Inhibition of superoxide generation and of increase in intracellular Ca2+ concentration by zinc in rat neutrophils stimulated with zymosan

The effect of Zn2+ on the O2- generation and change in intracellular Ca2+ concentration ([Ca2+]i) of rat peritoneal neutrophils was studied. Zymosan (serum-treated zymosan (STZ))-induced O2- generation was inhibited by Zn2+ at concentrations as low as 10 microM. A large amount of the inhibition was observed in the absence of extracellular Ca2+ but the inhibition could not be restored by increasing the extracellular Ca2+ concentration, indicating that Zn2+ does not necessarily inhibit the O2- generation competitively with extracellular Ca2+. In the absence of extracellular Ca2+, Zn2+ inhibited STZ-induced transient increase in [Ca2+]i in the concentration range that evoked a marked inhibition in the O2- generation. On the other hand, Zn2+ did not inhibit significantly STZ-induced uptake of 45Ca2+ from extracellular medium by the cells. From these results, it is suggested that Zn2+ inhibits STZ-induced release of Ca2+ from intracellular storage sites, resulting in the suppression of the activation mechanism of neutrophils.     

Na+-Ca2+ exchanger modulates Ca2+ content in intracellular Ca2+ stores in rat osteoblasts

Na+-Ca2+ exchanger (NCX) transports Ca2+ coupled with Na+ across the plasma membrane in a bi-directional mode. Ca2+ flux via NCX mediates osteogenic processes, such as formation of extracellular matrix proteins and bone nodules. However, it is not clearly understood how the NCX regulates cellular Ca2+ movements in osteogenic processes. In this study, the role of NCX in modulating Ca2+ content of intracellular stores ([Ca2+]ER) was investigated by measuring intracellular Ca2+ activity in isolated rat osteoblasts. Removal of extracellular Na+ elicited a transient increase of intracellular Ca2+ concentration ([Ca2+]i). Pretreatment of antisense oligodeoxynucleotide (AS) against NCX depressed this transient Ca2+ rise and raised the basal level of [Ca2+]i. In AS-pretreated cells, the expression and activity of alkaline phosphatase (ALP), an osteogenic marker, were decreased. However, the cell viability was not affected by AS-pretreatment. Suppression of NCX activity by the AS-pretreatment decreased ATP-activated Ca2+ release from intracellular stores and significantly enhanced Ca2+ influx via store operated calcium influx (SOCI), compared to those of S-pretreated or control cells. These results strongly suggest that NCX has a regulatory role in cellular Ca2+ pathways in osteoblasts by modulating intracellular Ca2+ content.     

Impaired intracellular Ca2+ dynamics in live cardiomyocytes revealed by rapid line scan confocal microscopy.

Altered intracellular Ca2+ dynamics are characteristically observed in cardiomyocytes from failing hearts. Studies of Ca2+ handling in myocytes predominantly use Fluo-3 AM, a visible light excitable Ca2+ chelating fluorescent dye in conjunction with rapid line-scanning confocal microscopy. However, Fluo-3 AM does not allow for traditional ratiometric determination of intracellular Ca2+ concentration and has required the use of mathematic correction factors with values obtained from separate procedures to convert Fluo-3 AM fluorescence to appropriate Ca2+ concentrations. This study describes methodology to directly measure intracellular Ca2+ levels using inactivated, Fluo-3-AM-loaded cardiomyocytes equilibrated with Ca2+ concentration standards. Titration of Ca2+ concentration exhibits a linear relationship to increasing Fluo-3 AM fluorescence intensity. Images obtained from individual myocyte confocal scans were recorded, average pixel intensity values were calculated, and a plot is generated relating the average pixel intensity to known Ca2+ concentrations. These standard plots can be used to convert transient Ca2+ fluorescence obtained with experimental cells to Ca2+ concentrations by linear regression analysis. Standards are determined on the same microscope used for acquisition of unknown Ca2+ concentrations, simplifying data interpretation and assuring accuracy of conversion values. This procedure eliminates additional equipment, ratiometric imaging, and mathematic correction factors and should be useful to investigators requiring a straightforward method for measuring Ca2+ concentrations in live cells using Ca2+-chelating dyes exhibiting variable fluorescence intensity.     

Rhodamine B degradation and reactive oxygen species generation by a ZnSe-graphene/TiO2 sonocatalyst

Nanostructured ZnSe-graphene/TiO2 was synthesized by a hydrothermal-assisted approach. ZnSe-graphene/TiO2 exhibited favorable adsorption of rhodamine B, a wide wavelength absorption range, and efficien...     

Nanoelectrodes for intracellular measurements of reactive oxygen and nitrogen species in single living cells

Reactive oxygen and nitrogen species (ROS and RNS) play important roles in various physiological processes (e.g. phagocytosis) and pathological conditions (e.g. cancer). The primary ROS/RNS, viz., hydrogen peroxide, peroxynitrite ion, nitric oxide, and nitrite ion, can be oxidized at different electrode potentials and therefore detected and quantified by electroanalytical techniques. Nanometer-sized electrochemical probes are especially suitable for measuring ROS/RNS in single cells and cellular organelles. In this article, we survey recent advances in the localized measurements of ROS/RNS inside single cells and discuss several methodological issues, including optimization of nanoelectrode geometry, precise positioning of an electrochemical probe inside a cell, and interpretation of electroanalytical data.     

Trichosanthin induced calcium-dependent generation of reactive oxygen species in human choriocarcinoma cells

The type-I ribosome-inactivating protein trichosanthin (TCS) has a broad spectrum of biological and pharmacological activities, including abortifacient, anti-tumor and anti-HIV. We found for the first time that TCS induced the generation of reactive oxygen species (ROS) in human choriocarcinoma cells (JAR cells) at the level of the single cell by using the fluorescent probe 2',7'-dichlorofluorescein diacetate with confocal laser scanning microscopy. TCS-induced ROS formation was shown to be dependent on the presence of extracellular Ca2+ and was further reduced when cytosolic Ca2+ was chelated by BAPTA-AM. The production of ROS increased rapidly after the application of TCS, which paralleled TCS-induced increase in intracellular calcium monitored using fluo 3-AM. Simultaneous observation of the nuclear morphological changes via two-photon laser scanning microscopy and production of ROS via confocal laser scanning microscopy revealed that ROS were involved in the apoptosis of JAR cells. The contribution of ROS was confirmed by experiments in which the antioxidant alpha-tocopherol prevented TCS-induced ROS formation and cell death. The finding that TCS induced calcium-dependent generation of ROS in JAR cells and that ROS were involved in the apoptosis of JAR cells might provide new insight into the anti-tumor and anti-HIV mechanism of TCS.     

Regulation of pro-inflammatory responses by lipoxygenases via intracellular reactive oxygen species in vitro and in vivo

Reactive oxygen species (ROS) performs a pivotal function as a signaling mediator in receptor-mediated signaling. However, the sources of ROS in this signaling have yet to be determined, but may include lipoxygenases (LOXs) and NADPH oxidase. The stimulation of lymphoid cells with TNF-alpha, IL-1beta, and LPS resulted in significant ROS production and NF-kappaB activation. Intriguingly, these responses were markedly abolished via treatment with the LOXs inhibitor nordihydroguaiaretic acid (NDGA). We further examined in vivo anti-inflammatory effects of NDGA in allergic airway inflammation. Both intraperitoneal and intravenous NDGA administration attenuated ovalbumin (OVA)-induced influx into the lungs of total leukocytes, as well as IL-4, IL-5, IL-13, and TNF-alpha levels. NDGA also significantly reduced serum levels of OVA-specific IgE and suppressed OVA-induced airway hyperresponsiveness to inhaled methacholine. The results of our histological studies and flow cytometric analyses showed that NDGA inhibits OVA-induced lung inflammation and the infiltration of CD11b+ macrophages into the lung. Collectively, our findings indicate that LOXs performs an essential function in pro-inflammatory signaling via the regulation of ROS regulation, and also that the inhibition of LOXs activity may have therapeutic potential with regard to the treatment of allergic airway inflammation.     

Photochemical behavior of 6-methylpterin in aqueous solutions: generation of reactive oxygen species

Pterins are a family of heterocyclic compounds present in a wide range of living systems that participate in relevant biological functions and are involved in different photobiological processes. 6-Methylpterin (MPT) was investigated for its efficiency of singlet-oxygen (1O2) production and quenching in aqueous solution. The quantum yields of 1O2 production (phi(delta)) was determined by measurements of the 1O2 luminescence in the near-infrared upon continuous excitation of the sensitizer. Values of phi(delta) were found to be 0.10 +/- 0.02 and 0.14 +/- 0.02 in acidic and alkaline media, respectively. Studies of the photooxidation of MPT in acidic (pH = 5.0-6.0) and alkaline (pH = 10.2-10.8) aqueous solutions at 350 nm and room temperature have been performed. The photochemical reactions were followed by UV-visible spectrophotometry, high-performance liquid chromatography and an enzymatic method for H2O2 determination. MPT is not light sensitive in the absence of oxygen, but it undergoes a photooxidation reaction in the presence of oxygen, yielding several nonpteridinic products. The quantum yields of MPT disappearance were determined and values of 2.4 (+/-0.5) x 10(-4) and 8.1 (+/-0.8) x 10(-4) were obtained in acidic and alkaline media, respectively. H2O2 was detected and quantified in irradiated solutions of MPT. The rate constant of the chemical reaction between 1O2 and MPT (k(r)) was determined to be 4.9 x 10(6) M(-1) s(-1) in alkaline medium and the role of 1O2 in the photooxidation of MPT is discussed.     

Loading effects of silver oxides upon generation of reactive oxygen species in semiconductor photocatalysis

Superoxide anion radical (O(2)(-*)) and OH radical generations in suspensions of Ag metal-, Ag(2)O-, or AgO-loaded TiO(2) and BiVO(4) photocatalysts in alkaline conditions (pH 12.0) were examined by means of a luminol chemiluminescence (CL) technique and a spin-trapping fluorescence one in which terephthalic acid reacts with an OH radical to afford the highly fluorescent 2-hydroxyterephthalic acid (TAOH), respectively. The observed luminol CL intensity was remarkably enhanced by the AgO loading on TiO(2) as well as BiVO(4). This can be explained by enhancement of O(2)(-*) production on the AgO-loaded photocatalysts caused by the synergetic effects on the thermocatalytic activity upon the AgO surface and the efficient electron-hole separation at the photocatalyst/AgO interface. On the other hand, loading effects of AgO on the TAOH formation were not so significant compared to those on the CL observation, though the TAOH formation rates for the TiO(2) samples were much larger than those for the BiVO(4) ones by about three orders of magnitude. The properties of O(2)(-*) and OH radical generations on these photocatalysts are discussed on the basis of the luminol CL kinetics and approximate band edge positions of TiO(2), BiVO(4), and silver oxides.     

利用AgVO3改性MoS2增强活性氧产生及其光催化性能的提高

随着全球工业化进程的发展,环境污染问题日益严重,已经成为21世纪影响人类生存与发展的重要问题.光催化氧化技术被认为是解决环境问题最有应用前景的技术之一,已经成为环境领域的研究热点.众所周知,二硫化钼(MoS2)可以被可见光激发产生电子-空穴对,但是由于其氧化还原电势并不高,抑制了氧分子活化的量子效率,且激发后的光生载流子容易复合,导致光催化效率不高.因此,迫切需要对MoS2光催化材料进行修饰与改性,采用提高光催化过程中活性氧(ROSs)的量来提高其光催化活性.银钒氧化物(AgVO3,Ag2V4O11,Ag3VO4和Ag4V2O7等)因其在锂电池、传感器和光催化剂领域的应用而引起了人们的关注.其中,AgVO3具有较窄的带隙和高度分散的价带,具有潜在的应用价值.本文采用水热法成功制备了AgVO3/MoS2复合光催化剂,并采用X射线粉末衍射、扫描电子显微、透射电子显微镜和紫外-可见漫反射光谱等表征技术研究了所制光催化剂的物相结构、样品形貌和光学性能.以四环素为研究对象,将其应用于AgVO3/MoS2复合光催化剂的降解实验.结果表明,随着AgVO3质量比从1.0 wt%增加到3.0 wt%,所得催化剂的光催化活性不断提高;当进一步增加AgVO3的质量时,复合催化剂的活性逐渐降低.这是由于过多的AgVO3的引入导致在光催化剂表面形成电子-空穴对复合中心,增加了载流子复合几率.因此,AgVO3/MoS2复合光催化剂中AgVO3的最佳质量比为3.0 wt%,其降解速率常数为0.0087 min–1,分别是MoS2(0.00509 min–1)和AgVO3(0.00495 min–1)的1.71和1.76倍.由于AgVO3改性后的MoS2具有优异的光催化性能,能促进O2的吸附/活化,加速MoS2表面生成H2O2的双电子氧还原反应,从而产生更多的ROSs.利用电子自旋共振光谱、POPHA荧光检测和自由基捕获实验相结合的方法来阐明ROSs的形成机理.同时,ROSs的产生会加速消耗AgOV3导带上的电子,为降解污染物留下更多的空穴.本文为表面催化工程促进ROSs生成的合理设计提供了新的思路,有望在环境治理中得到实际应用.     

Involvement of reactive oxygen species and reactive nitrogen species in the wound response of Dasycladus vermicularis

We investigated the signaling events involved in the wound response of the marine macroalga Dasycladus vermicularis, finding nitric oxide (NO) production in relation to injury. The addition of exogenous H2O2 to aliquots of injured algae accelerated the kinetics of NO production in the wounded region. Similarly, the addition of an NO donor caused an increase in detectable H2O2 around the site of injury. By wounding or incubating uninjured algae with an NO donor, peroxidase activity was enhanced. Based on the use of selected pharmacological probes, our results indicate that H2O2 production involves the upstream activation of signaling events similar to those observed in the physiology of higher plants.     

Reactive oxygen species are involved in nitric oxide-induced apoptosis in rat cortical neurons

Nitric oxide donor SNAP induced apoptosis in primary rat cerebral cortical neurons, which was characterized morphologically by chromatin condensation and the formation of apoptotic bodies. With redox-sensitive fluorescence probes DCFH-DA and DHR123, the formation of endogenous reactive oxygen species (ROS) inside cells during the apoptosis process was monitored by laser confocal scanning microscopy (LCSM). SNAP treatment also caused the accumulation of extracellular hydrogen peroxide. Pretreatment with the nitric oxide scavenger hemoglobin could effectively inhibit the formation of endogenous ROS and protect neurons from apoptosis. The results suggested that ROS might be involved in NO-induced apoptosis in neuronal cells.     

Photo-induced reactive oxygen species generation by different water-soluble fullerenes (C) and their cytotoxicity in human keratinocytes

Abstract In this study we report the phototoxicity toward HaCaT keratinocytes that results from the photogeneration of superoxide and singlet oxygen ((1)O(2)) by four different "water-soluble" fullerene (C(60)) preparations-monomeric (gamma-CyD)(2)/C(60) (gamma-cyclodextrin bicapped C(60)) and three aggregated forms-THF/nC(60) (prepared by solvent exchange from THF solution); Son/nC(60) (prepared by sonication of a toluene/water mixture); and gamma-CyD/nC(60) (prepared by heating the [gamma-CyD](2)/C(60) aqueous solution). Our results demonstrate that all four C(60) preparations photogenerate (1)O(2) efficiently. However, the properties of C(60)-generated (1)O(2), including its availability for reactions in solution, are markedly different for the monomeric and aggregated forms. (1)O(2) produced by monomeric (gamma-CyD)(2)/C(60) is quenchable by NaN(3) and its quantum yield in D(2)O, which is only weakly dependent on oxygen concentration, is as high as C(60) in toluene. In contrast, (1)O(2) generated from aggregated C(60) is not quenchable by NaN(3), exhibits a solvent-independent short-lived lifetime (ca 2.9 mus), is highly sensitive to oxygen concentration while its phosphorescence is redshifted. All these features indicate that (1)O(2) is sequestered inside the C(60) aggregates, which may explain why these preparations were not phototoxic toward HaCaT cells. Electron paramagnetic resonance studies demonstrated the generation of the C(60) anion radical (C(60)(*-)) when (gamma-CyD)(2)/C(60) was irradiated (lambda > 300 nm) in the presence of a reducing agent (NADH); spin trapping experiments (lambda > 400 nm) with 5,5-dimethyl-1-pyrroline N-oxide clearly showed the generation of superoxide resulting from the reaction of C(60)(*-) with oxygen. In vitro tests with HaCaT keratinocytes provided evidence that (gamma-CyD)(2)/C(60) phototoxicity is mainly mediated by (1)O(2) (Type II mechanism) with only a minor contribution from free radicals (Type I mechanism).     

Defensive effects of fullerene-C60/liposome complex against UVA-induced intracellular reactive oxygen species generation and cell death in human skin keratinocytes HaCaT,associated with intracellular uptake and extracellular excretion of fullerene-C60

The UVA-irradiation of 10 J/cm² on HaCaT keratinocytes increased 59.1% of the intracellular reactive oxygen species (ROS) by NBT assay and the cell viability decreased to 31.5% by WST-1 assay, comparing to the non-irradiated control. In the presence of fullerene-C60 (C60) incorporated in phospholipid membrane vehicle (LiposomeFullerene: Lpsm-Flln) of 250–500 ppm, they were restored to ?9.1% to  + 2.3% of the ROS and 83.0–84.8% of the cell viability, but scarcely restored by the liposome without C60 (Lpsm). In HaCaT cells administered with Lpsm-Flln (150 ppm), C60 was ingested at the intracellular concentrations of 1.4–21.9 ppm for 4–24 h, and, intracellular C60 was excreted by 80% at 4 h after rinsing-out, and decreased to 2–10% after 24–48 h. C60 was predominantly distributed around the outside of nuclear membrane without deterioration of intact cell morphology according to fluorescent immunostain. Thus Lpsm-Flln is found to be an effective antioxidant that could preserve HaCaT keratinocytes against UVA-induced cellular injury. Lpsm-Flln has a potential to serve as a cosmetic material for skin protection against UVA.     

还原氧化石墨烯-硒化银纳米复合材料的水热合成与表征及其活性氧物种产生(英文)

A visible-light photocatalyst containing Ag2Se and reduced graphene oxide(RGO) was synthesized by a facile sonochemical-assisted hydrothermal method. X-ray diffraction, scanning electron mi-croscopy with energy-dispersive X-ray analysis, and ultraviolet-visible diffuse reflectance spectros-copy results indicated that the RGO-Ag2Se nanocomposite contained small crystalline Ag2Se nano-particles dispersed over graphene nanosheets and absorbed visible light. The high crystallinity of the nanoparticles increased photocatalytic activity by facilitating charge transport. N2 adsorp-tion-desorption measurements revealed that the RGO-Ag2Se nanocomposite contained numerous pores with an average diameter of 9 nm, which should allow reactant molecules to readily access the Ag2Se nanoparticles. The RGO-Ag2Se nanocomposite exhibited higher photocatalytic activity than bulk Ag2Se nanoparticles to degrade organic pollutant rhodamine B and industrial dye Texbrite BA-L under visible-light irradiation(λ 420 nm). The generation of reactive oxygen spe-cies in RGO-Ag2Se was evaluated through its ability to oxidize 1,5-diphenylcarbazide to 1,5-diphenylcarbazone. The small size of the Ag2Se nanoparticles in RGO-Ag2Se was related to the use of ultrasonication during their formation, revealing that this approach is attractive to form po-rous RGO-Ag2Se materials with high photocatalytic activity under visible light.     

Modified hydrothermal synthesis and characterization of reduced graphene oxide-silver selenide nanocomposites with enhanced reactive oxygen species generation

A visible‐light photocatalyst containing Ag2Se and reduced graphene oxide (RGO) was synthesized by a facile sonochemical‐assisted hydrothermal method. X‐ray diffraction, scanning electron mi‐croscopy w...     

Reactive oxygen species are involved in lysophosphatidic acid-induced apoptosis in rat cerebellar granule cells

Lysophosphatidic acid (LPA) induced apoptosis in primary rat cerebellar granule cells, which was characterized morphologically by chromatin condensation and the formation of apoptotic bodies. With redox-sensitive fluorescence probes DCFH-DA and DHR123, the formation of endogenous reactive oxygen species (ROS) inside cells during the apoptosis process was monitored by laser confocal scanning microscopy (LCSM). Pretreatment with the antioxidant tetramethylpyrazine (TMP) could effectively inhibit the formation of endogenous ROS and protect neurons from apoptosis. The results suggest that ROS might be involved in LPA-induced apoptosis in neurons.     

TDAG51 deficiency promotes oxidative stress-induced apoptosis through the generation of reactive oxygen species in mouse embryonic fibroblasts

Apoptosis has an important role in maintaining tissue homeostasis in cellular stress responses such as inflammation, endoplasmic reticulum stress, and oxidative stress. T-cell death-associated gene 51 (TDAG51) is a member of the pleckstrin homology-like domain family and was first identified as a pro-apoptotic gene in T-cell receptor-mediated cell death. However, its pro-apoptotic function remains controversial. In this study, we investigated the role of TDAG51 in oxidative stress-induced apoptotic cell death in mouse embryonic fibroblasts (MEFs). TDAG51 expression was highly increased by oxidative stress responses. In response to oxidative stress, the production of intracellular reactive oxygen species was significantly enhanced in TDAG51-deficient MEFs, resulting in the activation of caspase-3. Thus, TDAG51 deficiency promotes apoptotic cell death in MEFs, and these results indicate that TDAG51 has a protective role in oxidative stress-induced cell death in MEFs.     

Multifunctional peptide-assembled micelles for simultaneously reducing amyloid-β and reactive oxygen species

The excessive production and deposition of amyloid-β (Aβ) is one of the most important etiologies of Alzheimer''s disease (AD). The interaction between Aβ and metal ions produces aberrant reactive oxygen species (ROS), which induce oxidative stress and accelerate the progression of AD. To reduce Aβ plaques and ROS to maintain their homeostasis is an emerging and ingenious strategy for effective treatment of AD. Herein, we report the rational design of multifunctional micelles (MPGLT) based on a polymer-grafted peptide to simultaneously clear Aβ and ROS for AD therapy. The MPGLT integrating three functional peptides as a ROS scavenger (tk-GSH), β-sheet breaker (LP) and an autophagy activator (TK) respectively, could capture and degrade Aβ. Meanwhile, the tk-GSH on the surface of MPGLT effectively scavenges the intracellular ROS. Consequently, MPGLT reduced the cytotoxicity of Aβ and ROS. In vivo animal studies using an AD mouse model further showed that MPGLT could transport across the blood–brain barrier for decreasing the Aβ plaque and eliminating ROS in vivo. This peptide micelle-based synergistic strategy may provide novel insight for AD therapy.

Multifunctional micelles based on a peptide–polymer for simultaneously targeting Aβ degradation and ROS scavenging for AD therapy.