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Bacterial infection is a life-threatening situation, and its rapid diagnosis is essential for treatment. Apart from medical applications, rapid identification of bacteria is vital in the food industry or the public health system. There are various bacterial identification techniques, including molecular-based methods, immunological approaches, and biosensor-based procedures. The most commonly used methods are culture-based methods, which are time-consuming. The objective of this study is to find a fingerprint of bacteria to identify them. Three strains of bacteria were selected, and seven different concentrations of each bacterium were prepared. The bacteria were then treated with two different molar concentrations of the fluorescent fluorophore, dichlorodihydrofluorescein diacetate for 30 minutes. Then, using the fluorescence mode of a multimode reader, the fluorescence emission of each bacterium is scanned twice during 60 minutes. Plotting the difference between two scans versus the bacteria concentration results in a unique fluorescence pattern for each bacterium. Observation of the redox state of bacteria, during 90 minutes, results in a fluorescence pattern that is clearly a fingerprint of different bacteria. This pattern is independent of fluorophore concentration. Mean Squares Errors (MSE) between the fluorescence patterns of similar bacteria is less than that of different bacteria, which shows the method can properly identify the bacteria. In this study, a new label-free method is developed to detect and identify different species of bacteria by measuring the redox activity and using the fluorescence fluorophore, dichlorodihydrofluorescein diacetate. This robust and low-cost method can properly identify the bacteria, uses only one excitation and emission wavelength, and can be simply implemented with current multimode plate readers. • The pattern of the redox state of bacterium is a fingerprint of that bacterium. • The mechanism of H2-DCF-DA conversion depends on the redox state of the bacteria and its enzymes. • Using H2-DCF-DA and scanning the sample twice, results in a fingerprint of each bacterium. [ABSTRACT FROM AUTHOR]

—Action potential (AP) of excitable plant cells is an important signaling event that can differentially alter physicochemical and physiological processes in various parts of the same cell. In giant cells of characean algae, the AP propagation has minor effect on photosynthetic electron transport in areas with high activity of plasmalemmal H+-pump but inhibits linear electron flow in regions featuring high passive H+/OH− conductance of the plasma membrane (PM). Uneven spatial distributions of local periplasmic and cytoplasmic pH facilitate the operation of distinct (CO 2 -dependent and O 2 -mediated) pathways of photoinduced electron flow, which presumably accounts for differential influence of AP on photosynthesis. The excitation of Chara australis cell in the presence of methyl viologen (MV), a redox mediator with the prooxidant action, provides a convenient model system to clarify the influence of voltage-dependent ion fluxes across PM on photosynthetic activity of chloroplasts. This study shows that permeation of MV to their target sites in chloroplasts is restricted by PM in resting cells, but MV easily passes through ionic channels opened during the PM depolarization. This gated permeation of MV gives rise to strong non-photochemical quenching, decrease in the effective quantum yield of linear electron flow, apparent O 2 uptake, and, finally, the enhanced ROS production, as detected by the fluorescent probe dichlorofluorescein. Taken together, the results indicate that the AP generation in the presence of MV acts as trigger for instant redirection of photosynthetic linear electron flow from CO 2 -dependent route to the path of O 2 reduction with the eventual formation of H 2 O 2 as a dominant and most stable ROS form. • Excitation of Chara cell opens membrane channels permeable to methyl viologen (MV2+). • Entry of MV upon excitation redirects electron flow and quenches the F m ′ fluorescence. • Inhibition of thylakoid ΔpH by nigericin mitigated the F m ′ quenching upon cell excitation. • Switching of linear electron flow to MV-mediated path is further evidenced by O 2 uptake. • Cell excitation in the presence of MV promotes ROS accumulation after a lag period. [ABSTRACT FROM AUTHOR]

Depletion of reactive oxygen species and reduction of oxidative stress have been identified as key parameters in the prevention of cellular aging. In previous in vitro studies, the tea catechin epigallocatechin gallate (EGCG) was found to have both pro- and antioxidant properties, disregarding the low stability under cell culture conditions. Besides hydrogen peroxide, theasinensin dimers amongst other oxidation products are formed. Exact quantities, cellular uptake and antioxidant capacities of these dimeric oxidation products remain unknown. Via high-performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (MS/MS), formation kinetics and cellular uptake of EGCG and its major oxidation products are quantified. The antioxidant capacity is determined on a cellular level using a modified dichlorofluorescein (DCF) approach. As a first result, oxidation product quantities of up to 21 µM each are measured after incubation of 50 µM EGCG. While EGCG is taken up equimolarly, its major oxidation products are accumulated in hepatocarcinoma HepG2 cells at millimolar concentrations, especially theasinensin A (TSA). Lastly, the oxidation products show higher antioxidant properties than the monomer EGCG. In correlation with cellular uptake, TSA displays the highest capacity of all tested analytes. The findings reveal the strong influence of EGCG oxidation products on its bioactivity in vitro. [ABSTRACT FROM AUTHOR]

Reactive oxygen species (ROS) are under scrutiny as a participant in the pathophysiology of myelodysplastic syndrome (MDS) and the progression of MDS to acute myeloid leukemia (AML). Measurement of intracellular ROS (iROS) is particularly important since iROS is a direct indicator of cellular health and integrity. We developed a technique to measure standardize iROS (siROS) level in lymphocytes and bone marrow (BM) CD34+ hematopoietic progenitors using the fluorescent probe dichlorofluorescein (DCF). We then quantified the siROS in 38 consecutive BM specimens from 27 MDS patients over the course of 10 months. Disease outcome of these patients were also assessed. High serum ferritin, high blast count and poor IPSS were associated with inferior survival and AML progression in this cohort. High blast MDS patients had lower siROS in their BM CD34+ cells than those of low blast patients, consistent with increased reliance on glycolysis and enhanced ROS defense in high blast MDS. We also observed narrower siROS distribution in the BM CD34+ cells of high blast patients, suggesting that loss of heterogeneity in ROS content accompanies the clonal evolution of MDS. Furthermore, we observed a strong correlation between CD34+ cells siROS and serum ferritin level in high blast patients. In one case, iron chelation therapy (ICT) resulted in parallel decreases in serum ferritin and CD34+ cells siROS. Our findings established the siROS profile in early hematopoietic cells of MDS patients and its relationship with blast count and iron overload. [ABSTRACT FROM AUTHOR]

Depletion of reactive oxygen species and reduction of oxidative stress have been identified as key parameters in the prevention of cellular aging. In previous in vitro studies, the tea catechin epigallocatechin gallate (EGCG) was found to have both pro- and antioxidant properties, disregarding the low stability under cell culture conditions. Besides hydrogen peroxide, theasinensin dimers amongst other oxidation products are formed. Exact quantities, cellular uptake and antioxidant capacities of these dimeric oxidation products remain unknown. Via high-performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (MS/MS), formation kinetics and cellular uptake of EGCG and its major oxidation products are quantified. The antioxidant capacity is determined on a cellular level using a modified dichlorofluorescein (DCF) approach. As a first result, oxidation product quantities of up to 21 µM each are measured after incubation of 50 µM EGCG. While EGCG is taken up equimolarly, its major oxidation products are accumulated in hepatocarcinoma HepG2 cells at millimolar concentrations, especially theasinensin A (TSA). Lastly, the oxidation products show higher antioxidant properties than the monomer EGCG. In correlation with cellular uptake, TSA displays the highest capacity of all tested analytes. The findings reveal the strong influence of EGCG oxidation products on its bioactivity in vitro.

The inherent biochemical, anatomical and physiological characteristics of the brain make it especially vulnerable to insult. Specifically, some of these characteristics such as myelin and a high energy requirement provide for the introduction of free radical-induced insult. Recently, the biochemistry of free radicals has received considerable attention. It also has become increasingly apparent that many drug and chemical-induced toxicities may be evoked via free radicals and oxidative stress. Major points addressed in this work are the regulation of neural free radical generation by antioxidants and protective enzymes, xenobiotic-induced disruption of cerebral redox status, and specific examples of neurotoxic agent-induced alterations in free radical production as measured by the fluorescent probe dichlorofluorescein. This article considers the thesis that free radical mechanisms may contribute significantly to the properties of several diverse neurotoxic agents and proposes that excess production of free radicals may be common phenomena of neurotoxicity.

A new postchemiluminescence (PCL) reaction was observed when urea was injected into the reaction mixture after the CL reaction of N-bromosuccinimide and dichlorofluorescein. A possible reaction mechanism was proposed based on the studies of the CL kinetic characteristics, CL spectra, fluorescence spectra, and other experiments. A new flow injection CL method for the determination of urea was established based on the PCL reaction. The relative standard deviation for the determination of urea was 1.3% (n = 11, c = 5.0 x 10-8 g/mL). The CL intensity responded linearly to the concentration of urea in the range 2.0 x 10-9-1.0 x 10-6 g/mL (r = 0.9992). The detection limit was 7 x 10-10 g/mL. The method had been applied to the determination of urea in milk with satisfactory results. [ABSTRACT FROM AUTHOR]

A novel hypochlorite-selective signaling probe based on the oxidative transformation of phenylselenylated 2′,7′-dichlorofluorescein 1 to its selenoxide analogue was investigated. In a pH 7.0 phosphate buffer system (containing 1% DMF as a solubilizer), the designed probe 1 exhibited pronounced colorimetric and fluorogenic hypochlorite signaling behavior over other representative oxidants such as hydrogen peroxide, tert -butyl hydroperoxide, perborate, and percarbonate as well as a range of environmentally relevant metal ions and anions. The signaling was due to the hypochlorite-assisted oxidation of the probe’s selenyl moiety to selenoxide. Hypochlorite signaling was unaffected by the presence of common metal ions and anions as a background. The hypochlorite-selective fluorescence signaling of probe 1 has a detection limit of 3.9 × 10 −8 M (0.002 ppm). Finally, the determination of hypochlorite concentration in tap water using a smartphone as a stand-alone portable signaling detection and processing tool was successfully demonstrated. [ABSTRACT FROM AUTHOR]

Highly selective chemosignaling behavior of thiophosphinated dichlorofluorescein towards fluoride ions was investigated. Prominent chromogenic and fluorescence turn-on type signaling was realized by employing fluoride-selective cleavage of the latent thiophosphinated probe in mixed aqueous media. The signaling process was confirmed by the UV–vis, fluorescence measurements as well as 1 H and 31 P NMR spectroscopy. Possible interferences from Hg 2+ and Cu 2+ ions were readily suppressed by using diethylenetriaminepentaacetic acid as a masking agent. With the aid of the masking agent, the probe also showed potentially useful sensing ability for the determination of fluoride ions in practical samples of tap water and simulated wastewater. The detection limit of the probe in the determination of fluoride ions was 9.8 nM. [ABSTRACT FROM AUTHOR]

The expression of phospholipase D1 (PLD1) and PLD2 were found to decrease at the transcription level during both replicative and premature senescence in human lung fibroblast IMR‐90 cells. Knockdown of PLD2 dramatically induced senescent phenotype in proliferating IMR‐90 cells and wild‐type HCT116 colon cancer cells, whereas this response was nearly abolished in p53‐ or p21Cip1/WAF1‐null HCT116 cells. PLD2 knockdown increased the intracellular reactive oxygen species (ROS). Antioxidant N‐acetyl‐l‐cysteine, NADPH oxidase inhibitor apocynin, and p22phox small interfering RNA (siRNA) reduced ROS generation and thus suppressed the appearance of senescence markers. Elevated CK2 α subunit (CK2α) expression repressed PLD2 downregulation‐mediated senescence. PLD2 overexpression increased protein kinase CK2 (also known as casein kinase 2) (CK2) activity. Taken together, these results show that PLD2 downregulation causes senescence through the p53–p21Cip1/WAF1 pathway by stimulating ROS production, which is induced by CK2 inhibition.PLD1 and PLD2 expression apparently decreased during replicative and premature senescence in IMR‐90 cells. PLD2 knockdown induced premature senescence in IMR‐90 and HCT116 cells. PLD2 knockdown increased the levels of p53 and reactive oxygen species. Elevated CK2 activity almost suppressed PLD2 downregulation‐mediated senescence. Thus, PLD2 downregulation promotes senescence through a ROS–p53 pathway, which is induced by CK2 inhibition. [ABSTRACT FROM AUTHOR]

Abstract: Background: Small molecule fluorescent probes are vital tools for monitoring reactive oxygen species in cells. Scope of review: The types of probe available, the extent to which they are specific or quantitative and complications in interpreting results are discussed. Major conclusions: Most commonly used probes (e.g. dihydrodichlorofluorescein, dihydrorhodamine) have some value in providing information on changes to the redox environment of the cell, but they are not specific for any one oxidant and the response is affected by numerous chemical interactions and not just increased oxidant generation. These probes generate the fluorescent end product by a free radical mechanism, and to react with hydrogen peroxide they require a metal catalyst. Probe radicals can react with oxygen, superoxide, and various antioxidant molecules, all of which influence the signal. Newer generation probes such as boronates act by a different mechanism in which nucleophilic attack by the oxidant on a blocking group releases masked fluorescence. Boronates react with hydrogen peroxide, peroxynitrite, hypochlorous acid and in some cases superoxide, so are selective but not specific. They react with hydrogen peroxide very slowly, and kinetic considerations raise questions about how the reaction could occur in cells. General significance: Data from oxidant-sensitive fluorescent probes can provide some information on cellular redox activity but is widely misinterpreted. Recently developed non-redox probes show promise but are not generally available and more information on specificity and cellular reactions is needed. We do not yet have probes that can quantify cellular production of specific oxidants. 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. [Copyright &y& Elsevier]

The article presents a study on the promising approach of gene therapy in the treatment of diseases using chitosan (CS) and its derivatives as cationic polymeric carriers for gene delivery. The researchers have used a range of techniques to monitor the self-assembly process including a gel electrophoresis of carcinogenic ethidium bromide. They have developed a green and low-cost alternative method using inexpensive dichlorofluorescein (DCF) for the estimation of CS and natural cationic polymer.

ABSTRACT A rapid and sensitive flow-injection chemiluminescence (FI-CL) method is described for the determination of diazepam based on its reaction with N-bromosuccinimide (NBS) in alkaline medium in the presence of dichlorofluorescein (DCF) as an effective energy-transfer agent. Under optimum conditions, the proposed method allowed the measurement of diazepam over the range of 2.0 × 10−6 to 2.0 × 10−4 mol/L with a detection limit of 5.0 × 10−7 mol/L. The relative standard deviation for 11 parallel measurements of 2.0 × 10−5 mol/L diazepam was 2.1%. The method was applied satisfactorily for the determination of diazepam in pharmaceutical preparations, and the results agree well with those obtained by spectrophotometry. The use of the proposed system for the determination of diazepam in urine and plasma samples was also tested. The possible mechanism of the chemiluminescence reaction is discussed briefly. Copyright © 2012 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Abstract: A new method was developed for the estimation of polyethylenimine (PEI) to nucleic acid ratio at which the polyplex was completely formed. The assay relied on the attraction of dichlorofluoresceinate dye to adsorb on self-assembling particles as counterions, as induced by the surface charge of the polyplex which became positive once PEI associated equivalently with nucleic acid. This phenomenon resulted in the appearance of pink colored pellets of the polyplex after centrifugation. By the other means, sodium hydroxide solution might be added to free the adsorbed dye into the solution, producing conspicuous green fluorescence under UV light (366nm). The assay was well applied to the polyplex formulations of PEI and plasmid DNA or siRNA with satisfactory detectability and gave the results in agreement with those from gel retardation method and zeta potential analysis. Importantly, the proposed method required no sophisticated instruments, time-consuming gel electrophoresis, carcinogenic ethidium bromide as well as costly dyes and the analysis could be accomplished within less than 10min. Hence, it was a fast, facile, cost-effective and safe-for-operator alternative method, suited for the investigation of the optimal PEI to nucleic acid ratio for gene delivery. [Copyright &y& Elsevier]

Highlights: [•] A novel fluorescence quenching method for the determination of torasemide is established. [•] A sensitive, simple and quick method has been developed for determining torasemide. [•] Some dihalogenated fluorescein dyes are used as fluorescence probes. [•] The fluorescence quenching of dihalogenated fluorescein dyes by torasemide is a static quenching process. [ABSTRACT FROM AUTHOR]

Summary: Photodynamic therapy (PDT), mediated by a sensitizer exposed to light to produce singlet oxygen (1O2), induces tumor responses varying from one person to another. Cancer growth induces oxidative stress at any step of its development from induction to treatment, which could also modify response to PDT. After the initial amount of 1O2 delivered, secondary oxidative species (SOS) are also generated inducing additional damages. Using an in vitro assay we saw variations among mice strains concerning their serum capability to generate SOS after 1O2 production. Nude mice had a higher capability to generate SOS as compared to the non mutated strain. Capability to generate SOS evolved during growth of orthotopically-grafted pulmonary cancers (A549), with either values corrected for hemolysis or not. Immediately after graft SOS production decreased, then increased again, reaching a plateau phase after 10 days which lasted for 20 days and finally increased steeply during the last phase of tumor growth, preceding cachexia and death. This profile differed profoundly from the one observed after heterotopic tumor grafts for which hemolysis induced artifacts masking important variations in SOS production. Our results demonstrate experimentally a relationship between the general health status of an individual, cancer progression and serum capability to generate SOS during PDT. These findings could explain some PDT failures as well as some unexpected successes on large tumors and should be taken into account when determining treatment parameters. They may also explain why different effects are observed on different experimental models with similar sensitizers. [Copyright &y& Elsevier]

ABSTRACT Exposure to air pollution leads to adverse pulmonary and systemic vascular effects. High levels of plasma high-density lipoproteins (HDL) cholesterol reduce cardiovascular risk. We explored whether HDL could protect against the prooxidative effects of an organic extract of diesel exhaust particles (DEP) in vascular cells. We used a cell-free fluorescent assay to evaluate DEP oxidation by air, estimated by the degree of dichlorofluorescein (DCF) fluorescence and tested the ability of HDL to inhibit it. We also evaluated DEP prooxidative effects in bovine aortic endothelial cells and RAW264.7 macrophages by DCF fluorescence. DEP oxidation and prooxidative cellular effects occurred in concentration- and time-dependent manners. Normal HDL inhibited DEP oxidation and prooxidative cellular effects, whereas dysfunctional HDL failed to inhibit DEP oxidation and instead, it promoted further oxidation. In conclusion, DEP prooxidative effects in endothelial cells and macrophages are inhibited by normal HDL. Therefore, HDL may protect against air pollution mediated adverse vascular effects. © 2013 Wiley Periodicals, Inc. J BiochemMol Toxicol 27:172-183, 2013; View this article online at wileyonlinelibrary.com. DOI 10.1002/jbt.21475 [ABSTRACT FROM AUTHOR]

Abstract: A strong post chemiluminescence (PCL) phenomenon was observed when ammonium was injected into the reaction mixture after the finish of CL reaction of N-bromosuccinimide (NBS) and dichlorofluorescein. Based on this, a sensitive flow injection PCL method was established for the determination of ammonium. The possible CL mechanism of the reaction was proposed based on a series of experiments. The PCL intensity responded linearly to the concentration of ammonium in the range 3.0×10−11–1.0×10−7 gmL−1 with a detection limit of 1×10−11 gmL−1. The relative standard deviation (R.S.D.) was 1.4% for 1.0×10−9 gmL−1 ammonium (n =11). This method had been applied to the determination of ammonium in samples of mineral water, tap water and river water. [Copyright &y& Elsevier]

Abstract: Singlet oxygen (1O2), produced during photodynamic therapy, deactivates during its interaction with tissues by producing reactive oxygen species (ROS) and peroxides as well as other degradation products. Here we investigated the role of parameters of light delivery, O2, and temperature on the ROS and peroxides production, secondary to 1O2. A series of simple in vitro experiments has been performed with Rose Bengal (RB) as a 1O2 producer, human serum (HS) as a target and dichlorofluorescein (DCFH) as a nonspecific marker, becoming fluorescent when oxidized. The overall secondary production of ROS and peroxides in HS had also been compared to fetal calf serum (FCS) or mice sera. Increasing power but with a same delivered energy decreased secondary ROS and peroxides when increasing power with a same duration for light delivery increased them. Increasing delivered energy increased linearly secondary ROS or peroxides. Delivering O2 by bubbling before light delivery increased secondary ROS or peroxides, when Ar decreased them. Delivering gases after light delivery had no influence on secondary ROS or peroxides production. Increasing temperature from 20 to 40°C increased secondary ROS or peroxides production but freezing after light delivery either before or after measurement had only a mild influence. Secondary ROS or peroxides production was 2 or 4 times lower in HS than in FCS or nude mice sera respectively. PDT seems to consist of two subsequent phases, both linked but developing independently. The intensity of photo-reactions varied with the model, human sera producing less secondary ROS than fetal calf or mouse sera. Search for new sensitizers should consider secondary ROS-induced pathways in addition to 1O2 production. [Copyright &y& Elsevier]

A flow injection technique combined with a chemiluminescence method was established for the determination of bovine serum albumin (BSA). Strong chemiluminescence was observed when BSA-dichlorofluoresce (DCF) complex was oxidized by sodium hypochlorite (NaClO) in an alkaline medium and in the presence of cetyltrimethylammonium bromide (CTAB). The reaction conditions of the chemiluminescence were carefully optimized. Under the optimal conditions, the method had a linear range of 0.01-20.0 μg/mL, with a detection limit of 0.007 μg/mL for BSA (3σ). The relative standard deviation of 1.0 μg/mL BSA (n = 8) is 1.4%. The method was applied to determine BSA in milk samples and it worked well. [ABSTRACT FROM AUTHOR]

Dichlorofluorescein derivatives with two aza-crown ether binding units were prepared by the Mannich reaction and their chemosensing behaviors toward transition metal ions were investigated. An 18-crown-6 ether derivative exhibited pronounced Cu2+-selective fluorescence signaling, with selectivity over other common physiologically important alkali, alkaline earth and transition metal ions. The compound also displayed 1:1 complex formation with Cu2+ ion, with a detection limit of 2.9×10−6 M in an aqueous 50% DMSO solution at pH 7, showing that it may offer potential as a chemosensor for the detection of submillimolar Cu2+ ions in physiological environments. A 15-Crown-5 ether analogue also revealed selective Cu2+ signaling, although with somewhat diminished selectivity compared with its 18-crown-6 counterpart. [Copyright &y& Elsevier]

Abstract: Melatonin is a modified tryptophan with potent biological activity, exerted by stimulation of specific plasma membrane (MT1/MT2) receptors, by lower affinity intracellular enzymatic targets (quinone reductase, calmodulin), or through its strong anti-oxidant ability. Scattered studies also report a perplexing pro-oxidant activity, showing that melatonin is able to stimulate production of intracellular reactive oxygen species (ROS). Here we show that on U937 human monocytes melatonin promotes intracellular ROS in a fast (<1 min) and transient (up to 5–6 h) way. Melatonin equally elicits its pro-radical effect on a set of normal or tumor leukocytes; intriguingly, ROS production does not lead to oxidative stress, as shown by absence of protein carbonylation, maintenance of free thiols, preservation of viability and regular proliferation rate. ROS production is independent from MT1/MT2 receptor interaction, since a) requires micromolar (as opposed to nanomolar) doses of melatonin; b) is not contrasted by the specific MT1/MT2 antagonist luzindole; c) is not mimicked by a set of MT1/MT2 high affinity melatonin analogues. Instead, chlorpromazine, the calmodulin inhibitor shown to prevent melatonin–calmodulin interaction, also prevents melatonin pro-radical effect, suggesting that the low affinity binding to calmodulin (in the micromolar range) may promote ROS production. [Copyright &y& Elsevier]

Abstract: A post-chemiluminescence (PCL) phenomenon was observed when pentoxyverine citrate solution was injected into the reaction mixture after the finish of chemiluminescence (CL) reaction of N-chlorosuccinimide (NCS) and alkaline dichlorofluorescein. The possible reaction mechanism was proposed based on the studies of the CL kinetic characteristics, the CL spectra and the fluorescence spectra of some related substances. Based on the PCL reaction, a rapid and sensitive method for the determination of pentoxyverine citrate was established. The linear response range of this method was from 6.0×10−9 to 1.0×10−6 gmL−1 with a linear correlation coefficient of 0.9998. The relative standard deviation for 2.0×10−8 gmL−1 pentoxyverine citrate was 2.1% (n =11). The detection limit was 9×10−10 gmL−1. This method has been applied to the determination of pentoxyverine citrate in human plasma and pharmaceutical samples with the satisfactory results. [Copyright &y& Elsevier]

A method for the determination of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in macroscopic sections of vessels has been developed on the basis of the dichlorofluorescein (DCF) assay. DCF was measured by fluorescence in extracts of vessels. The main artifact of the method is the oxidation of dichlorodihydrofluorescein (DCFH2) which is released from vessels together with DCF during the extraction procedure. This problem was resolved by decreasing pH during the extraction. The optimal conditions and the time for aorta incubation with DCFH2-DA and for the extraction of DCF from aorta have been determined. The ROS/RNS production in different aorta segments and the dependence of ROS/RNS production on rat age have been studied. It was shown that thoracic aorta sections produced the same amounts of ROS/RNS and the intermediate between the thoracic and the abdominal aorta part produced ROS and RNS by 14% more than the thoracic aorta. It was found that ROS/RNS production in aorta increases with rat age: the doubling time of ROS/RNS production rate is 113 days from birth. [ABSTRACT FROM AUTHOR]

The oxidation of 2',7'-dichlorodihydrofluorescein (2',7'-dichlorofluorescin, DCFH) to a fluorescent product, 2',7'-dichlorofluorescein (DCF), is commonly used to quantitatively measure oxidative stress in cells using a fluorescence microplate reader. However, many cell lines tend to grow non-uniformly in the wells. This non-uniform distribution results in a high degree of variability in the fluorescence signal and decreases the precision of the method. Also, samples treated in large culture plates, dishes or flasks cannot be assayed directly in fluorescence microplate readers. This study reports an improved DCF assay method that lyses cells with DMSO/PBS (90% dimethyl sulphoxide/10% phosphate buffered saline). Oxidative stress was induced with either hydrogen peroxide or an hypoxia-reoxygenation treatment. Cell lysis with DMSO/PBS resulted in highly stable fluorescence signals in comparison to Triton X-100/PBS lysed cells. The precision of DCF fluorescence measurements of DMSO/PBS lysed cells was much better than for attached cells measured directly in 96-well plates. While DCF fluorescence in PBS was strongly quenched by albumin, no quenching occurred in DMSO/PBS. In conclusion this study describes a more convenient and accurate method for measuring cellular oxidative stress that also makes it possible to assay cells treated in large culture plates. [ABSTRACT FROM AUTHOR]

The effects of the administration of water soluble coenzyme Q_{10} (25 mg/kg per day) over 30 days, after 50 days feeding on a high-fat diet (3% lard + 1.3% cholesterol), were investigated in the plasma and liver mitochondria of rabbits. Results showed that this atherogenic diet enhanced lipid levels both in plasma and liver mitochondria, reduced plasma and mitochondrial concentrations of retinol and coenzyme Q_{10}, led to higher DNA damage in peripheral blood lymphocytes and reactive oxygen species concentration in liver mitochondria. The treatment of animals with coenzyme Q_{10} reduced (to the healthy group levels) lipid concentration in liver mitochondria with no effect on plasma lipids, increased mitochondrial levels of α-tocopherol, restored mitochondrial coenzyme Q_{10} and improved α-tocopherol levels in plasma. Moreover, coenzyme Q_{10} supplementation reduced mitochondrial reactive oxygen species levels and decreased DNA damage in peripheral blood lymphocytes. The findings suggest that antioxidant therapy with coenzyme Q_{10} may be used in the treatment of liver pathologies associated to the intake of high-fat, atherogenic, diets. [ABSTRACT FROM AUTHOR]

A post-chemiluminescence (PCL) was observed when loperamide hydrochloride solution was injected into the reaction mixture after the finish of CL reaction of alkaline N-Chlorosuccinimide (NCS) and dichlorofluorescein. Based on this phenomenon, a simple, sensitive and fast flow injection PCL method was established for the determination of loperamide hydrochloride. The possible mechanism for the PCL reaction was discussed via the investigation of the CL kinetic characteristics, the CL spectra, the fluorescence spectra. The PCL intensity responded linearly to the concentration of loperamide hydrochloride in the range 8.0×10-10 to 6.0×10-7 g · ml-1 with a linear correlation of 0.9995. The detection limit was 4×10-10 g · ml-1. The relative standard deviation was 2.4% for 4.0×10-8 g · ml-1 loperamide hydrochloride (n=11). This method has been applied to the determination of loperamide hydrochloride in human plasma and pharmaceutical samples with satisfactory results. [ABSTRACT FROM AUTHOR]

Abstract: Introduction: Type 2 diabetes mellitus shows a characteristic altered platelet function that can be due to several mechanisms such as oxidative stress. Hyperhomocysteinemia, considered as a risk factor for various arterial thrombosis, may have a role in generating oxidative damage, even if the pathogenic mechanisms are still not clear. In this report we aimed to determine the role of plasma homocysteine in inducing oxidative stress in type 2 diabetes mellitus. Materials and methods: The study was performed on a group of 34 males with type 2 diabetes and 36 healthy subjects matched for sex and age. Patients and healthy subjects were undergone to laboratory evaluation for plasma homocysteine levels and other metabolic parameters. In both groups of subjects platelet reactive oxygen species, nitric oxide and guanosine 3′,5′ cyclic monophosphate levels were measured. Moreover the reduced glutathione content in platelets of patients and of healthy subjects was assayed. Results: Plasma homocysteine levels were significantly increased in patients compared with healthy subjects. The basal level of reactive oxygen species was significantly higher in patients than in controls. In addition platelets of patients stimulated with thrombin produced more reactive oxygen species than healthy subjects ones. The nitric oxide, guanosine 3′,5′ cyclic monophosphate and reduced glutathione content were decreased in platelets of patients. Conclusions: As homocysteine stimulates oxidative stress and inhibits nitric oxide formation, hyperhomocysteinemia measured in type 2 diabetic patients, promoting platelet hyperactivity, could have a role in the atherogenic effects described in type 2 diabetes. [Copyright &y& Elsevier]

Purpose: To evaluate the protective effects of antioxidant agents against space radiation-induced oxidative stress in cultured human epithelial cells.Methods and Materials: The effects of selected concentrations of N-acetylcysteine, ascorbic acid, sodium ascorbate, co-enzyme Q10, alpha-lipoic acid, l-selenomethionine, and vitamin E succinate on radiation-induced oxidative stress were evaluated in MCF10 human breast epithelial cells exposed to radiation with X-rays, gamma-rays, protons, or high mass, high atomic number, and high energy particles using a dichlorofluorescein assay.Results: The results demonstrated that these antioxidants are effective in protecting against radiation-induced oxidative stress and complete or nearly complete protection was achieved by treating the cells with a combination of these agents before and during the radiation exposure.Conclusion: The combination of antioxidants evaluated in this study is likely be a promising countermeasure for protection against space radiation-induced adverse biologic effects. [ABSTRACT FROM AUTHOR]

The oxidations of dichlorodihydrofluorescein and dihydrorhodamine by peroxynitrite are zero-order in the indicator between pH 3 and 10. The yield of the oxidized products, dichlorofluorescein and rhodamine, significantly increased at pH values > 7, and the maximal molar yields were 0.47 ± 0.04 mol rhodamine and 0.54 ± 0.06 mol, dichlorofluorescein per mol peroxynitrite at pH 8.5. The increase in yield of oxidized products as a function of pH indicates that the peroxynitrite anion may form an adduct with the indicator, followed by protonation and oxidation of the indicator. Carbon dioxide decreased the yield of fluorescent products to about 5%, relative to peroxynitrite, and the rate of product formation is again zero-order in the indicator. Given this yield, it is proposed that nitrogen dioxide and trioxocarbonate (•1−) are the reactive species that oxidize the indicators. [Copyright &y& Elsevier]

Broccoli can positively impact human health in a number of ways including acting as a vehicle for the delivery of promising antioxidants. Studies in non-cellular assay systems indicate that broccoli extracts have a high capacity for scavenging free radicals or interrupting free radical reactions. Here we report the effect of broccoli extracts on oxidative stress in HepG2 cells using the dichlorofluorescein-diacetate (DCFH-DA) assay. Our results confirm the association between broccoli extracts and enhanced antioxidant activity while providing additional evidence for protection against reactive oxygen species (ROS) at the cellular level. In HepG2 cells, the level of protection against AAPH-induced ROS differed among broccoli genotypes tested. We found that the HepG2 cell assay provided more information about the antioxidative lipid soluble fraction than the commonly reported ORAC assay. [ABSTRACT FROM PUBLISHER]

Non-fluorescent dichlorofluorescin (DCFH) was converted to fluorescent products by photo-irradiation during observations with spectrofluorometer and fluorescence microscopy. Photo-irradiation of DCFH at 250, 300, 330, 400, 500, or 600 nm generated fluorescent dichlorofluorescein (DCF), an oxidation product of DCFH, and an unrecognized fluorescent product. The ratio of the unknown product to DCF varied from 0.15 to 8.21 depending on wavelength. Although reactive oxygen species scavengers, such as catalase, superoxide dismutase, and sodium azide, did not suppress the increase in non-specified fluorescence, reagents such as ascorbic acid, mercaptopropionyl glycine, and methoxycinnamic acid, in a cell-free system, almost completely suppressed it with little effect on the fluorescence of DCF. Meanwhile, ascorbic acid also suppressed non-specified fluorescence in cells, but not completely. At low concentrations of DCFH, the speed of increasing fluorescence was considerably retarded, to such a degree that the fluorescence increase in cells during fluorescence microscopic observation was negligible. The addition, at the time of evaluation, of the above reagents to cell-free systems and, in cell systems, reducing the concentration of DCFH, effectively suppressed the photoreaction of DCFH. [Copyright &y& Elsevier]

Four simple, selective, accurate and reproducible procedures are described for the assay of methdilazine in bulk form and in formulations. One titrimetric and three spectrophotometric methods are based on the oxidation of the drug with potassium iodate, and determination of either excess iodate or iodine released in the reaction. In the titrimetric method (Method A) the drug is reacted with a known excess of iodate in sulphuric acid medium followed by the iodometric determination of residual oxidant. The residual oxidant is determined by reacting it with variamine blue and measuring the absorbance of the oxidised dye at 540 nm (Method B). The second spectrophotometric method (Method C) is based on the oxidation of the drug in sulphuric acid medium in the presence of chloride ions by a large excess of iodate and the iodate being reduced to iodine. The ICl2− generated in this reaction is used to iodinate 2′,7′-dichlorofluorescein dye, and the red colour of the iodinated dye is measured at 525 nm. The other spectrophotometric method (Method D) also involves the oxidation of the drug in acid medium by a large excess of iodate with the liberation of iodine and its subsequent extraction with carbon tetrachloride followed by measuring the absorbance 520 nm. The methods were successfully applied to the determination of methdilazine in tablets and syrup and the results obtained in agreement with the label claim. [Copyright &y& Elsevier]

In order to understand the mechanism by which advanced glycation endproducts (AGEs) elicit oxidative stress, macrophage-like RAW264.7 cells were exposed to various AGE-albumins, and oxidant stress was estimated from the fluorescence of oxidized dichlorofluorescein using the microtiter plate assay. Strongest fluorescence was observed with methylglyoxal modified albumin (MGO–BSA) compared with native albumin. Similar effects that were prevented by arginine coincubation were seen with phenylglyoxal-BSA. MGO–BSA had increased affinity for Cu2+ and Ca2+, but was conformationally similar to native albumin. Surprisingly, the mere addition of unmodified albumin to cells suppressed the fluorescence of oxidized DCF. While, several site-directed mutants of human serum albumin (HSA), including C34S and recombinant domains II and III retained fluorescence suppressing activity, proteolytic digests, recombinant domain I, and several nonalbumin proteins failed to suppress. Kinetic and ANS binding studies suggested albumin quenches DCF fluorescence by binding to hydrophobic pockets in domains II and III and that MGO–BSA is less hydrophobic than BSA. Finally, BSA also prevented H2O2 catalyzed DCF fluorescence more potently than MGO–BSA. These studies reveal important caveats of the widely used dichlorofluorescein assay and suggest methods other than the microtiter plate assay are needed to accurately assess cellular oxidant stress in presence of native or modified albumin. [Copyright &y& Elsevier]

[Display omitted] [ABSTRACT FROM AUTHOR]

Intracellular production of active oxygen in the brown alga Fucus evanescens C. Ag. was studied by measuring the capacity for in vivo conversion of 2′,7′-dichlorohydro-flurescein diacetate (DCFH-DA) to the fluorescent dye 2′-7′-dichlorofluorescein (DCF), both in emersed and immersed seaweeds. Algae were inclubated in sewawter containing DCFH-DA under a range of conditions, and it was also possible to load algae with DCFH-DA and then follow subsequent DCF production in enmersed tissue. DCF formation was linear for at least 2 in both darkness and light, with the rate of formation incrasing with the lgiht level. DCF formation was temperature dependent. It also increased when algae were trated with H[SUB2]O[SUB2] or methyl viologen (paraquat), which disrupts photosystem I electron transport and increased O[SUB2] production. Exogenous catalase reduced in vivo DCF production, presumably by lowering cellular concentration of H[SUB2]O[SUB2]. Hydrogen peroxide was released into the seawater by illuminated algae resulting in extrernal dye conversion to DCF. However, this does not interfere with in vivo measurement of DCF by loaded, washed algae becasue DCF leakage appearred to be negligible. Internal DCF did not affect photosynthetic oxygen production relative to untreated controls. Overall, our data suggest that DCFH-DA is a potentially very useful probe for studying acive oxygen metabolism is seaweeds subected to environmental stresses. [ABSTRACT FROM AUTHOR]

Temporal and spatial changes due to oxidative stress in the rat gastric mucosa were visualized and quantified during the process of mucosal hemorrhagic change. The fluorescence associated with dichlorofluorescein (DCF), a hydroperoxide-sensitive fluorochrome, increased 30 min after repeated electrical stimuli to the gastric artery. The increase in the fluorescence was enhanced in the area between two adjacent collecting venules. The content of platelet-activating factor (PAF), the activity of myeloperoxidase (MPO) in the gastric mucosa, the area of mucosal lesions, and the luminol-dependent chemiluminescence activity in zymosan-treated blood samples, obtained from the gastric vein, were measured and found to increase significantly 30 min after the stimuli. The intravenous injection of CV-6209, a PAF antagonist, 5 min prior to the stimuli significantly inhibited the DCF activation, the increases in PAF level and MPO activity, the mucosal hemorrhagic change, and the elevation in chemiluminescence activity. In addition, continuous infusion of superoxide dismutase also inhibited all these changes, except for chemiluminescence activity. These results suggest that oxygen radicals derived from PAF-activated granulocytes induce oxidative stress, and that oxidative changes are actually implicated in the pathogenesis of gastric mucosal injury. [ABSTRACT FROM AUTHOR]

The oxidation of 2′,7′-dichlorofluorescin (DCFH) to a fluorescent product is currently used to evaluate oxidant stress in cells. However, there is considerable uncertainty as to the enzymatic and nonenzymatic pathways that may result in DCFH oxidation. Iron/hydrogen peroxide-induced DCFH oxidation was inhibited by catalase or by the hydroxyl radical scavenger dimethylsulfoxide; however, superoxide dismutase (SOD) had no effect on DCFH oxidation. The formation of hydroxyl radical (indicated by the oxidation of salicylic acid to 2,3-dihydroxybenzoic acid) was proportional to DCFH oxidation, suggesting that the hydroxyl radical is responsible for the iron/peroxide-mediated oxidation of DCFH. Utilizing a superoxide generating system consisting of hypoxanthine/xanthine oxidase, oxidation of DCFH was unaffected by SOD, catalase or desferoxamine, and stimulated by removing hypoxanthine from the reaction mixture. In contrast, SOD or elimination of hypoxanthine abolished superoxide formation. In addition, potassium superoxide did not support the oxidation of DCFH. Thus, superoxide is not involved in DCFH oxidation. Boiling xanthine oxidase eliminated its concentration-dependent oxidation of 1 μM DCFH, indicating that xanthine oxidase ian enzymatically utilize DCFH as a high affinity substrate. Kinetic studies of the oxidation of DCFH by xanthine oxidase indicated a K of 0.62μM. Hypoxanthine competed with DCFH with a K of 1.03 mM. These studies suggest that DCFH oxidation may be a useful indicator of oxidative stress. However, other types of cellular damage may produce DCFH oxidation. For example, conditions or chemicals that damage intracellular membranes may release to the cytoplasm oxidases or peroxidases that might use DCFH as a substrate, similar to xanthine oxidase [ABSTRACT FROM AUTHOR]

Muscle pain is the most prevalent type of pain in the world, but treatment remains ineffective. Thus, it is relevant to develop trustable animal models to understand the involved pain mechanisms. Therefore, this study characterised the nociception and inflammation in a traumatic muscle injury model in rats. A single blunt trauma impact on the right gastrocnemius muscle of male Wistar rats (250–350 g) was used as model for muscle pain. Animals were divided into four groups (sham/no treatment; sham/diclofenac 1%; injury/no treatment; injury/diclofenac 1%) and the topical treatment with a cream containing 1% monosodium diclofenac (applied at 2, 6, 12, 24, and 46 h after muscle injury; 200 mg/muscle) was used as an anti-inflammatory control. Nociception (mechanical and cold allodynia, or nociceptive score) and locomotor activity were evaluated at 26 and 48 h after injury. Also, inflammatory and oxidative parameters were evaluated in gastrocnemius muscle and the creatine kinase (CK) activity and lactate/glicose levels in rat's serum and plasma, respectively. Muscle injury caused mechanical and cold allodynia, and increased nociceptive scores, without inducing locomotor impairment. This model also increased the inflammatory cells infiltration (seen by myeloperoxidase and N-acetyl-β-D-glucosaminidase activities and histological procedure), nitric oxide, interleukin (IL)-1β, IL-6, and dichlorofluorescein fluorescence in muscle samples; and CK activity and lactate/glicose ratio. The treatment with 1% monosodium diclofenac reduced inflammatory cells infiltration, dichlorofluorescein fluorescence and lactate/glicose levels. Thus, we characterised the traumatic muscle injury as a reproducible model of muscle pain, which makes it possible to evaluate promising antinociceptive and anti-inflammatory therapies. [ABSTRACT FROM AUTHOR]

2',7'-dichlorofluorescein (DCF) and derivatives are commonly used as fluorescent indicators of a broad spectrum of reactive oxygen species (ROS) generation in cell-based assays. However, there are numerous challenges inherent to the utilization of DCF probes for intracellular microscopic analysis, including photostability and probe efflux. Plate spectroscopy is comparatively simple and scalable compared to microscopy or flow cytometry-based acquisition, however is often subject to artefacts, including those introduced by thermal gradients and normalization methods. In this protocol we demonstrate a simple and sensitive plate spectrometry-based protocol utilizing the probes H 2 DCFDA and sulforhodamine B. The rapid sulforhodamine B assay (SRB) for cellular protein allows for a stable endpoint measurement of total cell population while also preserving morphology, can be combined or run in parallel with any other assay for normalization of readout to cell mass, and complemented by microscopic scoring of cell number and nuclear count. The oxidative stress and normalisation methods may enhance fields of research investigating cell differentiation, stress, or toxicity.. Graphical abstract: Graphical overview for quantification of ROS generation and cellular protein ., Competing Interests: Competing interestsThe authors declare they have no competing interests., (Copyright © 2021 The Authors; exclusive licensee Bio-protocol LLC.)

A new postchemiluminescence (PCL) reaction was observed when urea was injected into the reaction mixture after the CL reaction of N-bromosuccinimide and dichlorofluorescein. A possible reaction mechanism was proposed based on the studies of the CL kinetic characteristics, CL spectra, fluorescence spectra, and other experiments. A new flow injection CL method for the determination of urea was established based on the PCL reaction. The relative standard deviation for the determination of urea was 1.3% (n = 11, c = 5.0 × 10 -8  g/mL). The CL intensity responded linearly to the concentration of urea in the range 2.0 × 10 -9 -1.0 × 10 -6  g/mL (r = 0.9992). The detection limit was 7 × 10 -10  g/mL. The method had been applied to the determination of urea in milk with satisfactory results., (Copyright © 2016. Published by Elsevier B.V.)

The study presents the possibility of localization of reactive oxygen species (ROS) in cytological preparations of seminal roots and coleoptiles of eleven-day-old seedlings of recombinant inbred lines (RILs) of rye, subjected to the influence of abiotic stress caused by nitrogen and potassium deficiencies in a medium in in vitro cultures of mature embryos. Fifty-one recombinant inbred lines of rye (F[sub 9]) considered as tolerant and susceptible to stress caused by nitrogen and potassium deficiencies in a medium were selected for the research. The plants were derived from the F[sub 2] generation, obtained from a combination of crossings of inbred lines of rye Ot0-6 and Ot1-3. The response of each line to stress was assessed under the conditions of high and decreased level of N and K in a medium. In the medium with high N and K content the seedlings developed on average longer coleoptiles, shorter the longest roots and less numerous roots in comparison with the medium with low content of N and K. Other described lines included lines which developed shorter coleoptiles and significantly shorter the longest roots on the medium with deficient nitrogen and potassium in comparison with the medium with a high content of nitrogen and potassium. Depending on individual morphological responses of the seedlings, the lines were clustered with the use of Ward's agglomerative method with regard to the response of three seedling traits: coleoptile length, the longest root length and root number. RILs with extreme responses to nutrient stress were identified in the outermost groups of Ward's dendrogram. ROS , mainly H[sub 2]O[sub 2], were visualized in the epidermal part of the elongation zone of the seminal roots of seedlings subjected to the influence of nutrient stress. More intense fluorescence of the fluorescence dye (2',7'-dichlorofluorescein) was detected in the seedlings of the lines considered as susceptible to nutrient deficiencies as compared to tolerant lines. The generation of ROS , as important signal molecules provesfunctioning of the molecular mechanisms of sensing and plant cells response to nutrient stress. [ABSTRACT FROM AUTHOR]

Increasing evidence reveals a large dependency of epithelial cancer cells on oxidative phosphorylation (OXPHOS) for energy production. In this study we tested the potential of epigallocatechin-3-gallate (EGCG), a natural polyphenol known to target mitochondria, in inducing OXPHOS impairment and cell energy deficit in human epitheliod (REN cells) and biphasic (MSTO-211H cells) malignant pleural mesothelioma (MMe), a rare but highly aggressive tumor with high unmet need for treatment. Due to EGCG instability that causes H2O2 formation in culture medium, the drug was added to MMe cells in the presence of exogenous superoxide dismutase and catalase, already proved to stabilize the EGCG molecule and prevent EGCG-dependent reactive oxygen species formation. We show that under these experimental conditions, EGCG causes the selective arrest of MMe cell growth with respect to normal mesothelial cells and the induction of mitochondria-mediated apoptosis, as revealed by early mitochondrial ultrastructure modification, swelling and cytochrome c release. We disclose a novel mechanism by which EGCG induces apoptosis through the impairment of mitochondrial respiratory chain complexes, particularly of complex I, II and ATP synthase. This induces a strong reduction in ATP production by OXPHOS, that is not adequately counterbalanced by glycolytic shift, resulting in cell energy deficit, cell cycle arrest and apoptosis. The EGCG-dependent negative modulation of mitochondrial energy metabolism, selective for cancer cells, gives an important input for the development of novel pharmacological strategies for MMe., (© 2013 Elsevier B.V. All rights reserved.)

Sulfite oxidase (SO) deficiency is biochemically characterized by tissue accumulation and high urinary excretion of sulfite, thiosulfate and S-sulfocysteine. Affected patients present severe neurological symptoms and cortical atrophy, whose pathophysiology is still poorly established. Therefore, in the present work we investigated the in vitro effects of sulfite and thiosulfate on important parameters of energy metabolism in the brain of young rats. We verified that sulfite moderately inhibited the activity of complex IV, whereas thiosulfate did not alter any of the activities of the respiratory chain complexes. It was also found that sulfite and thiosulfate markedly reduced the activity of total creatine kinase (CK) and its mitochondrial and cytosolic isoforms, suggesting that these metabolites impair brain cellular energy buffering and transfer. In contrast, the activity of synaptic Na(+),K(+)-ATPase was not altered by sulfite or thiosulfate. We also observed that the inhibitory effect of sulfite and thiosulfate on CK activity was prevented by melatonin, reduced glutathione and the combination of both antioxidants, as well as by the nitric oxide synthase N(ω)-nitro-l-arginine methyl ester, indicating the involvement of reactive oxygen and nitrogen species in these effects. Sulfite and thiosulfate also increased 2',7'-dichlorofluorescin oxidation and hydrogen peroxide production and decreased the activity of the redox sensor aconitase enzyme, reinforcing a role for oxidative damage in the effects elicited by these metabolites. It may be presumed that the disturbance of cellular energy and redox homeostasis provoked by sulfite and thiosulfate contributes to the neurological symptoms and abnormalities found in patients affected by SO deficiency., (© 2013.)

N-(3-oxododecanoyl)-l-homoserine lactone (3OC(12)-HSL) is a quorum-sensing molecule produced by gram-negative microbial pathogens such as Pseudomonas aeruginosa (PAO1). 3OC(12)-HSL is involved in the regulation of bacterial virulence factors and also alters the function of the host immune cells. Others and we have previously shown that paraoxonase 2 (PON2), a member of the paraoxonase gene family expressed in immune cells, hydrolyzes 3OC(12)-HSL. In this study, we examined i) whether macrophage PON2 participates in 3OC(12)-HSL hydrolysis, ii) the effect of PON2 deficiency in acute PAO1 infection in mice and iii) the effect of 3OC(12)-HSL on PON2 deficient (PON2-def) macrophages. When compared to wild type macrophages, both intact cells and membrane-enriched protein lysates obtained from PON2-def macrophages show a marked impairment in their ability to hydrolyze 3OC(12)-HSL. PON2 expression (message and protein) is not altered in response to 3OC(12)-HSL in macrophages. 3OC(12)-HSL treated PON2-def macrophages showed i) an increase in ER stress and oxidative stress, ii) defective phosphatidylinositol 3-kinase (PI3 kinase)/AKT activation, and iii) reduced phagocytosis function. Moreover, the nitration to phosphorylation ratio of Tyr458 in p85 protein, the regulatory subunit of PI3-kinase that has been correlated with the phagocytosis function of macrophages, was increased in PON2-def macrophages. Antioxidant treatment reversed the effects of PON2 deficiency in macrophage phagocytosis function. Furthermore, following administration of 1.6 × 10(7) CFU of PAO1, bacterial clearance was significantly reduced in the lungs (5.7 fold), liver (2.5 fold), and spleen (14.8 fold) of PON2-def mice when compared to wild type mice. Our results suggest that PON2 plays an important role in innate immune defense against PAO1 infection., (© 2013.)

A rapid and sensitive flow-injection chemiluminescence (FI-CL) method is described for the determination of diazepam based on its reaction with N-bromosuccinimide (NBS) in alkaline medium in the presence of dichlorofluorescein (DCF) as an effective energy-transfer agent. Under optimum conditions, the proposed method allowed the measurement of diazepam over the range of 2.0 × 10(-6) to 2.0 × 10(-4) mol/L with a detection limit of 5.0 × 10(-7) mol/L. The relative standard deviation for 11 parallel measurements of 2.0 × 10(-5) mol/L diazepam was 2.1%. The method was applied satisfactorily for the determination of diazepam in pharmaceutical preparations, and the results agree well with those obtained by spectrophotometry. The use of the proposed system for the determination of diazepam in urine and plasma samples was also tested. The possible mechanism of the chemiluminescence reaction is discussed briefly., (Copyright © 2012 John Wiley & Sons, Ltd.)