the results are not three dimensional. Artifacts associated with thin-section preparation are minimal for natural porous media composed mostly of sand, such as aquifer materials. Also, CLSM images are affected minimally by changes to biological material due to sample preparation, whereas artifacts associated with SEM images are very prominent, due to the higher magnification and resolution. CLSM of thin-sections and SEM are very powerful methods for viewing microbial growth in natural porous media, but CLSM is preferable because it allows three-dimensional visualization and measurements of cells and aggregates with few artifacts.     

非培养细胞的贴壁方法及细胞内Ca^2＋观测

本文介绍非培养细胞贴壁的简易方法和细胞内Ｃａ＾２＋观察。以ｆｌｕｏ－３／ＡＭ进行染色,在３７℃孵育箱内孵育６０分钟,用激光共聚焦显微镜监测细胞内Ｃａ＾２＋的荧光信号。结果表明本方法可成功地应用于共聚焦显微镜测定细胞的研究。     

Bacillus cereus B-02对Botrytis cinerea拮抗机理的研究

采用电子显微镜(扫描、透射)和激光扫描共聚焦显微镜,从细胞形态学和生理学水平上研究蜡样芽孢杆菌Bacillus cereus B-02过滤液对灰葡萄孢菌Botrytis cinerea的拮抗机理。结果表明,处理菌丝表面形态受到严重破坏,发生强烈变形;菌丝细胞核、线粒体和细胞壁等亚细胞结构发生了明显改变,细胞内出现大量无膜透明内含物,并产生较大液泡。此外,处理菌丝DNA、线粒体膜电位和活性氧荧光强度均低于对照组,且差异极显著;说明B-02菌株对病原真菌菌丝细胞DNA的合成、线粒体膜电位和活性氧水平有重要影响。     

Bacillus cereus B-02对Botrytis cinerea 拮抗机理的研究

采用电子显微镜(扫描、透射)和激光扫描共聚焦显微镜,从细胞形态学和生理学水平上研究蜡样芽孢杆菌Bacillus cereus B-02过滤液对灰葡萄孢菌Botrytis cinerea的拈抗机理.结果表明,处理菌丝表面形态受到严重破坏,发牛强烈变形;荫丝细胞核、线粒体和细胞壁等哑细胞结构发生了明显改变,细胞内出现大量无膜透明内含物,并产生较人液泡.此外,处理菌丝DNA、线粒体膜电位和活性氧荧光强度均低于对照组,且差异极显著;说明B-02菌株对病原真菌菌丝细胞DNA的合成、线粒体膜电位和活性氧水平有重要影响.     

Cell adaptations of Rhodococcus rhodochrous IEGM 66 to betulin biotransformation

The findings of a comprehensive study on R. rhodochrous IEGM 66 and triterpenoid betulin interactions during its biotransformation were reported. In the presence of betulin, rhodococci were shown to form heterogeneous cell aggregates. The enhanced size of the aggregates from 12–15 μm to 25– 35 μm was consistent with the increase in betulin concentration from 0.5 to 3.0 g/L. The confocal laser scanning microscopy indicated a high (80.0%) level of rhodococcal viability during betulin biotransformation regardless of the betulin concentration. Experiments employing the combined confocal laser scanning and atomic force microscopy system confirmed that interactions between actinobacterial cells and betulin occur by direct contact. Transforming activities of the crude cell extracts from R. rhodochrous IEGM 66 were compared, and localization of enzymes catalyzing betulin oxidation to betulone was determined. Additionally the effects of betulin on fatty acid composition of rhodococci and their morphometric and morphofunctional characteristics during biotransformation were studied. Our findings could be used to develop approaches for enhanced betulin bioavailability, thus leading to improved biotransformation efficiency.