Study of molecular events in cells by fluorescence correlation spectroscopy

To understand processes in a living cell, sophisticated and creative approaches are required that can be used for gathering quantitative information about large number of components interacting across temporal and spatial scales without major disruption of the integral network of processes. A physical method of analysis that can meet these requirements is fluorescence correlation spectroscopy (FCS), which is an ultrasensitive and non-invasive detection method capable of single-molecule and real-time resolution. Since its introduction about 3 decades ago, this until recently emerging technology has reached maturity. As commercially built equipment is now available, FCS is extensively applied for extracting biological information from living cells unattainable by other methods, and new biological concepts are formulated based on findings by FCS. In this review, we focus on examples in the field of molecular cellular biology. The versatility of the technique in this field is illustrated in studies of single-molecule dynamics and conformational flexibility of proteins, and the relevance of conformational flexibility for biological functions regarding the multispecificity of antibodies, modulation of activity of C5a receptors in clathrin-mediated endocytosis and multiplicity of functional responses mediated by the p53 tumor suppressor protein; quantitative characterization of physicochemical properties of the cellular interior; protein trafficking; and ligand-receptor interactions. FCS can also be used to study cell-to-cell communication, here exemplified by clustering of apoptotic cells via bystander killing by hydrogen peroxide.Received 15 July 2004; received after revision 13 October 2004; accepted 12 November 2004     

乙酰胆碱对大鼠离体输精管平滑肌细胞膜电位的作用

采用细胞内微电极技术对大鼠离体输精管平滑肌细胞进行记录,观察其静息电位、膜阻抗等被动膜电性质及ACh对平滑肌细胞膜电位的作用,在此基础上运用微电泳技术对所记录细胞进行标记,从而分析不同走行方向的平滑肌细胞的电生理学特性。实验测得输精管平滑肌细胞静息电位为-48.56±0.46mV(n=1141),膜输入阻抗为1638.70±205.20MΩ(n=45)。实验用ACh(0.3～100μmol L)灌流平滑肌细胞,可引起膜发生浓度依赖性超极化。10μmol LACh引起超极化幅值为13.67±0.98mV(n=116),膜电导平均增加2.31%(n=10)。标本经1μmol L硫酸阿托品(n=10)和100nmol L2' deoxyadenosine5' monophosphate(n=11)预处理后,ACh引起的超极化幅值明显减小。经荧光染料(0.1%propidiumiodide)标记鉴别后,统计分析外纵走行细胞的静息电位为-53.56±3.88mV(n=37),膜输入阻抗为2245.60±372.50MΩ(n=13);内环走行细胞的静息电位为-51.62±4.27mV(n=17),膜输入阻抗为2101.50±513.50MΩ(n=10);并且2种走形方向的平滑肌细胞对ACh的反应形式亦无显著性差异。实验表明:输精管平滑肌细胞的电生理特性以及对ACh的反应与其走形方向无关。     

异丙嗪对下丘脑细胞内游离钙浓度的影响

目的：观察异丙嗪（ｐｒｏｍｅｔｈａｚｉｎｅ,ＰＭＺ）对下丘脑细胞内游离钙浓度（［Ｃａ２＋］ｉ）的影响。方法：以酶法制备家兔下丘脑细胞悬液,运用钙指示剂Ｆｕｒａ－２／ＡＭ作为细胞内游离钙的荧光探针测定下丘脑细胞［Ｃａ２＋］ｉ。结果：１）ＰＭＺ（０４６ｍｍｏｌ／Ｌ）使下丘脑［Ｃａ２＋］ｉ显著升高,且在一定的剂量范围内呈量效关系。２）事先向细胞悬液中加入钙通道阻滞剂维拉帕米可明显抑制ＰＭＺ诱导的［Ｃａ２＋］ｉ升高,但不能完全阻断ＰＭＺ的这种作用。结论：上述结果提示ＰＭＺ可引起下丘脑［Ｃａ２＋］ｉ升高,钙通道开放导致细胞外钙内流是ＰＭＺ引起下丘脑［Ｃａ２＋］ｉ升高的机制之一。     

红细胞膜变形能力与胞内血红蛋白聚合状态及细胞形态的关系

以显微激光散射光谱和图像分析技术,对单个活态红细胞在无扰、在位、实时的情况下,测量了人血红细胞胞膜的变形能力,以及与之同时对应的胞内血红蛋白的聚合状态和细胞的形态结构.并据此研究了它们的相关关系以及随各种生理条件而交互变化的情况.     

UV/固定化TiO2对太湖源水中藻类胞内有机物的去除效能

采用溶胶-凝胶法,以玻璃纤维网为载体负载TiO2薄膜,X射线衍射分析表明,其薄膜中TiO2晶相为锐钛矿型.基于UV/TiO2体系,研究其对太湖源水中藻类胞内有机物(IOM)的降解机制及其影响因素.与腐殖酸(HA)对比研究结果表明,IOM较难被光催化氧化工艺降解,反应60 min时,UV254和DOC的降解率只有33.3％和19.1％,而HA的UV254和DOC降解率分别达到96.6％和57.3％;原因在于IOM主要是由大分子亲水性有机物组成,含有较多的氨基结构,而腐殖酸是强疏水性有机物,含有较多的芳香结构.同时,UV/TiO2体系的光强增强在一定程度上能提高IOM的降解效果;在pH=6.7时,IOM降解率达到最佳.     

伤寒沙门菌非编码RNA ArpH表达特征及功能初探

通过研究σ因子和RNase对伤寒沙门菌非编码RNA ArpH表达的影响，并结合全基因组芯片的结果，初步探讨ArpH对细菌侵袭力和胞内生存力的影响.应用qRT-PCR检测了rpoE和rpoS对ArpH转录的影响，以及RNase III、RNase G和RNase E分别对ArpH降解的影响.另外还利用全基因组芯片技术对ArpH高表达菌株在氧应激下的基因表达谱进行分析.最后观察了ArpH对伤寒沙门菌侵袭力的影响和对胞内生存力的作用.结果显示，rpoE在酸、氧及高渗应激下、rpoS在氧应激下调节ArpH的转录.细菌中RNase III主要参与了ArpH的降解.氧应激下高表达ArpH后，基因表达上调的有89个，基因表达下调的有24个.ArpH高表达可以减弱伤寒沙门菌对上皮细胞的侵袭力，并能增强细菌的胞内生存力.     

Effect of Aβ1-40 on membrane permeability and intracellular free Ca2+ of nerve cells

The effects of soluble and fibrillar Aβ1-40 on membrane permeability and intracellular free Ca2+ of nerve cells were investigated by the laser confocal microscopy. Results indicate that: i) Effects of soluble and fibrillar Aβ1-40 on cell membrane permeability are both concentration-de- pendent. Soluble Aβ1-40 increases membrane permeability only at concentration of 3 ?mol/L, while the toxic effect of fibrillar Aβ1-40 is much stronger, its evident effect begins from 1 ?mol/L. When its concentration rose to 3 ?mol/L, not only the membrane permeability increased, but also the nuclear membrane broke seriously. ii) Both soluble and fibrillar Aβ1-40 at high concentrations increased the intracellular free Ca2+, and the increased amplitudes are concentration-dependent. However, the fibrillar one induces the increase of intracellular Ca2+ much quicker and synchronously. These results indicate that some correlation exists between the neurotoxicity of high concentration soluble and fibrillar Aβ1-40 and the change of physico-chemical properties and intracellular Ca ion imbalance.     

支气管哮喘患者吸入白细胞介素-5所致细胞间粘附因子增高的研究

采用随机、双盲及设置对照组的办法进行研究。在吸入ＩＬ５之前，对８例不吸烟的哮喘患者及６例非哮喘过敏性疾病患者测定其痰及血清的ｓＩＣＡＭ１；吸入ＩＬ５后，于２ｈ、２４ｈ、４８ｈ及７２ｈ取上述标本分别测定其ｓＩＣＡＭ１的水平。结果示对照组患者的ｓＩＣＡＭ１在整个研究过程中都没有明显的改变。但吸入ＩＬ５后支气管哮喘患者于痰中ｓＩＣＡＭ１出现明显的增高，于４８ｈ达最高峰，但持续不超过７２ｈ。同样的结果亦出现在血清中。研究表明ＩＬ５可能是导致支气管哮喘患者痰及血的ｓＩＣＡＭ１增高的原因，对非哮喘过敏疾病患者则无此作用。     

Multi-Target Recognition in Intracellular Regulation Networks

This paper presents an algorithm for identifying desirable multiple targets in an intracellular regula- tion network. The algorithm is based on constrained state feedback and Monte-Carlo simulations. The com- putational complexity of the algorithm increases linearly with increasing the number of species in a gene regulation system. An estimate is derived for the confidence level of the predicted minimum required pertur- bation strength when targets are prescribed a priori. The algorithm has been used to analyze the cell cycle of Xenopus frog eggs. The results agree well with available results for single target perturbations, and multi- target interference is usually not equal to the summation of the single-target interferences.     

Nitric oxide: a synchronizing chemical messenger

Nitric oxide (NO) has been recognized as a ubiquitous chemical messenger in a large number of different biological systems. Its chemical properties make it less specific and less controllable than practically any other neurotransmitter or hormone. In view of this, its extensive biological role as a chemical messenger seems surprising. It is suggested that the biological function of NO evolved early in the anaerobic stage of evolution. In view of its low molecular weight, limited interaction with water, and its electrical neutrality, which allow it to diffuse rapidly through the cytoplasm and biomembranes, it is suggested that the need for NO has been retained by and maintained in eukaryote cells because of its ability to affect many biochemical functions simultaneously, acting primarily as an intracellular synchronizing chemical messenger.