Human erythrocyte damage at the initial stages of oxidative stress

Specific fluorescent probes have been used to monitor changes in erythrocyte membranes in the first stages of the hemolytic process induced by irradiation with visible light in the presence of protoporphyrin IX. Although no change, or even a slight increase of fluorescence anisotropy, occurred with two probes having a preferential binding to membrane proteins, such as fluorescamine and 3-pyrene maleimide, the fluorescence anisotropy of two lipophilic probes, namely diphenyl-hexatriene and anilino-naphtalene sulfonate, underwent a substantial decrease upon irradiation.     

He-Ne激光对小麦ROP GTPase经增强UV-B辐射造成的损伤的修复研究

采用He-Ne激光辐照对增强UV-B辐射后小麦幼苗ROP GTPase损伤修复作用进行了研究。采用了SDS-PAGE电泳法检测各组ROP GTPase的含量和激光共聚焦显微镜对小麦微丝进行FITC荧光强度标记的测定。研究结果表明：经增强UV-B辐射后,小麦幼苗的Rop GTPase含量降低,LCSM扫描细胞原生质体形状发生改变,细胞骨架受到破坏,其被标记的荧光变暗,强度减弱,在整个UV-B处理期间均低于对照组（CK）,再以He-Ne激光处理后,其含量,形状和强度均有所提高,但仍低于对照组。由此说明,UV-B辐射能使小麦幼苗的Rop GTPase含量下降,微丝骨架受到破坏,一定剂量的激光对UV-B辐射后小麦的蛋白含量和细胞骨架有一定修复作用。Rop GTPase参与了微丝骨架重组的过程。     

He-Ne激光和增强UV-B辐射对小麦幼苗叶绿素荧光特性的影响

选用"ML7113"小麦幼苗为材料,分别采用He-Ne激光(5mW·mm-2)和增强UV-B(10.8 kJ/m-2·d-1)辐射以及两者的复合辐照进行处理,利用PAM-2100便携式叶绿素荧光仪测定小麦幼苗在不同处理天数(5、6、7、8)下叶绿素荧光特性的变化。结果表明:增强UV-B辐射后,随着处理时间的延长,小麦幼苗的Fo、Fm、Fv/Fm、qP、ФPSⅡ、叶绿素含量的值均逐渐下降,qN值均逐渐升高,从而不断减弱小麦幼苗的叶绿体荧光特性;而低剂量的He-Ne激光辐照后能够在一定程度上修复经UV-B辐射后对小麦幼苗叶绿素荧光所造成的损伤。     

Human erythrocyte damage at the initial stages of oxidative stress

Specific fluorescent probes have been used to monitor changes in erythrocyte membranes in the first stages of the hemolytic process induced by irradiation with visible light in the presence of protoporphyrin IX. Although no change, or even a slight increase of fluorescence anisotropy, occurred with two probes having a preferential binding to membrane proteins, such as fluorescamine and 3-pyrene maleimide, the fluorescence anisotropy of two lipophilic probes, namely diphenyl-hexatriene and anilino-naphthalene sulfonate, underwent a substantial decrease upon irradiation. Concomitantly, a dramatic decrease of ATPase activity and an increase of thiobarbituric-reacting substances were observed in erythrocyte membranes. Instead, there was no effect on the activities of the intracellular enzymes glucose-6-phosphate dehydrogenase and pyruvate kinase. These findings are consistent with the hypothesis that protoporphyrin-sensitized irradiation induces, primarily in the erythrocyte membrane, the peroxidation of the lipid component, which results in an increase of the fluidity of the bilayer. Hemolysis eventually occurs because of an osmotic imbalance resulting from the combination of increased passive diffusion and decreased active ion transport.     

增强UV-B辐射和He-Ne激光对小麦原生质体微管骨架的影响

以小麦叶片原生质体为材料,采用间接免疫荧光定位法标记其微管系统,并利用激光共聚焦扫描显微系统进行观察。研究了低剂量He-Ne激光(5mW.mm-2)、增强UV-B辐射(10.08kJ.m-2.d-1)及二者的复合处理对小麦幼苗叶肉细胞中微管骨架的影响。结果表明,增强UV-B辐射后,小麦叶片细胞中微管骨架发生解聚,呈短棒状或点状分布,微管束弥散且荧光强度减弱;而增强UV-B辐射后再施以He-Ne激光处理,小麦叶肉细胞微管骨架有部分断裂,但较单独UV-B处理组的损伤程度轻,说明低剂量的He-Ne激光可以部分修复增强UV-B辐射对微管骨架的损伤,且对微管的聚合有促进作用。     

Effects of microwave radiation (340 and 900 MHz) on different structural levels of erythrocyte membranes

By use of fluorescence probes 1-anilinonaphthalene-8-sulfonic acid, 2-toluidinylnaphthalene-6-sulfonate, pyrene, perylene and chemical label phosphatidylethanolamine 2,4,6-trinitrobenzele sulfonic acid, the effect of microwave radiation on the erythrocyte membrane was studied. The studies with the fluorescence probes were carried out on erythrocyte ghosts and with 2,4,6-trinitrobenzene sulfonic acid on whole erythrocytes. The fluorescence was measured during irradiation of the membranes with 340-MHz microwaves at an SAR of 100 W/kg. Trinitrophenylation of phosphatidylethanolamine from whole erythrocytes was performed simultaneously with microwave irradiation at 900 MHz (10 mW/cm2). It was shown that the microwave field decreased lipid viscosity, altered the structural state of lipid-protein contact regions, and decreased the protein shielding of lipids. These changes corresponded to those produced by thermal action of microwaves.     

Membrane oxidative damage induced by ionizing radiation detected by fluorescence polarization

Effects of ionizing radiation on biological membranes include alterations in membrane proteins, peroxidation of unsaturated lipids accompanied by perturbations of the lipid bilayer polarity. We have measured radiation-induced membrane modifications using two fluorescent lipophilic membrane probes (TMA-DPH and DPH) by the technique of fluorescence polarization on two different cell lines (Chinese hamster ovary CHO-K1 and lymphoblastic RPMI 1788 cell lines). γ-Irradiation was performed using a ⁶⁰Co source with dose rates of 0.1 and 1 Gy/min for final doses of 4 and 8 Gy. Irradiation induced a decrease of fluorescence intensity and anisotropy of DPH and TMA-DPH in both cell lines, which was dose-dependent but varied inversely with the dose rate. Moreover, the fluorescence anisotropy measured in lymphoblastic cells using TMA-DPH was found to decrease as early as 1 h after irradiation, and remained significantly lower 24 h after irradiation. This study indicates that some alterations of membrane fluidity are observed after low irradiation doses and for some time thereafter. The changes in membrane fluidity might reflect oxidative damage, thus confirming a radiation-induced fluidization of biological membranes. The use of membrane fluidity changes as a potential biological indicator of radiation injury is discussed. Received: 14 May 1996 / Accepted in revised form: 30 September 1996     

The reaction of rat blood plasma to gamma radiation studied by the use of laser photon-correlation spectroscopy]

Using the method of laser photon correlation spectroscopy it was shown that the typical pattern of distribution of blood plasma (BP) particles according to their sizes, characteristic of intact animals, was asymmetric with regard to a mean value. Gamma irradiation with sublethal doses was shown to change essentially the pattern of BP distribution. For instance, in BP of exposed animals, the number of particles of much smaller sizes increased as compared to that in intact animals. The smaller BP particle size was not the result of the direct effect of radiation on the blood. The in vitro irradiation of BP with various doses results usually in a considerable growth of particle sizes.     

He-Ne激光和增强UV-B辐射对小麦幼苗类囊体捕光色素的影响

采用5mW.mm-2He-Ne激光辐照、10.08kJ.m-2d-1UV-B辐射及二者组合对冬小麦幼苗进行处理。通过测定叶绿体捕光色素含量和色素蛋白组成的变化,进一步探讨He-Ne激光对增强UV-B辐射后小麦幼苗类囊体捕光色素损伤的修复效应。循环处理小麦幼苗4d,利用90%乙醇和80%丙酮分别提取各处理组小麦幼苗叶片中的叶绿素,通过纸层析和分光光度法检测捕光色素含量的变化,并探讨不同处理对叶绿素与蛋白质结合牢固性的影响。利用柱层析法测定色素蛋白的主要成分。研究表明:与对照组相比,增强UV-B辐照后小麦幼苗捕光色素总含量降低了17.76%,叶绿素和蛋白质结合牢固度显著降低,色素蛋白的组成也发生变化,D1和D2蛋白质条带消失;而一定剂量He-Ne激光辐照可使增强UV-B辐射后的叶绿体色素含量增加约10.64%,但仍低于ck组约8.12%,叶绿素和蛋白质结合牢固度也显著高于B组,色素蛋白的组成与对照组相似。因此,低剂量的He-Ne激光辐照对增强UV-B辐射后小麦幼苗类囊体捕光色素的损伤具有促进修复效应。     

Direct stochastic optical reconstruction microscopy with standard fluorescent probes

Direct stochastic optical reconstruction microscopy (dSTORM) uses conventional fluorescent probes such as labeled antibodies or chemical tags for subdiffraction resolution fluorescence imaging with a lateral resolution of ～20 nm. In contrast to photoactivated localization microscopy (PALM) with photoactivatable fluorescent proteins, dSTORM experiments start with bright fluorescent samples in which the fluorophores have to be transferred to a stable and reversible OFF state. The OFF state has a lifetime in the range of 100 milliseconds to several seconds after irradiation with light intensities low enough to ensure minimal photodestruction. Either spontaneously or photoinduced on irradiation with a second laser wavelength, a sparse subset of fluorophores is reactivated and their positions are precisely determined. Repetitive activation, localization and deactivation allow a temporal separation of spatially unresolved structures in a reconstructed image. Here we present a step-by-step protocol for dSTORM imaging in fixed and living cells on a wide-field fluorescence microscope, with standard fluorescent probes focusing especially on the photoinduced fine adjustment of the ratio of fluorophores residing in the ON and OFF states. Furthermore, we discuss labeling strategies, acquisition parameters, and temporal and spatial resolution. The ultimate step of data acquisition and data processing can be performed in seconds to minutes.     

Effect of aminazine and triftazin on the synaptosome membranes of the rat cerebral cortex

Fluorescent probes 1,6-diphenyl-1,3,5-hexatriene (DPH) and pyrene were employed in studying the effect of aminazine and triftazin versus that of imipramine on microviscosity of rat brain cortex synaptosomal membranes. Unlike imipramine, the neuroleptics decrease microviscosity of membrane's lipid bilayer. All drugs decrease fluorescence of endogenous tryptophan, but fail to change fluorescence of L-tryptophan in the solution. It is concluded that neuroleptics induce conformational perturbations in membrane-bound proteins modifying microviscosity of lipid bilayer whereas imipramine changes the surface electric charge of lipid bilayer of synaptosomal membranes.     

The action of helium-neon laser radiation on the growth of Mycobacterium tuberculosis

The growth properties of M. tuberculosis subjected to the action of helium-neon laser radiation was studied. Laser radiation was shown to change the quantitative and qualitative composition of mycobacterial population. Disturbances in the viability of mycobacteria appear as a consequence of changes in the morphological structure of mycobacterial cells. The maximum effect of helium-neon laser radiation was achieved after the irradiation of M. tuberculosis culture on days 2-3 after inoculation. These results made it possible to suggest that the effect of helium-neon laser radiation was most pronounced in cells at the stage of mitosis (the logarithmic stage of growth) with the highest degree of metabolism.     

Fluorescence studies of the blood platelet membranes associated with fibrinogen

To follow microviscosity changes in membranes associated with fibrinogen binding to human platelets, specific fluorescent probes were used and their fluorescence anisotropy was analysed. The degree of fluorescence anisotropy of diphenylhexatriene, anilinonaphthalene sulfonate (ANS) and fluorescamine increased significantly when fibrinogen reacted with its membrane receptors. Fluorescence polarization analyses showed that fibrinogen binding to platelet membranes is accompanied by an increase in the membrane lipid rigidity. On the other hand, changes in the fluorescence anisotropy of membrane tryptophans and N-(3-pyrene)maleimide suggest augmented mobility of the membrane proteins. The binding of fibrinogen to the membrane receptors is not accompanied by any change in the fluorescence intensity of ANS attached to the membranes. This may suggest that covering of platelets with fibrinogen molecules does not influence the surface membrane charge.     

低功率He-Ne激光照射下细胞内活性氧自由基的产生和游离Ca^2＋浓度的研究

在临床应用中,低功率He-Ne激光(632.8 nm)能促进骨骼肌修复,加速创伤愈合,降低牙齿的超敏感性,减缓疼痛等.大量研究表明:低功率He-Ne激光能调节细胞的众多行为,如细胞增殖、分泌、迁移、粘附、蛋白质合成和基因表达等.但低功率He-Ne激光调节细胞行为的分子机制并未阐明,考察低功率He-Ne激光照射后细胞内活性氧自由基的产生水平和游离ca2 浓度是否会发生变化,通过激光扫描共聚焦显微镜,分别利用H:DCFDA和Fluo-3/AM这两种荧光探针,检测到经He-Ne激光照射后,肺腺癌细胞内活性氧自由基的水平上调以及游离Ca2 浓度增加.该研究为低功率He-Ne激光的生物光刺激效应提供了可能的分子机理.     

Protein-DNA conjugates produced by UV irradiation and their use as probes for hybridization.

Several proteins have been crosslinked to DNA by low dose uv irradiation. The principle of the method is based on an efficient and fast radiation induced reaction of amino acid residues with DNA at low pH. The method seems to be of general applicability for crosslinking proteins to DNA in a very simple one step procedure. Some of such DNA-protein conjugates have been used as probes for hybridization experiments. DNA-protein A probes were found to be most useful.     

Effect of gamma radiation on membrane fluidity of MOLT-4 nuclei

These experiments measured the effect of gamma radiation on the nuclear envelope using doxyl-fatty acid spin-label probes. Nuclei were isolated from cultured MOLT-4 cells, a radiation-sensitive human T-cell lymphocyte. Membrane fluidity was measured from the electron paramagnetic resonance spectra of the probes. MOLT-4 cells were grown under standard conditions, and suspensions were exposed to 60Co gamma radiation at room temperature. The spectra of 5-doxylstearic acid in the nuclei were those of a strongly immobilized label. A difference in the membrane fluidity was detected in a series of experiments comparing labeled irradiated and nonirradiated nuclei. The change in fluidity was measured by comparing the changes in the order parameter, S, of the spin label in irradiated nuclei with those in control nuclei. The change in the S ratio is dependent on radiation dose, increasing with doses up to 15 Gy. The maximum change of the order parameter with time after irradiation occurs 16-20 h after radiation exposure. These observations are correlated with changes in cell viabilities.     

He-Ne激光照射对血液及其组分荧光光谱影响的实验研究

为研究弱激光照射对人血液携氧能力的影响及机制,我们用荧光仪分别测量了He-Ne激光照射前后正常血液及其组分（血浆、红细胞）的荧光光谱,研究了激光照射导致的光谱变化,并分析了光谱变化与血液携氧能力改变的关系。实验结果显示：全血液标本在490nm及614nm附近有荧光峰值;血浆的荧光则主要分布在420－500nm之间;红细胞在500nm及614nm附近有荧光。He-Ne激光照射后,全血液及红细胞在614nm处的荧光谱都有较明显的变化,且较相似。由此可得出结论,He-Ne激光照射可影响血液的携氧能力。     

He-Ne激光对增强UV-B辐射后小麦幼苗光合作用的影响

以"晋麦8号"小麦幼苗为研究材料,分别采用He-Ne激光(辐照剂量为5 mW·mm-2)、增强UV-B(辐射剂量为10.08 kJ·m-2·d-1)以及二者的复合辐照进行处理.循坏处理不同天数(4、5、6、7、8 d)后,利用电导仪、低温荧光测定法检测了小麦叶绿体电子传递速率、膜透性和荧光发射光谱的变化;采用紫外分光光...     

Fluorescent analysis of irradiation-induced changes in chromatin state

A fluorescent technique with Hoechst-33258 and acridine orange staining was used to assess changes in chromatin state induced by radiation. Fluorochromes with different modes of binding to DNA were chosen. In T lymphocytes chronic irradiation caused a rearrangement of binding between nonhiston proteins and lipids accompanied by conformational changes in DNA, resulting in chromatin condensation. The decrease in fluorescence probably resulted from a reduction in the number of sites accessible for dye binding. After acute irradiation, the fluorescence intensity decreases predominantly due to double-strand breaks.     

Use of fluorescence probes 1-aniline-8-naphthalene sulfonate and pyrene for studying the localisation of proteins in inner membranes from wheat etioplasts

The fluorescence probes 1-aniline-8-naphthalene sulfonate (ANS) and pyrene were applied for characterisation of the light-induced changes in etioplast inner membranes (EPIMs) from 7 d-old dark-grown wheat seedlings (Triticum aestivum L. cv. Pobeda). The major aim was to obtain information about the localisation of membrane proteins in the EPIMs, using probes situated in different regions of the membranes. The quenching of tryptophan fluorescence showed tha the main parts of proteins were accessible to the pyrene buried in the lipid bilayer which suggests that most of the proteins also enter the lipid bilayer. The substantial quenching of the tryptophan fluorescence by the surface-situated ANS demonstrated that a part of the tryptophan residues was probably localised close to the membrane surface. The registered changes after irradiation could be explained by the presence of large aggregates of NADPH-protochlorophyllide oxidoreductase (POR), protochlorophyllide (PChlide) and NADPH in membranes that start to disconnect and redistribute along the prothylakoids. This revised version was published online in August 2006 with corrections to the Cover Date.