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1.
It is increasingly apparent that nature evolved peroxiredoxins not only as H 2O 2 scavengers but also as highly sensitive H 2O 2 sensors and signal transducers. Here we ask whether the H 2O 2 sensing role of Prx can be exploited to develop probes that allow to monitor intracellular H 2O 2 levels with unprecedented sensitivity. Indeed, simple gel shift assays visualizing the oxidation of endogenous 2-Cys peroxiredoxins have already been used to detect subtle changes in intracellular H 2O 2 concentration. The challenge however is to create a genetically encoded probe that offers real-time measurements of H 2O 2 levels in intact cells via the Prx oxidation state. We discuss potential design strategies for Prx-based probes based on either the redox-sensitive fluorophore roGFP or the conformation-sensitive fluorophore cpYFP. Furthermore, we outline the structural and chemical complexities which need to be addressed when using Prx as a sensing moiety for H 2O 2 probes. We suggest experimental strategies to investigate the influence of these complexities on probe behavior. In doing so, we hope to stimulate the development of Prx-based probes which may spearhead the further study of cellular H 2O 2 homeostasis and Prx signaling. 相似文献
2.
Hydrazine (N 2H 4) is a highly toxic and harmful chemical reagent. Fluorescent probes are simple and efficient tools for sensitive monitoring of N 2H 4 enrichment in the environment, humans, animals, and plants. In this work, a ratiometric fluorescent probe (FP-1) containing coumarin was used for hydrazine detection. The proposed FP-1 probe had a linear detection range of 0–250 μM and a limit of detection (LOD) of 0.059 μM (1.89 ppb). A large red Stokes shift was observed in fluorescence and UV–vis absorption spectra due to the hydrolysis of ester bonds between FP-1 and hydrazine. The hydrazine detection mechanism of FP-1 was also investigated using density functional theory (DFT) calculations. Finally, FP-1 could sensitively and selectively monitor hydrazine in actual water samples and BEAS-2B cells. Therefore, it has great application potential in environmental monitoring and disease diagnosis. 相似文献
3.
Hydrogen peroxide (H 2O 2) is an incompletely reduced metabolite of oxygen that has a diverse array of physiological and pathological effects within
living cells depending on the extent, timing, and location of its production. Characterization of the cellular functions of
H 2O 2 requires measurement of its concentration selectively in the presence of other oxygen metabolites and with spatial and temporal
fidelity in live cells. For the measurement of H 2O 2 in biological fluids, several sensitive methods based on horseradish peroxidase and artificial substrates (such as Amplex
Red and 3,5,3’5’-tetramethylbenzidine) or on ferrous oxidation in the presence of xylenol orange (FOX) have been developed.
For measurement of intracellular H 2O 2, methods based on dihydro compounds such as 2’,7’-dichlorodihydrofluorescein that fluoresce on oxidation are used widely
because of their sensitivity and simplicity. However, such probes react with a variety of cellular oxidants including nitric
oxide, peroxynitrite, and hypochloride in addition to H 2O 2. Deprotection reaction-based probes (PG1 and PC1) that fluoresce on H 2O 2-specific removal of a boronate group rather than on nonspecific oxidation have recently been developed for selective measurement
of H 2O 2 in cells. Furthermore, a new class of organelle-targetable fluorescent probes has been devised by joining PG1 to a substrate
of SNAP-tag. Given that SNAP-tag can be genetically targeted to various subcellular organelles, localized accumulation of
H 2O 2 can be monitored with the use of SNAP-tag bioconjugation chemistry. However, given that both dihydro- and deprotection-based
probes react irreversibly with H 2O 2, they cannot be used to monitor transient changes in H 2O 2 concentration. This drawback has been overcome with the development of redox-sensitive green fluorescent protein (roGFP)
probes, which are prepared by the introduction of two redox-sensitive cysteine residues into green fluorescent protein; the
oxidation of these residues to form a disulfide results in a conformational change of the protein and altered fluorogenic
properties. Such genetically encoded probes react reversibly with H 2O 2 and can be targeted to various compartments of the cell, but they are not selective for H 2O 2 because disulfide formation in roGFP is promoted by various cellular oxidants. A new type of H 2O 2-selective, genetically encoded, and reversible fluorescent probe, named HyPer, was recently prepared by insertion of a circularly
permuted yellow fluorescent protein (cpYFP) into the bacterial peroxide sensor protein OxyR. 相似文献
4.
Mounting evidence supports the role of hydrogen peroxide (H 2O 2) in physiological signaling as well as pathological conditions. However, the subtleties of peroxide-mediated signaling are not well understood, in part because the generation, degradation, and diffusion of H 2O 2 are highly volatile within different cellular compartments. Therefore, the direct measurement of H 2O 2 in living specimens is critically important. Fluorescent probes that can detect small changes in H 2O 2 levels within relevant cellular compartments are important tools to study the spatial dynamics of H 2O 2. To achieve temporal resolution, the probes must also be photostable enough to allow multiple readings over time without loss of signal. Traditional fluorescent redox sensitive probes that have been commonly used for the detection of H 2O 2 tend to react with a wide variety of reactive oxygen species (ROS) and often suffer from photostablilty issues. Recently, new classes of H 2O 2 probes have been designed to detect H 2O 2 with high selectivity. Advances in H 2O 2 measurement have enabled biomedical scientists to study H 2O 2 biology at a level of precision previously unachievable. In addition, new imaging techniques such as two-photon microscopy (TPM) have been employed for H 2O 2 detection, which permit real-time measurements of H 2O 2in vivo. This review focuses on recent advances in H 2O 2 probe development and optical imaging technologies that have been developed for biomedical applications. 相似文献
5.
A highly sensitive and selective fluorescence method for the detection of acetylcholine (ACh) based on enzyme-generated hydrogen peroxide (H 2O 2) and a new boronate intramolecular charge transfer (ICT) fluorescence probe, 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)- N-butyl-1,8-naphthalimide (BN), was developed. This strategy involves the reaction of ACh with acetylcholinesterase (AChE) to produce choline, which is further oxidized by choline oxidase (ChOx) to obtain betaine and H 2O 2. The enzyme-generated H 2O 2 reacts with BN and results in hydrolytic deprotection of BN to generate fluorescent product (4-hydroxyl- N-butyl-1,8-naphthalimide, ON). Two consecutive linear response ranges allow determining ACh in a wide concentration range with a low detection limit of 2.7 nM (signal/noise = 3). Compared with other fluorescent probes based on the mechanism of nonspecific oxidation, this reported boronate probe has the advantage of no interference from other biologically relevant reactive oxygen species (ROS) on the detection of ACh. This study provides a new method for the detection of ACh with high selectivity and sensitivity. 相似文献
6.
A dual‐function fluorescent probe (Probe 1 ) was developed for discriminative detection of Hg 2+ and N 2H 4. Probe 1 could discriminatively detect Hg 2+ and N 2H 4 through two different reaction sites, with the mechanism for Probe 1 for Hg 2+ depending on a desulfurization reaction and for N 2H 4 depending on the Schiff‐base reaction. N 2H 4 had minimal effect on Hg 2+ detection in dimethyl sulfoxide (DMSO)/H 2O solution, but Hg 2+ could interfere with N 2H 4 detection in DMSO/buffer solution. Different concentrations of Hg 2+ and N 2H 4 resulted in different blue shades of Probe 1 test strips, and the shade of blue was different with the same concentration of Hg 2+ or N 2H 4, as observed under ultraviolet light at 365 nm wavelength. 相似文献
7.
Protein carbonylation is a major form of protein oxidation and is widely used as an indicator of oxidative stress. Carbonyl groups do not have distinguishing UV or visible, spectrophotometric absorbance/fluorescence characteristics and thus their detection and quantification can only be achieved using specific chemical probes. In this paper, we review the advantages and disadvantages of several chemical probes that have been and are still being used for protein carbonyl analysis. These probes include 2,4-dinitrophenylhydazine (DNPH), tritiated sodium borohydride ([ 3H]NaBH 4), biotin-containing probes, and fluorescence probes. As our discussions lean toward gel-based approaches, utilizations of these probes in 2D gel-based proteomic analysis of carbonylated proteins are illustrated where applicable. Analysis of carbonylated proteins by ELISA, immunofluorescent imaging, near infrared fluorescence detection, and gel-free proteomic approaches are also discussed where appropriate. Additionally, potential applications of blue native gel electrophoresis as a tool for first dimensional separation in 2D gel-based analysis of carbonylated proteins are discussed as well. 相似文献
8.
This review aims at holistically analyzing the environmental problems associated with nitrous oxide (N 2O) emissions by evaluating the most important sources of N 2O and its environmental impacts. Emissions from wastewater treatment processes and the industrial production of nitric and adipic acid represent nowadays the most important anthropogenic point sources of N 2O. Therefore, state-of-the-art strategies to mitigate the generation and release to the atmosphere of this greenhouse and O 3-depleting gas in the waste treatment and industrial sectors are also reviewed. An updated review of the end-of-the-pipe technologies for N 2O abatement, both in the waste treatment and industrial sectors, is herein presented and critically discussed for the first time. Despite the consistent efforts recently conducted in the development of cost-efficient and eco-friendly N 2O abatement technologies, physical/chemical technologies still constitute the most popular treatments for the control of industrial N 2O emissions at commercial scale. The recent advances achieved on biological N 2O abatement based on heterotrophic denitrification have opened new opportunities for the development of eco-friendly alternatives for the treatment of N 2O emissions. Finally, the main limitations and challenges faced by these novel N 2O abatement biotechnologies are identified in order to pave the way for market implementation. 相似文献
9.
UV light has been the most important energy source on the primitive Earth. However, very few experiments have been performed to test directly the possible role of this energy source on the chemical evolution of the primitive atmosphere, mainly on account of experimental difficulties. Experiments are generally performed with other excitations, mainly electric discharge, and it is frequently assumed that UV irradiation would give similar results.As theoretical considerations make this assumption questionable, direct experimental controls have been undertaken: Model primitive atmospheres have been submitted to 147 nm UV light and the gaseous phase has been analysed. Preliminary qualitative results concerning CH 4–NH 3 atmospheres are reported.Irradiation of pure CH 4 gives rise to the synthesis of a large number of hydrocarbons, mainly saturated hydrocarbons but including also unsaturated ones as, C 2H 2, C 2H 4, C 3H 6, C 3H 4. These insaturated hydrocarbons are synthetized at a very low rate when ammonia is present in the medium.Irradiations of CH 4–NH 3 mixtures give rise, in addition to hydrocarbons, to important amounts of HCN (about 0.1%) and to lesser amounts of CH 3CN and C 2H 5CN. No unsaturated nitriles such as acrylonitrile and cyanoacetylene have been detected. Search for amines is in progress.These results evidence that UV irradiation may contribute largely to synthesis of HCN in CH 4–NH 3 atmospheres and, consequently to the synthesis of many biochemical compounds that can be derivated from HCN. However, synthesis of other compounds, such as pyrimidines, which can derivate from other nitriles, such as cyanoacetylene, cannot be initiated only by UV light, contrary to electric discharges. In addition, if electric discharges are very efficient for synthesis of nitriles in CH 4–N 2 atmospheres, there is not yet evidence that UV light is able to do so.Presented at the 2nd ISSOL Meeting and the 5th ICOL in Kyoto, 5–10 April, 1977. 相似文献
10.
A new procedure for fluorescent detection of intracellular H 2O 2 in cells transiently expressing the catalyst Horseradish Peroxidase (HRP) is setup and validated. More specific reaction with HRP largely amplifies oxidation of the redox probes used (2′,7′-dichlorodihydrofluorescein and dihydrorhodamine). Expression of HRP does not affect cell viability. The procedure reveals MAO activity, a primary intracellular H 2O 2 source, in monolayers of intact transfected cells. The probes oxidation rate responds specifically to the MAO activation/inhibition. Their oxidation by MAO-derived H 2O 2 is sensitive to intracellular H 2O 2 competitors: it decreases when H 2O 2 is removed by pyruvate and it increases when the GSH-dependent removal systems are impaired. Specific response was also measured after addition of extracellular H 2O 2. Oxidation of the fluorescent probes following reaction of H 2O 2 with endogenous HRP overcomes most criticisms in their use for intracellular H 2O 2 detection. The method can be applied for direct determination in plate reader and is proposed to detect H 2O 2 generation in physio-pathological cell models. 相似文献
11.
The O-O-N-N-O-type pentadentate ligands H 3ed3a, H 3pd3a and H 3pd3p (H 3ed3a stands ethylenediamine- N, N, N′-triacetic acid; H 3pd3a stands 1,3-propanediamine- N, N, N′-triacetic acid and H 3pd3p stands 1,3-propanediamine- N, N, N′-tri-3-propionic acid) and the corresponding novel octahedral or square-planar/trigonal-bipyramidal copper(II) complexes have been prepared and characterized. H 3ed3a, H 3pd3a and H 3pd3p ligands coordinate to copper(II) ion via five donor atoms (three deprotonated carboxylate atoms and two amine nitrogens) affording octahedral in case of ed3a 3− and intermediate square-pyramidal/trigonal-bipyramidal structure in case of pd3a 3− and pd3p 3−. A six coordinate, octahedral geometry has been established crystallographically for the [Mg(H 2O) 6][Cu(ed3a)(H 2O)] 2 · 2H 2O complex and five coordinate square-pyramidal for the [Mg(H 2O) 5Cu(pd3a)][Cu(pd3a)] · 2H 2O. Structural data correlating similar chelate Cu(II) complexes have been used for the better understanding the pathway: octahedral → square-pyramidal ↔ trigonal- bipyramid geometry. An extensive configuration analysis is discussed in relation to information obtained for similar complexes. The infra-red and electronic absorption spectra of the complexes are discussed in comparison with related complexes of known geometries. Molecular mechanics and density functional theory (DFT) programs have been used to model the most stable geometric isomer yielding, at the same time, significant structural data. The results from density functional studies have been compared with X-ray data. 相似文献
12.
Even though a given mineral, for instance olivine, may contain only traces of dissolved H 2O, CO 2 and N 2 the gases which evolve from its surface during heating comprise (a) highly reduced molecules such as H 2, CH 4, C mH n and more complex hydrocarbons, HCN and other N-bearing compounds (b) oxidized species in various degrees of oxidation from formaldehyde and CO to oxygen. These gases evolve sequentially besides H 2O, CO 2 and possibly N 2, their relative amounts being controlled by experimental parameters such as the rate of heating. Preliminary indications of amino acids have been obtained by liquid extraction. The chemical complexity is a consequence of radical reactions between different solute species in the surface and the bulk of the mineral grains. Data for synthetic MgO and for mantle-derived olivine are presented. 相似文献
13.
Hydrophobic anions of dipicrylamine and of sodium tetraphenylborate have been employed as probes of interfacial dipole potential variations in lipid bilayer membranes. Systematic variation of dipole potentials has been achieved by introduction of compounds incorporating N + and B ? charge centers. Distribution of hydrophilic and and hydrophobic groups relative to these charge centers has been shown to control the orientation in the membrane/solution interface of the electric dipole moment formed by these centers. Thus triphenyl-[4-trimethylphenylammonium] borate orients with the B ? center, surrounded by phenyl groups, embedded in the membrane, while the smaller methylated N + center is directed toward the aqueous phases. This orientation has been confirmed using dipicrylamine probe ions. Results obtained in this system have been interpreted quantitatively using a previously developed model incorporating discrete charge effects. A second class of compounds, borane (T nAB) complexes having the generic formula (C nH ) 3N +B ?H 3, have also been synthesized for this study, using even-carbon alkyls ranging from ethyl to decyl. Molecular orientation of the complex is with the N + center and its associated alkyl groups directed into the membranes, while the protonated B ? center is directed toward the aqueous phases, as confirmed by use of tetraphenylborate ions as probes. 相似文献
15.
Members of the green fluorescent protein (GFP) family are widely used in experimental biology as genetically encoded fluorescent tags. Chromophores of GFP-like proteins share a common structural core: 3,5-dihydro-4 H-imidazol-4-one. This review covers synthetic approaches to 3,5-dihydro-4 H-imida-zol-4-ones, substituted at different positions. General, as well as specific methods, represented by single examples are considered. The most popular synthetic route to substituted 3,5-dihydro-4 H-imidazol-4-ones includes synthesis of azlactones, followed by transformation into N-acyldehydroamino acids and, finally, cyclization into target heterocycles. Accordingly, the review is divided into three parts: the first part covers syntheses of azlactones, the second part covers main approaches to N-acyldehydroamino acids, and in the third part we summarize cyclizations of N-acyldehydroamino acids, as well as all other approaches to 3,5-dihydro-4 H-imidazol-4-ones. 相似文献
16.
Studying the implication of hydrogen peroxide in biological processes in plants remains a challenge due to the current shortcomings of H 2O 2-responsive probes. The use of ContPY1, a new fluorescent probe, which is highly selective and sensitive for H 2O 2, was investigated. To validate the use of ContPY1 on plants, we have generated protocols employing cells suspensions and leaves, and measured specifically H 2O 2 production by plants using spectrofluorometry and microscopy. 相似文献
17.
The methodology, characteristics and application of the sensitive C 2H 2-C 2H 4 assay for N 2 fixation by nitrogenase preparations and bacterial cultures in the laboratory and by legumes and free-living bacteria in situ is presented in this comprehensive report. This assay is based on the N 2ase-catalyzed reduction of C 2H 2 to C 2H 4, gas chromatographic isolation of C 2H 2 and C 2H 4, and quantitative measurement with a H 2-flame analyzer. As little as 1 μμmole C 2H 4 can be detected, providing a sensitivity 10 3-fold greater than is possible with 15N analysis. A simple, rapid and effective procedure utilizing syringe-type assay chambers is described for the analysis of C2H2-reducing activity in the field. Applications to field samples included an evaluation of N2 fixation by commercially grown soybeans based on over 2000 analyses made during the course of the growing season. Assay values reflected the degree of nodulation of soybean plants and indicated a calculated seasonal N2 fixation rate of 30 to 33 kg N2 fixed per acre, in good agreement with literature estimates based on Kjeldahl analyses. The assay was successfully applied to measurements of N2 fixation by other symbionts and by free living soil microorganisms, and was also used to assess the effects of light and temperature on the N2 fixing activity of soybeans. The validity of measuring N2 fixation in terms of C2H2 reduction was established through extensive comparisons of these activities using defined systems, including purified N2ase preparations and pure cultures of N2-fixing bacteria. With this assay it now becomes possible and practicable to conduct comprehensive surveys of N2 fixation, to make detailed comparisons among different N2-fixing symbionts, and to rapidly evaluate the effects of cultural practices and environmental factors on N2 fixation. The knowledge obtained through extensive application of this assay should provide the basis for efforts leading to the maximum agricultural exploitation of the N2 fixation reaction. 相似文献
18.
Aromatic amines and nitroarenes are important antioxidants and intermediates in the synthesis of dyes, pesticides and plastics. In the present paper we introduce methods for the synthesis of deuterated standards: 3-[ 2H 8]aminofluoranthene, 3,3′-dimethyl-[ 2H 4]benzidine, [ 2H 4]benzidine, N′-acetyl-[ 2H 4]benzidine, 2,4-[ 2H 6]toluenediamine, 2,6-[ 2H 6]toluenediamine. These standards have been used for the quantification of haemoglobin adducts of diamines and polyaromatic amines. Haemoglobin was hydrolysed in 0.1 M sodium hydroxide and the hydrolysate extracted with dichloromethane. The extracts were derivatised with heptafluorobutyric anhydride and analysed by GC–MS with negative chemical ionisation. In one run up to 15 aromatic amines can be determined: 6-aminochrysene, 3-aminofluoranthene, 2-aminofluorene, 1-aminopyrene, benzidine, 3,3′-dichlorobenzidine, 3,3′-dimethoxybenzidine, 3,3′-dimethylbenzidine, 3,3′-methylenedianiline, 4,4′-methylenedianiline, N′-acetyl-benzidine, N′-acetyl-4,4′-methylenedianiline, 4,4′-methylene bis(2-chloroaniline), 2,4-toluenediamine and 2,6-toluenediamine. 相似文献
19.
Syntheses, structural studies from single-crystal X-ray diffraction and thermal behaviour of (C 4H 12N 2)[M II(H 2O) 6](SO 4) 2 with M II = Mn, Ni, Fe and Cu are reported. All compounds crystallise in monoclinic system, space group P2 1/ n. The two isotypical compounds (C 4H 12N 2)[Mn(H 2O) 6](SO 4) 2 ( I) and (C 4H 12N 2)[Ni(H 2O) 6](SO 4) 2 ( II), are isostructural with the related cobalt and zinc phases, while the isotypical sulfates (C 4H 12N 2)[Fe(H 2O) 6](SO 4) 2 ( III) and (C 4H 12N 2)[Cu(H 2O) 6](SO 4) 2 ( IV) belong to another structure type. The three-dimensional structure networks for the four compounds consist of isolated [M II(H 2O) 6] 2+ and (C 4H 12N 2) 2+ cations and (SO 4) 2− anions linked by hydrogen-bonds only. The thermal behaviour of the precursors has been studied by powder thermodiffractometry and thermogravimetric analyses. The first stages of dehydration are discussed with respect to the hydrogen bonds within the compounds. 相似文献
20.
BackgroundLife is a constant flow of electrons via redox couples. Redox reactions determine many if not all major cellular functions. Until recently, redox processes remained hidden from direct observation in living systems due to the lack of adequate methodology. Over the last years, imaging tools including small molecule probes and genetically encoded sensors appeared, which provided, for the first time, an opportunity to visualize and, in some cases, quantify redox reactions in live cells. Genetically encoded fluorescent redox probes, such as HyPer, rxYFP and roGFPs, have been used in several models, ranging from cultured cells to transgenic animals, and now enough information has been collected to highlight advantages and pitfalls of these probes. Scope of reviewIn this review, we describe the main types of genetically encoded redox probes, their essential properties, advantages and disadvantages. We also provide an overview of the most important, in our opinion, results obtained using these probes. Finally, we discuss redox-dependent photoconversions of GFP and other prospective directions in redox probe development. Major conclusionsFluorescent protein-based redox probes have important advantages such as high specificity, possibility of transgenesis and fine subcellular targeting. For proper selection of a redox sensor for a particular model, it is important to understand that HyPer and roGFP2-Orp1 are the probes for H 2O 2, whereas roGFP1/2, rxYFP and roGFP2-Grx1 are the probes for GSH/GSSG redox state. Possible pH changes should be carefully controlled in experiments with HyPer and rxYFP. General significanceGenetically encoded redox probes are the only instruments allowing real-time monitoring of reactive oxygen species and thiol redox state in living cells and tissues. We believe that in the near future the palette of FP-based redox probes will be expanded to red and far-red parts of the spectrum and to other important reactive species such as NO, O 2 and superoxide. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn. 相似文献
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