Chemiluminescent Probe Research Articles

Novel Mycotoxin from Acremonium exuviarum Is a Powerful Inhibitor of the Mitochondrial Respiratory Chain Complex III

A novel mycotoxin named acrebol, consisting of two closely similar peptaibols (1726 and 1740 Da), was isolated from an indoor strain of the mitosporic ascomycete fungus Acremonium exuviarum. This paper describes the unique mitochondrial toxicity of acrebol, not earlier described for any peptaibol. Acrebol inhibited complex III of the respiratory chain of isolated rat liver mitochondria (1 mg of protein mL(-1)) with an IC(50) of approximately 80 ng mL(-1) (50 nM) after a short preincubation, and 350 ng mL(-1) caused immediate and complete inhibition. Acrebol thus is a complex III inhibitor almost as potent as antimycin A and myxothiazol but completely different in structure. Similarly to myxothiazol but in contrast to antimycin A, acrebol decreased the level of mitochondrial superoxide anion detectable by chemiluminescent probe 3,7-dihydro-2-methyl-6-(4-methoxyphenyl)imidazol[1,2-a]pyrazine-3-one. Unlike other peptaibols, acrebol in toxic concentrations did not increase the ionic and solute permeability of membranes of isolated rat liver mitochondria, did not induce disturbance of the ionic homeostasis or the osmotic balance of mitochondria, and did not release apoptogenic proteins like cytochrome c from the intermembrane space of mitochondria. In boar spermatozoa, acrebol inhibited the respiratory chain and caused ATP depletion by activation of the oligomycin-sensitive F(0)F(1)-ATPase, which resulted in the inhibition of the progressive movement. In mouse insulinoma MIN-6 cells, whose energy supply solely depends on oxidative phosphorylation, acrebol induced necrosis-like death. The pathophysiological relevance of these findings is discussed.

A dicopper complex chemiluminescence probe for the determination of thiols in the extracts of murine P388 lymphocytic leukemia cell

A chemiluminescence (CL) probe of peroxidase-like dicopper complex (Cu2L2) for the determination of glutathione (GSH) and related cellular thiols was developed for the first time.

Self-cleavable chemiluminescent probes suitable for protease sensing

A new generation of dioxetane-based chemiluminescent substrates suitable for detecting protease activities is described. Our strategy involves the use of a self-cleavable spacer as the key molecular component of these protease-sensitive chemiluminescent probes. Among the assayed strategies, the PABA (para-aminobenzylic alcohol) linker associated with an ether linkage enables the release of the light-emitting phenolic 1,2-dioxetane moiety through an enzyme-initiated domino reaction. To validate this strategy, two proteolytic enzymes were chosen: penicillin amidase and caspase-3, and the corresponding self-cleavable chemiluminescent substrates were synthesised. Their evaluation using an in vitro assay has enabled us to prove the decomposition of the linker under physiological conditions and the selectivity for the targeted enzyme.

A novel luciferin-based bright chemiluminescent probe for the detection of reactive oxygen species

This communication reports the synthesis, chemiluminescence properties, and biological application of KEIO-BODIPY-imidazopyrazine (KBI), a yellow-green chemiluminescent probe for the detection of reactive oxygen species (ROS) generated from living cells.

Free tyrosine and tyrosine-rich peptide-dependent superoxide generation catalyzed by a copper-binding, threonine-rich neurotoxic peptide derived from prion protein

Previously, generation of superoxide anion (O(2)(*-)) catalyzed by Cu-binding peptides derived from human prion protein (model sequence for helical Cu-binding motif VNITKQHTVTTTT was most active) in the presence of catecholamines and related aromatic monoamines such as phenylethylamine and tyramine, has been reported [Kawano, T., Int J Biol Sci 2007; 3: 57-63]. The peptide sequence (corresponding to helix 2) tested here is known as threonine-rich neurotoxic peptide. In the present article, the redox behaviors of aromatic monoamines, 20 amino acids and prion-derived tyrosine-rich peptide sequences were compared as putative targets of the oxidative reactions mediated with the threonine-rich prion-peptide. For detection of O(2)(*-), an O(2)(*-)-specific chemiluminescence probe, Cypridina luciferin analog was used. We found that an aromatic amino acid, tyrosine (structurally similar to tyramine) behaves as one of the best substrates for the O(2)(*-) generating reaction (conversion from hydrogen peroxide) catalyzed by Cu-bound prion helical peptide. Data suggested that phenolic moiety is required to be an active substrate while the presence of neither carboxyl group nor amino group was necessarily required. In addition to the action of free tyrosine, effect of two tyrosine-rich peptide sequences YYR and DYEDRYYRENMHR found in human prion corresponding to the tyrosine-rich region was tested as putative substrates for the threonine-rich neurotoxic peptide. YYR motif (found twice in the Y-rich region) showed 2- to 3-fold higher activity compared to free tyrosine. Comparison of Y-rich sequence consisted of 13 amino acids and its Y-to-F substitution mutant sequence revealed that the tyrosine-residues on Y-rich peptide derived from prion may contribute to the higher production of O(2)(*-). These data suggest that the tyrosine residues on prion molecules could be additional targets of the prion-mediated reactions through intra- or inter-molecular interactions. Lastly, possible mechanism of O(2)(*-) generation and the impacts of such self-redox events on the conformational changes in prion are discussed.

Synthesis and properties of novel chemiluminescent biological probes: 2- and 3-(2-Succinimidyloxycarbonylethyl)phenyl acridinium esters

Five new chemiluminescent aryl acridiniumcarboxylate esters 6– 10, with a linker group in an ortho- or meta-position of the phenoxy ring, have been synthesized. The ortho- derivatives 6, 7 and 9 show significant improvement in the quantum yield of chemiluminescence and slower chemiluminescence kinetics compared to the unsubstituted para- derivative, 1, while the ortho- derivative 8 shows quicker chemiluminescence kinetics and a lower quantum yield of chemiluminescence. The meta- derivative 10 shows similar chemiluminescent properties to that of its para-substituted analogue.

Copper(II)−Alizarin Red S Complex as an Efficient Chemiluminescent Probe for the Detection of Human Serum Proteins after Polyacrylamide Gel Electrophoresis

A novel chemiluminescent probe, copper(II)-Alizarin Red S (ARS) complex, for the detection of human serum proteins after polyacrylamide gel electrophoresis (PAGE) is described. The detection is based on the binding of the copper(II)-ARS complex to proteins and the catalytic activity of copper(II) in the luminol-hydrogen peroxide chemiluminescence (CL) system. Various proteins are directly detected in polyacrylamide gels, avoiding tedious transferring procedures. In the present study, the possible reaction mechanism and sensitivity evaluation are analyzed. The experimental conditions such as solution concentration, complex ratio, and washing reagents are likewise optimized. The proposed method offers simple, fast, and sensitive detection of serum proteins. As a novel chemiluminescent detection method, it shows significant analytical potential in biochemistry.

Abstract 410: Mitochondrial Uncoupling with CCCP Reverses Acute Hyperglycemia Induced Endothelial Dysfunction in Human Arterioles

Background : Acute hyperglycemia (HG) impairs endothelial function, which may be related to hyperpolarization of the inner mitochondrial membrane and excessive production of mitochondrial superoxide (ROS). Mitochondrial uncoupling agents could potentially reverse endothelial dysfunction due to HG in intact human arterioles. Methods : Human adipose arterioles were incubated in either euglycemic (NG, 5mM) or hyperglycemic (HG, 33mM) buffer for 4 hours. Vessels were then exposed to increasing doses of acetylcholine (ACh) in the presence and absence of NADPH oxidase inhibitor gp91ds-tat or CCCP, (100 nM), a mitochondrial membrane uncoupling agent. Vascular superoxide levels were also measured after incubation with NG and HG using a chemiluminescent probe (L-012) in the absence and presence of 1 and 2 above. Results : ACh-induced vasodilation was impaired by HG vs. NG control (P<0.001). This impairment was modestly reversed by gp91 ds-tat (P< 0.001), but endothelial function was completely restored to the level observed under euglycemic conditions by CCCP (P=0.008 vs. HG, P>0.90 vs. NG). Endothelium-dependent vasodilation in HG vessels was greater after CCCP compared to gp91ds-tat (P<0.001). L-NAME inhibited all dilation to ACh indicating endothelium-dependence (data not shown). HG induced an increase in superoxide relative to NG (P<0.001), which was similarly suppressed by gp91ds-tat (P = 0.001) and CCCP (P=0.002). Conclusion : HG-induced endothelial dysfunction is reversed with partial uncoupling of mitochondrial oxidative phosphorylation. Mitochondrial uncoupling more than NADPH oxidase blockade improves endothelial dysfunction due to HG.

Vitamin C depletion increases superoxide generation in brains of SMP30/GNL knockout mice

Vitamin C (VC) has a strong antioxidant function evident as its ability to scavenge superoxide radicals in vitro. We verified that this property actually exists in vivo by using a real-time imaging system in which Lucigenin is the chemiluminescent probe for detecting superoxide in senescence marker protein-30 (SMP30)/gluconolactonase (GNL) knockout (KO) mice, which cannot synthesize VC in vivo. SMP30/GNL KO mice were given 1.5 g/L VC [VC(+)] for 2, 4, or 8 weeks or denied VC [VC(−)]. At 4 and 8 weeks, VC levels in brains from VC(−) KO mice were <6% of that in VC(+) KO mice. Accordingly, superoxide-dependent chemiluminescence levels determined by ischemia-reperfusion at the 4- and 8 weeks test intervals were 3.0-fold and 2.1-fold higher, respectively, in VC(−) KO mice than in VC(+) KO mice. However, total superoxide dismutase activity and protein levels were not altered. Thus, VC depletion specifically increased superoxide generation in a model of the living brain.

Selective Production of Superoxide Ions and Hydrogen Peroxide over Nitrogen- and Sulfur-Doped TiO2Photocatalysts with Visible Light in Aqueous Suspension Systems

Superoxide ions (O2•−) and hydrogen peroxide (H2O2) produced by visible light irradiation were analyzed to investigate the photocatalytic reaction mechanism for commercially available nitrogen- and sulfur-doped TiO2 (BAPW25 and SJC). O2•− was detected by the chemiluminescence (CL) probe method with luminol, while the amount of H2O2 was measured as the CL intensity by the reaction with the luminol oxidized upon the addition of hemoglobin. By irradiation with 442-nm light, the S-doped TiO2 surpassed the N-doped TiO2 in the ability to produce O2•−, while the N-doped TiO2 surpassed the S-doped TiO2 in accumulating H2O2. The difference in the photocatalytic processes was attributed to the multivalence property of sulfur atoms which may promote the decomposition of H2O2.

Hydrogen-rich pure water prevents superoxide formation in brain slices of vitamin C-depleted SMP30/GNL knockout mice

Hydrogen is an established anti-oxidant that prevents acute oxidative stress. To clarify the mechanism of hydrogen’s effect in the brain, we administered hydrogen-rich pure water (H 2) to senescence marker protein-30 (SMP30)/gluconolactonase (GNL) knockout (KO) mice, which cannot synthesize vitamin C (VC), also a well-known anti-oxidant. These KO mice were divided into three groups; recipients of H 2, VC, or pure water (H 2O), administered for 33 days. VC levels in H 2 and H 2O groups were <6% of those in the VC group. Subsequently, superoxide formation during hypoxia-reoxygenation treatment of brain slices from these groups was estimated by a real-time biography imaging system, which models living brain tissues, with Lucigenin used as chemiluminescence probe for superoxide. A significant 27.2% less superoxide formed in the H 2 group subjected to ischemia–reperfusion than in the H 2O group. Thus hydrogen-rich pure water acts as an anti-oxidant in the brain slices and prevents superoxide formation.

Thermal ageing of poly(ethylene oxide)/poly(3,4-ethylenedioxythiophene) semi-IPNs

The thermal stability study of a conducting semi-IPN has been reported. The thermo-oxidation of poly(ethylene oxide) (PEO)/poly(3,4-ethylenedioxythiophene) (PEDOT) semi-Interpenetrating Polymer Network (semi-IPN) was studied at 80 °C in open air. The degradation was followed by spectrophotometry in the visible and near infrared range, cyclic voltamperometry and thermogravimetric analysis. Fluorescence spectrophotometry allowed for the identification of OH by-product originated in the PEO network degradation by the use of a chemiluminescent probe, typically terephthalic acid. The formation of hydroxyl radicals damaged the PEDOT chains as checked by infrared spectroscopy. The mechanism of degradation is further confirmed (i) by introducing a radical scavenger or (ii) by performing a thermal ageing under inert atmosphere; in both cases the semi-IPN life-time is tremendously increased.

New Approaches to In-Cell Detection of Protein Activity: Genetically Encoded Chemiluminescence Probes Pave the Way to Robust HTS Assays

New genetically encoded biosensors utilizing the modified firefly luciferase promise a great improvement in the signal-to-noise ratio and the dynamic range of response in living cells. These biosensors are particularly suitable for high-throughput screening assays that use large-well-capacity formats because of their excellent response characteristics. The biosensor design strategies are highly generalizable and will be extremely valuable for expanding the repertoire of screenable targets in living cell systems.

A novel probe for chemiluminescent image detection of proteins in two‐dimensional gel electrophoresis

Carrier ampholytes were found to enhance the chemiluminescence (CL) emission from the 3-aminophthalic hydrazide (luminol)-hydrogen peroxide system. They can be used as a chemiluminescent probe for rapid detection of major proteins in gels. This probe attracted much interest due to its ability to attach proteins, and to the possibility to combine it with separation techniques generating the CL emission directly. Increased signal intensity was achieved employing optimized concentrations of the carrier ampholyte enhancer. The binding of carrier ampholyte to proteins was found to occur at the pI of the proteins. Proteins from different regions of the gels were identified by their matrix-assisted TOF mass spectra and by appropriate database search, the results illustrating the possibility of major protein detection in human serum. Direct CL image detection with the carrier ampholyte probe can be applied for the detection of characteristic proteins in patients, i.e., proteins which cannot be detected without the probe.

Effect of CoQ homologues on reactive oxygen generation by mitochondria

Effect of CoQ compounds (Qs) on reactive oxygen (ROS) generation by mitochondrial complex I was studied using rat liver mitochondria and chemiluminescence probe L012. Kinetic analysis revealed that short chain Qs, such as Q2 and idebenone enhanced ROS generation by mitochondrial NADH oxidase system by a succinate-inhibitable mechanism. Lipid peroxidation in mitochondrial membranes induced by NADH and iron was inhibited by short chain Qs. The inhibitory activity was enhanced by co-oxidation of succinate as determined by chemiluminescence method and by electron spin resonance spectroscopy. These results suggested that the reduced form of short chain Qs inhibited mitochondrial ROS generation and lipid peroxidation.

Lucigenin-enhanced chemiluminescence of animal tissues

Lucigenin-enhanced chemiluminescence (LcCL) allows one to investigate the reactions of superoxide anion radical (*O2-) generated by mitochondria and is applied to study the superoxide production in enzymatic and membrane systems by isolated mitochondria and cells, and in whole organs. The application of lucigenin-enhanced chemiluminescence to estimate the respiration of human tissues involves the use of small tissue pieces, which can be obtained, for instance, by biopsia; however, no systematic investigations have been performed on these objects. In the present paper, a comparative study of lucigenin-enhanced chemiluminescence of tissues isolated from different organs of the rat was carried out to elucidate its dependence on the extent of tissue defragmentation, storage time, and access for oxygen. It was shown that the addition of lucigenin to a piece of tissue, a suspension of fine tissue fragments, and homogenates greatly enhanced chemiluminescence, and a whole piece of tissue possessed a much lesser (by 1-1.5 order of magnitude) intensity of chemiluminescence than homogenate or gruel. In the absence of stirring of the surrounding solution, the lucigenin-enhanced chemiluminescence of tissue quickly decreased, apparently due to a decrease in the level of oxygen in the tissue, as the result of its consumption. The chemiluminescence consisted of two components: a lucigenin-dependent and lucigenin-independent one (intrinsic chemiluminescence). Thus, the tissue was a source of lucigenin-enhanced chemiluminescence, and this luminescence was observed only at a sufficient access for oxygen. The lucigenin-independent component did not practically depend on oxygen and was determined by the components coming out of the tissue into the surrounding solution. Nitric oxide (NO) inhibited chemiluminescence as its concentration increased and did not affect considerably the rate of oxygen consumption by the tissue. The results obtained allow one to conclude that lucigenin can be used as a rather effective chemiluminescent probe for the production of superoxide radicals by tissue pieces.

On the identification of helical instabilities in a reacting swirling flow

An experimental study of unsteady reacting flow in a lean premixed swirl-stabilized combustor has been performed using a high speed camera, LDV, chemiluminescence and acoustic probes. To discern the role of fluid mechanics instability, isothermal and reacting (propane burning) tests were carried out. A distinct modulation of the combustion process by the hydrodynamical instability was revealed. It was confirmed that the flow unsteadiness is attributed to the precessing left-handed spiral (helical) vortical structure. The abnormal effect of a non-monotonic evolution of precession frequency vs. swirl number, with a region of inverted (decreasing) dependence, was observed regardless of the flow conditions (isothermal/reacting, confined/unconfined). The temporal frequency of the helical mode was treated as a superposition of two terms accounting for its pure rotation and axial translation but weighted with coefficients which are azimuthal and axial wavenumbers, respectively. Using cross-correlation measurements, the impact of swirl was confirmed to increase the helical structure’s axial wavelength. This effect naturally yields the observed decreasing trend in the frequency curve due to the second contributor.

High-Throughput Automated Luminescent Magnetic Particle-Based Immunoassay to Monitor Human Exposure to Pyrethroid Insecticides

We have developed a sensitive, automated, competitive chemiluminescent immunoassay for the detection of 3-phenoxybenzoic acid (3-PBA), a metabolite common to many pyrethroid insecticides. The system uses a competitive hapten-protein conjugate that has been labeled with an acridinium ester as the chemiluminescent probe and secondary antibody-coated paramagnetic particles for the separation. After the immunoassay reagents and samples are combined for the competitive incubation step, a fully automated system is used to load the postincubation mixture into a delivery cuvette, facilitating the subsequent magnetic separation of the immunocomplex and the measurement of chemiluminescent signal for quantification. The immunoassay format described here supports the requirement for high throughput necessary for monitoring large numbers of samples in population-based studies. The optimized immunoassay was more sensitive than the conventional enzyme immunoassay in buffer (IC(50) = 0.1 and 2 microg/L, respectively). The mixed-mode solid-phase extraction used for sample preparation to reduce possible urinary matrix effects allowed the accurate measurement of 3-PBA levels as low as 1 microg/L. The automated chemiluminescent immunoassay described here is sensitive, simple to use, and more rapid than the previously reported standard microplate assay.

Visualization and characterization of UVB-induced reactive oxygen species in a human skin equivalent model

Reactive oxygen species (ROS) play important roles in the process of ultraviolet-induced skin damage or photoaging. Although many enzymatic and chemical methods have been developed for evaluating ROS, evaluation methods for ROS generation in living systems are quite limited. Here we propose a unique system to visualize UVB-induced ROS and investigate the biological impact of ROS. In brief, a human skin equivalent model (HSEM) was exposed to UVB. Emitted luminescence from the HSEM was visualized and semi-quantified by using a chemiluminescent probe (CLA) and an ultra low-light imaging apparatus. The effects of anti-oxidative compounds such as ascorbate, beta-carotene, superoxide dismutase (SOD), and yeast ferment filtrate (YFF) on the HSEM were evaluated by semi-quantification of emitted chemiluminescence (CL) intensities, MTT assay and 8-hydroxy-2'-deoxyguanosine (8-OHdG) staining. Visualization of time- and space-dependent dynamics of ROS generation in the HSEM was successfully achieved by utilizing a sensitive two-dimensional ultra-low light luminograph. Treatments with beta-carotene and SOD effectively suppressed CL intensity, indicating the generation of 1O2 and O2 .- in the HSEM under UVB exposure. Tested anti-oxidative compounds also attenuated UVB-induced CL and ameliorated the induced skin damages in terms of 8-OHdG formation and cell death. As a conclusion, this model is useful for not only visualizing the production of UVB-induced ROS in real-time but also evaluating the efficacy of topically applied anti-oxidative compounds to suppress ROS generation and attenuate sequential chemical and biological responses.

On the Effect of 1,4-Diazabicyclo[2.2.2]octane on the Singlet-Oxygen Dimol Emission: Chemical Generation of (1O2)2 in Peroxide Reactions

The acetone-catalyzed decomposition of monoperoxysulfate ions, the molybdate ion-induced decay of hydrogen peroxide, and the reactions of N-chlorosuccinimide or N-bromosuccinimide with hydrogen peroxide and of dimethyldioxirane with tertiary amines as well as the thermal decomposition of 1,4-dimethylnaphthalene endoperoxide lead to the chemiluminescence of singlet-oxygen dimol species (1O2)2 emitting at 634 and 703 nm. In contrast to the expected enhancement of (1O2)2 chemiluminescence upon addition of 1,4-diazabicyclo[2.2.2]octane (DABCO) [Deneke, C.F.; Krinsky, N. I. J. Am. Chem. Soc. 1976, 98, 3041. Di Mascio, P.; Sies, H. J. Am. Chem. Soc. 1989, 111, 2909.], quenching has been observed. Our data show that enhancement of singlet-oxygen dimol chemiluminescence is not a general phenomenon and, consequently, DABCO is not a reliable chemiluminescent probe for the presence of (1O2)2 in chemical and biochemical systems.