Magnetic field effects on the fluorescence of mutant strains of Rhodopseudomonas capsulata

The magnetic field effects on bacteriochlorophyll fluorescence in six strains of Rhodopseudomonas capsulata were investigated. All strains exhibit an increase in fluorescence upon application of a magnetic field. Large magnetic field effects are shown to arise in mutants which contain the B800–850 complex as the only bacteriochlorophyll-containing protein. These fluorescence increases are observed only with carotenoid excitation and are best described by a carotenoid singlet heterofission mechanism. Variations in the magnitudes of the magnetic field effects for the Rps. capsulata strain arise from energy differences in the excited states of the molecules involved in the process. In order to determine the contribution from reaction centers to the magnetic field effects observed in the mutants which contain all three pigment-protein complexes, reaction centers were isolated from these strains. The reaction center contribution to the magnetic field effect on fluorescence in whole cells was determined to be smaller than the antenna contribution when carotenoid excitation was employed.     

Reagentless and regenerable immunosensor for monitoring of immunoglobulin G based on non-separation immunoassay

Based on the enhancement of fluorescein isothiocyanate (FITC) fluorescence caused by reactions between proteins, we developed a reagentless, regenerable and rapid immunosensing system to determine immunoglobulin G (IgG). Fluorescence intensity of the immobilized FITC depends on IgG concentration, ranging from 10 to 50 microg/ml, specifically, even with co-existing proteins. The response time is 30 min during steady-state measurement and is less than a minute during transient measurement. When the FITC-labeled protein A binds to IgG, the surrounding atmosphere of FITC becomes hydrophobic. Since the fluorescence intensity of fluorescent substances generally increases at a hydrophobic environment, FITC fluorescence intensity increases with the concentration of protein A bonding to IgG. This system is regenerable because the fluorescence enhancement repeatedly occurs every time the immobilized fluorescent reagent is immersed in sample solutions.     

环境参数变化对γ射线诱变微生物的影响

报告了在非自然环境中培养选育青霉菌菌株的一些结论。实验表明随着辐射剂量的增加菌株的致死率也相应增加;当辐照剂量相同时,与自然环境相比其致死率有所提高,且随着非自然环境参数值的增加而增加。当电场强度为３００ｋＶ／ｍ、磁场强度为６００Ｇｓ时,正变率有一极大值。     

Variation in the intensity of scattered light by solutions of DNA subjected to an electric field

Preliminary experiments have been carried out which measure the variation in the intensity of the light scattered by DNA solutions under the influence of an electric field. Data have been collected on the length of DNA molecules, their electric polarizability, and their dispersion behavior.     

The effect of a constant magnetic field on components of protein structures in human blood

A study on the effects of a constant magnetic field on the components of the protein structures in human blood was conducted by the methods of electron spectroscopy, optic aggregometry, and electrophoresis on an agarose gel. It was found that protein structures of different localizations experience the primary effect of a constant magnetic field. As a result, the total charge, form, and linear dimensions of the molecules change, as well as the rotation frequency of the components around valence bonds; this leads to changes in the spatial structures of the blood protein components. It was suggested as a working hypothesis that an increase in the intensity of oxidation processes under the effect of a magnetic field and, consequently, an increase in the negative activities of oxygen metabolites underlie these changes.     

One- and two-photon induced fluorescence of Pacific Blue-labeled human serum albumin deposited on different core size silver colloids

We studied one- and two-photon induced fluorescence of Pacific Blue (PB)-labeled human serum albumin (HSA) in the presence of different size silver colloids. The PB fluorescence emission intensity was observed with small (30-40 nm) and large (about 120 nm) colloids and compared with PB emission in absence of colloids. For the system with a small core size colloids we did not detect any fluorescence enhancement with one-photon excitation and the enhancement observed with two-photon excitation was about 2.5-fold. In contrast, for large silver colloids we observed about a 2-fold increase in PB fluorescence brightness for one-photon excitation, and the enhancement with two-photon excitation excided 13-folds. Much stronger increases in brightness observed with two-photon excitation, compared to one-photon excitation, indicate a dominant role of enhanced local field in fluorescence enhancement on silver colloids in solutions.     

Effect of weak magnetic fields on fluorescence of water and water-salt solutions. Partial characterization of fluorescing fractions

It has been shown that exposure to weak combined permanent (42 mT) and low-frequency (3-60 Hz) alternating (0.001-0.06 mT) magnetic fields changes the intensity of fluorescence of water and water-salt solutions. The gel filtration of solutions of inorganic salts treated with magnetic fields gives rise to intensively fluorescing fractions. Control solutions not exposed to electromagnetic waves do not exhibit these effects. The results obtained suggest that treatment with weak electromagnetic fields induces structural changes of water solutions, and the manifestations of these changes depend on the conditions of chromatography and chemical composition of solutions under study.     

Modification of albumin with different degrees of the oxidation of SH-groups in the reaction with glucose

A molecule of the major blood protein albumin contains 34 cysteine residues involved in disulfide bonds and one unpaired SH-group of residue Cys34. Normally, 20–30% of these SH-groups are oxidized and form disulfide bonds or the derivatives of sulfenic, sulfinic, and sulfonic acids. The goal of the present work was to study the influence of the degree of oxidation of sulfhydryl groups on the capacity of albumin for glycation. Commercially available human albumin containing 0.4 moles of sulfhydryl groups per 1 mole of the protein (nonmercaptalbumin) was used. Disulfide bonds in this preparation were reduced with dithiothreitol to 0.7 mole/mole to give mercaptalbumin. The preparations were incubated for three weeks with glucose at a concentration of 5 and 50 mM. The content of ketoamine, a glycation product, was determined by the colorimetric method, the content of pentosidine (glycation end product) was analyzed by fluorescence, and the content of SH-groups was determined using the Ellman’s reagent. Changes in the structure and properties of the protein during glycation were studied by fluorescence and HPLC. During the incubation of both albumin preparations with 5 mM glucose, no significant increase in the ketoamine content was observed, whereas the incubation with 50 mM glucose was accompanied by a considerable accumulation of ketoamine. It was found that the greatest amount of ketoamine under these conditions forms in nonmercaptalbumin; in this case, the intensity of tryptophan fluorescence decreases. The intensity of pentosidine fluorescence increases with increasing content of ketoamine. The results obtained enable the conclusion that the oxidation of free SH-groups of the protein changes its conformation; as a result, the glycation of earlier hidden sites becomes possible, and the degree of protein glycation increases.     

Synthesis and fluorescent properties of europium(III) complexes based on novel coumarin derivatives

Four novel coumarin fluorescence small-molecules were successfully prepared and validated by proton nuclear magnetic resonance (¹H-NMR), carbon-13 (¹³C)-NMR, and mass spectrometry (MS). Their corresponding europium(III) complexes were synthesized and characterized. The ligand can emit green fluorescence in solutions, and the best concentration was 40 μmol/L. The emission peak of ligand has a red-shift with the increase of concentration and solvent polarity. And the effect of various substituents in ligand was ordered using fluorescence intensity as standard: - NO₂ > -Cl > -OCH₃ > -OH. The order of fluorescence quantum yield is in line with the order of fluorescence intensity. The title europium complexes exhibit red fluorescence of europium ion (Eu³⁺) with good thermal stability. The effect of various substituents in ligand on the fluorescence intensity of title europium complexes was also consistent with the earlier results. This suggests that the prepared coumarins fluorescence small-molecules and their corresponding europium complexes have potential application prospects in the field of optical materials.     

Effect of magnetic resonance imaging on human respiratory burst of neutrophils

It is known that low intensity magnetic fields increase superoxide anion production during the respiratory burst of rat peritoneal neutrophils in vitro. We investigated whether the high intensity magnetic fields (1.5 T) during magnetic resonance imaging can influence the human neutrophil function under in vivo conditions. Blood samples were obtained from 12 patients immediately before and after magnetic resonance imaging (mean time 27.6(+/-11.4 min)). The induced respiratory burst was investigated by the intracellular oxidative transformation of dihydrorhodamine 123 to the fluorescent dye rhodamine 123 via flow cytometry. The respiratory burst was induced either with phorbol 12-myristate 13-acetate, Escherichia coli, N-formyl-methionyl-leucylphenylalanine or priming with tumor necrosis factor followed by FMLP stimulation. There was no significant difference between the respiratory burst before and after magnetic resonance imaging, irrespective of the stimulating agent. Short time exposure to a high intensity magnetic field during magnetic resonance imaging seems not to influence the production of radical species in living neutrophils.     

Effect of weak combined low frequency constant and alternative magnetic fields on intrinsic fluorescence of proteins in aqueous solutions]

It was shown that weak combined static (42 microT) and low-frequency variable (40 nT; 3-5 Hz) magnetic fields change the intensity of intrinsic fluorescence of some proteins (cytochrome c, bovine serum albumin, horseradish peroxidase, alkaline phosphatase). The effect can be interpreted as a change in the conformational state of the protein in water environment by the action of weak magnetic fields. The dynamics of the process, the concentration dependence, the binding of proteins to the fluorescence probe 1,8-ANS after treatment with magnetic fields, the frequency dependence of these reactions, and the dependence of the effect on the presence of the static constituent of the magnetic field were studied. It was shown that the changes in the intrinsic fluorescence of some enzymes (horseradish peroxidase, alkaline phosphatase) are related to changes in their functional activity. It was found that the effect is partially transferred via a solvent (water, 0.01 M NaCl) preliminarily treated with magnetic field. In the solvent, changes in its intrinsic fluorescence by the action of weak magnetic fields were also registered.     

Monitoring the inhibitive effect of the static magnetic field on the activity of lysozyme with acoustic wave impedance analysis technique

The inhibitive effect of static magnetic field on the activity of lysozyme was studied using acoustic wave impedance analysis technique. Equivalent circuit parameters of piezoelectric quartz crystal (PQC) were obtained and discussed. The results showed that the activity of lysozyme was inhibited due to the effect of static magnetic field and the inhibitive effect becomes greater with an increase in magnetization time or magnetic field intensity. According to the response characteristics of motional resistance change (deltaR1), which is related to the change in the bacterial number, a quantitative response model reflecting the activity of lysozyme was theoretically derived. By fitting deltaR1 versus time curves under a specific magnetic field intensity but different magnetic time to the model, the relationship between K1 reflecting the activity of lysozyme and magnetic time t(m) was established. Based on the relationship, a new impedance response model that indicates the inhibitive influence of the magnetization time on the activity of lysozyme was derived as follows: deltaR1 = R0((K4(exp[K0exp(-0.26t(m))]t - 1) + 1)1/2 - 1). Similarly, another response model that indicates the effect of magnetic field intensity was derived as follows: deltaR1 = R0((K4(exp(K0 exp(- 5.17B)t) - 1) + 1)1/2 - 1).     

Fluorescence of hemoproteins in reversed micelles of surfactants in octanes

The fluorescence of myoglobin, cytochromes b5 and c in the reversed aerosol OT (AOT) micelles in octane has been investigated. The fluorescence intensity of all the three hemoproteins is higher than that in aqueous solutions. The maxima and intensities of fluorescence in the AOT micelles depend on the [H2O]/[AOT] ratio and reflect the protein structure. Aliphatic alcohols and secondary amines (piperidine and morpholine) quench the cytochrome c fluorescence in the AOT micelles, whereas dipolar aprotic solvents (dimethylsulfoxide, dimethylformamide) significantly increase the intensity of cytochrome c fluorescence in the same micelles. The transformations of the proteins solubilized by the reversed micelles of a surfactant are discussed.     

汞化合物对红细胞膜作用的研究

本文研究了汞化合物对红细胞膜作用的光谱变化,观察了膜蛋白的荧光和磷光,膜上DPH的荧光偏振和ANS与红细胞膜的结合,以及他们与汞产生红血球溶血的关系。 在5P7.5缓冲液中红细胞膜蛋白的荧光随HgCl_2 Hg(AC)_2和PCMB的浓度加大而降低,表现为快和慢双相变化的过程,其淬灭作用的大小为HgCl_2>Hg(AC)_2PCMB,这是由于膜上形成了不发荧光的R—Trp—Hg~ 络合物以及能量从Trp转移到R—S—Hg~ 络合物上。HgCl_2对膜蛋白磷光的作用也是随汞离子浓度加大而降低,但磷光/荧光比则是增加的。标记红细胞膜的DPH偏振度是随HgCl_2浓度增加,表明膜流动性是随汞离子浓度加大而降低。标记膜上的ANS的荧光强度随HgCl_2和Hg(AC)_2的浓度加大而增加,这是由于ANS与膜的结合数随汞离子浓度加大而增加的缘故。上述各种变化是与汞离子对红血球溶血的作用一致的。     

Delivery of Short Interfering Ribonucleic Acid-Complexed Magnetic Nanoparticles in an Oscillating Field Occurs via Caveolae-Mediated Endocytosis

Gene delivery technologies to introduce foreign genes into highly differentiated mammalian cells have improved significantly over the last few decades. Relatively new techniques such as magnetic nanoparticle-based gene transfection technology are showing great promise in terms of its high transfection efficiency and wide-ranging research applications. We have developed a novel gene delivery technique, which uses magnetic nanoparticles moving under the influence of an oscillating magnetic array. Herein we successfully introduced short interfering RNA (siRNA) against green fluorescent protein (GFP) or actin into stably-transfected GFP-HeLa cells or wild-type HeLa and rat aortic smooth muscle cells, respectively. This gene silencing technique occurred in a dose- and cell density- dependent manner, as reflected using fluorescence intensity and adhesion assays. Furthermore, using endocytosis inhibitors, we established that these magnetic nanoparticle-nucleic acid complexes, moving across the cell surface under the influence of an oscillating magnet array, enters into the cells via the caveolae-mediated endocytic pathway.     

葡聚糖磁性纳米颗粒外加磁场对人树突状细胞基因转染效率的研究

目的研究葡聚糖磁性纳米颗粒（the dextran coated magnetic iron oxide nanoparticles,DMN）在外加钕一铁一硼稀土固定磁场的作用下对人树突状细胞转染效率以及安全性的影响。方法先通过磁力计对DMN进行分析;再将修饰有多聚赖氨酸（Poly-L—Lysine,PLL）的DMN携带绿色荧光蛋白pEGFP—Cl质粒报告基因,在钕-铁-硼稀土周定强磁场的作用下,体外转染人树突状细胞,用荧光显微镜直接观察和流式细胞仪检测来评价外加磁场对DMN作为人树突状细胞转染载体效率的影响;在转染后采用MTT比色法测定在磁场干预下的DMN对人树突状细胞增殖和功能的影响以了解其细胞毒性。结果DMN的核心直径〈30nm,具有明硅的超顺磁性,比饱和磁化强度也明显高于相同Fe3O4含量的普通磁块;DMN作为基因载体在外加磁场作用下,转染12h即可将报告基因转染至人树突状细胞内并成功表达,在荧光显微镜下可观察到绿色荧光细胞,24h转染率可达到最高（约为27％）,转染效率较未加磁场组提高了2～4倍。而且转染后的人树突状细胞增殖活性及功能未因DMN外加磁场及其作用时间的长短而受到影响。结论超顺磁性的DMN在外加磁场作用下可以明显、安全、有效地提高对人树突状细胞的转染效率。     

Effect of the bidistilled modified water on bovine serum albumin conformation. Fluorescent spectroscopy data

It was shown that bidistilled modified water induces a marked decrease in the intensity of intrinsic fluorescence of bovine serum albumin and increases the binding of this protein to the fluorescent probe 1.8 ANS. These effects can be interpreted as a denaturing action of bidistilled modified water on the protein and a change in its conformational state, which is probably caused by changes in the microenvironment of the protein molecule. In addition, a substantial increase in the intrinsic fluorescence of bidistilled modified water, as compared with that of distilled water, was found.     

A novel amphiphilic thiosemicarbazone derivative for binding and selective sensing of human serum albumin

The interaction of ligands and drug molecules with protein is of major interest in drug pharmacokinetics and pharmacodynamics. In this study, we synthesized a novel thiosemicarbazone‐based amphiphilic molecule for selective binding and detection of human serum albumin (HSA) with significant increase in fluorescence intensity. The compound 5‐(octyloxy) naphthalene substituted salicylaldehyde thiosemicarbazone was designed to interact with site I of HSA. The weak fluorescence of the probes in aqueous solution showed a dramatic increase in fluorescence intensity upon binding with HSA, while the responses to various other proteins and enzymes were negligible under similar experimental conditions. Changes in fluorescence intensity and formation of a new emission maximum of the compound in the presence of HSA as well as an increase in steady‐state anisotropy values reflected well the nature of binding and location of the probe inside the protein environment. Copyright © 2012 John Wiley & Sons, Ltd.     

Studies on the electrogenic action of acetylcholine with Torpedo marmorata electric organ: IV. Quinacrine: a fluorescent probe for the conformational transitions of the cholinergic receptor protein in its membrane-bound state

Quinacrine, like a typical local anaesthetic, blocks the response of Electrophorus electricus electroplaque in vivo in a non-competitive manner and enhances, in vitro, the affinity of the cholinergic receptor present in Torpedo marmorata membrane fragments for acetylcholine. The interaction of quinacrine with T. marmorata membrane fragments can be followed by differential fluorescence spectroscopy either upon direct illumination (λ_Ex = 350 nm) or by energy transfer from membrane proteins (λ_Ex = 290 nm). Carbamylcholine and most of the cholinergic ligands tested cause an increase of the light intensity emitted by membrane-bound quinacrine under conditions of direct excitation; all these effects are blocked by a preincubation of the membrane fragments with the α-toxin from Naja nigricollis. When quinacrine is excited by energy transfer, carbamylcholine, phenyltrimethylammonium and hexamethonium cause an increase of fluorescence but flaxedil, tetraethylammonium and the α-toxin give a much smaller fluorescence increase or none.Local anaesthetics like prilocaine or quotane cause a decrease of fluorescence intensity of membrane-bound quinacrine in both the presence and absence of carbamylcholine. Quantitative studies on quinacrine binding and fluorescence as a function of quinacrine concentration reveal at least two populations (saturable and non-saturable) of binding sites, the saturable one being identical or closely related to the specific site of action of local anaesthetics. It is concluded that binding of cholinergic ligands primarily increases the quantum yield of a fraction of bound quinacrine.The curves of variation of fluorescence intensity with agonist and antagonist concentrations determined under conditions of direct illumination, closely resemble the binding curves determined at equilibrium with radioactive ligands. Under these conditions quinacrine therefore enables us to determine the occupancy of the receptor site by cholinergic ligands. On the other hand, the change of quinacrine fluorescence observed by energy transfer, which takes place with some of the cholinergic ligands but not with others, and does not correlate with any variation of the intrinsic fluorescence of membrane proteins, most likely reflects a change of structure bearing a qualitative relationship to the pharmacological activity of the tested ligands.