Redox-sensitive Fluorescent Probe Research Articles

Exposure to Selenomethionine and Selenocystine Induces Redox-Mediated ER Stress in Normal Breast Epithelial MCF-10A Cells.

Selenium is an essential trace element co-translationally incorporated into selenoproteins with important biological functions. Health benefits have long been associated with selenium supplementation. However, cytotoxicity is observed upon excessive selenium intake. The aim of this study is to investigate the metabolic pathways underlying the response to the selenium-containing amino acids selenomethionine and selenocysteine in a normal human breast epithelial cell model. We show that both selenomethionine and selenocystine inhibit the proliferation of non-cancerous MCF-10A cells in the same concentration range as cancerous MCF-7 and Hela cells, which results in apoptotic cell death. Selenocystine exposure in MCF-10A cells caused a severe depletion of free low molecular weight thiols, which might explain the observed upregulation of the expression of the oxidative stress pathway transcription factor NRF2. Both selenomethionine and selenocystine induced the expression of target genes of the unfolded protein response (GRP78, ATF4, CHOP). Using a redox-sensitive fluorescent probe targeted to the endoplasmic reticulum (ER), we show that both selenoamino acids shifted the ER redox balance towards an even more oxidizing environment. These results suggest that alteration of the redox state of the ER may disrupt protein folding and cause ER stress-induced apoptosis in MCF-10A cells exposed to selenoamino acids.

Chemically Fueled Dissipative Cross-Linking of Protein Hydrogels Mediated by Protein Unfolding.

Cells assemble dynamic protein-based nanostructures far from equilibrium, such as microtubules, in a process referred to as dissipative assembly. Synthetic analogues have utilized chemical fuels and reaction networks to form transient hydrogels and molecular assemblies from small molecule or synthetic polymer building blocks. Here, we demonstrate dissipative cross-linking of transient protein hydrogels using a redox cycle, which exhibit protein unfolding-dependent lifetimes and mechanical properties. Fast oxidation of cysteine groups on bovine serum albumin by hydrogen peroxide, the chemical fuel, formed transient hydrogels with disulfide bond cross-links that degraded over hours by a slow reductive back reaction. Interestingly, despite increased cross-linking, the hydrogel lifetime decreased as a function of increasing denaturant concentration. Experiments showed that the solvent-accessible cysteine concentration increased with increasing denaturant concentration due to unfolding of secondary structures. The increased cysteine concentration consumed more fuel, which led to less direction oxidation of the reducing agent and affected a shorter hydrogel lifetime. Increased hydrogel stiffness, disulfide cross-linking density, and decreased oxidation of redox-sensitive fluorescent probes at a high denaturant concentration provided evidence supporting the unveiling of additional cysteine cross-linking sites and more rapid consumption of hydrogen peroxide at higher denaturant concentrations. Taken together, the results indicate that the protein secondary structure mediated the transient hydrogel lifetime and mechanical properties by mediating the redox reactions, a feature unique to biomacromolecules that exhibit a higher order structure. While prior works have focused on the effects of the fuel concentration on dissipative assembly of non-biological molecules, this work demonstrates that the protein structure, even in nearly fully denatured proteins, can exert similar control over reaction kinetics, lifetime, and resulting mechanical properties of transient hydrogels.

Combining Gallium Protoporphyrin and Gallium Nitrate Enhances In Vitro and In Vivo Efficacy against Pseudomonas aeruginosa: Role of Inhibition of Bacterial Antioxidant Enzymes and Resultant Increase in Cytotoxic Reactive Oxygen Species.

Pseudomonas aeruginosa is a highly antibiotic-resistant opportunistic pathogenic bacteria that is responsible for thousands of deaths each year. Infections with P. aeruginosa disproportionately impact individuals with compromised immune systems as well as cystic fibrosis patients, where P. aeruginosa lung infection is a leading cause of morbidity and mortality. In previous work, we showed that a combination of gallium (Ga) nitrate and Ga protoporphyrin worked well in several bacterial infection models but its mechanism of action (MOA) is unknown. In the current work, we have investigated the MOA of Ga combination therapy in P. aeruginosa and its analysis in the in vivo model. In P. aeruginosa treated with Ga combination therapy, we saw a decrease in catalase and superoxide dismutase (SOD) activity, key antioxidant enzymes, which could correlate with a higher potential for oxidative stress. Consistent with this hypothesis, we found that, following combination therapy, P. aeruginosa demonstrated higher levels of reactive oxygen species, as measured using the redox-sensitive fluorescent probe, H2DCFDA. We also saw that the Ga combination therapy killed phagocytosed bacteria inside macrophages in vitro. The therapy with low dose was able to fully prevent mortality in a murine model of P. aeruginosa lung infection and also significantly reduced lung damage. These results support our previous data that Ga combination therapy acts synergistically to kill P. aeruginosa, and we now show that this may occur through increasing the organism's susceptibility to oxidative stress. Ga combination therapy also showed itself to be effective at treating infection in a murine pulmonary-infection model.

Photophysical properties of alloxazine derivatives with extended aromaticity – Potential redox-sensitive fluorescent probe

The spectral and photophysical properties of two four-ring alloxazine derivatives, naphtho[2,3-g]pteridine-2,4(1H,3H)-dione (1a) and 1,3-dimethylnaphtho[2,3-g]pteridine-2,4(1H,3H)-dione, (1b) were studied. The propensity of 1a for excited-state proton transfer reactions in the presence of acetic acid as a catalyst was also studied, showing no signature of the reaction occurring. In addition, quenching of 1a fluorescence by acetic acid was investigated. Singlet and triplet states and spectral data for 1a and 1b were calculated using density functional theory TD-DFT at B3LYP/6-31G(d) and UB3LYP levels. Finally, fluorescence lifetime imaging microscopy (FLIM) using 1a and 1b as fluorescence probes was applied to in vitro human red blood cells (RBCs) with and without tert-butyl hydroperoxide (TB) as an oxidising agent. To evaluate and compare the effects of 1a and 1b on the redox properties of RBCs, the fluorescence lifetime, amplitude and fractional intensities were calculated, and phasor plot analysis was performed. The results obtained show the appearance of a new proximal cluster in the phasor fingerprint of RBCs in the presence of 1b and a shorter fluorescence lifetime of RBCs in the presence of 1a.

Xenopus gpx3 Mediates Posterior Development by Regulating Cell Death during Embryogenesis.

Glutathione peroxidase 3 (GPx3) belongs to the glutathione peroxidase family of selenoproteins and is a key antioxidant enzyme in multicellular organisms against oxidative damage. Downregulation of GPx3 affects tumor progression and metastasis and is associated with liver and heart disease. However, the physiological significance of GPx3 in vertebrate embryonic development remains poorly understood. The current study aimed to investigate the functional roles of gpx3 during embryogenesis. To this end, we determined gpx3’s spatiotemporal expression using Xenopus laevis as a model organism. Using reverse transcription polymerase chain reaction (RT-PCR), we demonstrated the zygotic nature of this gene. Interestingly, the expression of gpx3 enhanced during the tailbud stage of development, and whole mount in situ hybridization (WISH) analysis revealed gpx3 localization in prospective tail region of developing embryo. gpx3 knockdown using antisense morpholino oligonucleotides (MOs) resulted in short post-anal tails, and these malformed tails were significantly rescued by glutathione peroxidase mimic ebselen. The gene expression analysis indicated that gpx3 knockdown significantly altered the expression of genes associated with Wnt, Notch, and bone morphogenetic protein (BMP) signaling pathways involved in tailbud development. Moreover, RNA sequencing identified that gpx3 plays a role in regulation of cell death in the developing embryo. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and phospho-histone 3 (PH3) staining confirmed the association of gpx3 knockdown with increased cell death and decreased cell proliferation in tail region of developing embryos, establishing the involvement of gpx3 in tailbud development by regulating the cell death. Furthermore, these findings are inter-related with increased reactive oxygen species (ROS) levels in gpx3 knockdown embryos, as measured by using a redox-sensitive fluorescent probe HyPer. Taken together, our results suggest that gpx3 plays a critical role in posterior embryonic development by regulating cell death and proliferation during vertebrate embryogenesis.

Evaluation of Ebselen-azadioxatriangulenium as redox-sensitive fluorescent intracellular probe and as indicator within a planar redox optode

Redox homeostasis is essential for cellular survival and is therefore tightly regulated. Visualizing changes in the local redox state is essential for detecting oxidative stress, especially with the help of red, long emission lifetime probes. We synthesized a new reversible, redox-sensitive fluorescent probe, ebselen-azadioxatriangulenium (Ebselen-ADOTA), and characterized its potential use for intra- and extracellular redox sensing. The probe consists of a photostable triangulenium-based fluorophore (emission maximum at 564 nm) modulated by a redox-active ebselen motif. The reduced form only shows around 1/10 of the emission intensity of the oxidized form. The Ebselen-ADOTA probe exhibited reversible oxidation and reduction in solution under extracellular conditions. In addition, Ebselen-ADOTA can visualize an increase in oxidative stress within anaerobic bacteria when exposing them to air, the probe did not readily respond to changes in the redox environment inside mammalian cells, probably due to unfavorable localization, aggregation or binding to cysteine containing proteins. When incorporated inside a polymer matrix (hydrogel D4 mixed with poly(acrylic acid)), the probe showed a rapid and fully reversible response to redox changes, thus enabling the preparation of a redox-sensitive optode.

Mechanisms and Applications of Redox-Sensitive Green Fluorescent Protein-Based Hydrogen Peroxide Probes.

Genetically encoded hydrogen peroxide (H2O2) sensors, based on fusions between thiol peroxidases and redox-sensitive green fluorescent protein 2 (roGFP2), have dramatically broadened the available "toolbox" for monitoring cellular H2O2 changes. Recent Advances: Recently developed peroxiredoxin-based probes such as roGFP2-Tsa2ΔCR offer considerably improved H2O2 sensitivity compared with previously available genetically encoded sensors and now permit dynamic, real-time, monitoring of changes in endogenous H2O2 levels. The correct understanding and interpretation of probe read-outs is crucial for their meaningful use. We discuss probe mechanisms, potential pitfalls, and best practices for application and interpretation of probe responses and highlight where gaps in our knowledge remain. The full potential of the newly available sensors remains far from being fully realized and exploited. We discuss how the ability to monitor basal H2O2 levels in real time now allows us to re-visit long-held ideas in redox biology such as the response to ischemia-reperfusion and hypoxia-induced reactive oxygen species production. Further, recently proposed circadian cycles of peroxiredoxin hyperoxidation might now be rigorously tested. Beyond their application as H2O2 probes, roGFP2-based H2O2 sensors hold exciting potential for studying thiol peroxidase mechanisms, inactivation properties, and the impact of post-translational modifications, in vivo. Antioxid. Redox Signal. 29, 552-568.

In vitro antioxidant/prooxidant effects of combined use of flavonoids

The present study was undertaken to investigate the individual and combined antioxidant or prooxidant effects of genistein, daidzein and quercetin in human erythrocytes and rat microsomes in vitro. Their reducing potential against oxidation of a redox sensitive fluorescent probe, their protective effect against H2O2-induced membrane lipid peroxidation and their inhibitory effect on AAPH-induced hemolysis were evaluated. Genistein and daidzein were prooxidant in erythrocytes but antioxidant in microsomes where their metabolites might have been formed which suggests the importance of metabolic capacity in in vitro models to predict the physiological situation. Quercetin showed antioxidant effects in all models and conditions. Prooxidant effect of ‘genistein–daidzein mixture’, at their concentrations reflecting the real life, was suppressed by addition of quercetin to the mixture. Our study shows that flavonoids can exert prooxidant effects depending on the conditions, but the mixture effect should be considered while assessing their effects and safety in humans.

Novel indole-based melatonin analogues: Evaluation of antioxidant activity and protective effect against amyloid β-induced damage

Oxidative stress has been recognized as a contributing factor in ageing and various diseases including cancer and neuropathological disorders. Indole derivatives such as the neurohormone melatonin (MLT) constitute an important class of therapeutic agent in medicinal chemistry. MLT can scavenge different reactive oxygen species and can also stimulate the synthesis of antioxidant enzymes. As a part of our ongoing studies, a series of new indole-based hydrazide/hydrazone derivatives were synthesized as MLT analogues. Their antioxidant activity was investigated in human erythrocytes by evaluating their reducing effect against oxidation of a redox-sensitive fluorescent probe. Possible inherent cytotoxicity of the compounds was investigated in CHO-K1 cells by lactate dehydrogenase leakage test. Protection of neuronal PC12 cells against amyloid β-induced damage was examined by MTT assay and their ability in reduction of ROS generation induced by amyloid β was tested. MLT analogues having an o-halogenated aromatic moiety exhibited effective antioxidant properties without having any membrane-damaging effect. Moreover, derivatives having o-halogenated and dihalogenated aromatic side chain significantly protected neuronal cells at concentrations of 10 and 100μM. In conclusion, MLT derivatives represent promising scaffolds for discovery of effective antioxidant and neuroprotective agents.

Editorial: Redox Regulation in Skeletal Muscle Aging and Exercise.

Editorial: Redox Regulation in Skeletal Muscle Aging and Exercise.

Novel indole-based melatonin analogues substituted with triazole, thiadiazole and carbothioamides: studies on their antioxidant, chemopreventive and cytotoxic activities

Melatonin (MLT) is a well-known free-radical scavenger, involving in the prevention of cellular damage that can lead to cancer, ageing and a variety of neurodegenerative diseases. Research on MLT-related compounds has been required to optimise the maximum pharmaceutical activity with the lowest side effects. In our ongoing research, we have synthesized new indole-based MLT analogues as potential antioxidant agents by modifying the MLT molecule. In this study, we build on previous findings, through the synthesis, characterization and in vitro antioxidant profiling of a series of new indole-based MLT analogues which possess triazole, thiadiazole and carbothioamides on the third position on the indole ring. In vitro antioxidant activity was investigated by evaluating their reducing effect against oxidation of a redox sensitive fluorescent probe and their radical scavenging activity was assessed via the DPPH assay. In addition, in vitro cytotoxic effects of newly synthesized compounds were investigated in CHO-K1 cells using the MTT assay.

Abstract 915: Effects of black raspberry extract (BRB) and related compounds on mutagenesis induced by metabolites of the tobacco carcinogen, dibenzo(a,l)pyrene in cultured rat oral fibroblasts; and on oxidative stress in human oral cancer cells

Abstract BRB powder, extracts and metabolites have been shown to inhibit tumorigenesis in oral and other sites in animal models. Here we examined the effects of an anthocyanin-enriched BRB extract, and protocatechuic acid (PA), a major BRB anthocyanin metabolite, on mutagenesis and DNA adduct formation by DBP-dihydrodiol, and DBP diolepoxide (DBPDE) (the proximate and ultimate carcinogenic metabolites, resp. of DBP) in lacI rat oral fibroblasts. DBP was poorly metabolized to genotoxic metabolites by the oral fibroblasts, but DBP-dihydrodiol was mutagenic and produced DNA adducts. DBP-dihydrodiol (0.5 uM) induced a mutant fraction of 14.1 +/- 3.9 mutants/10E5 pfu in the target cII gene. This was reduced in a concentration-dependent fashion to 9.1 +/- 2.4 by 0.1-1uM PA– a 35% reduction. Mutagenesis by DBP-dihydrodiol was also reduced in a concentration-dependent fashion by BRB extract and at the highest concentration (20ug/ml) mutagenesis was reduced by 68%. The major DNA adduct produced by 0.5 uM DBP-dihydrodiol was anti-DBPDE-dA, as assessed by L.C.-M.S.-M.S. Its levels were reduced from 13.7 +/- 1.2 to 7.8 +/- 1.9 adducts/10E6 dA by 3uM protocatechuic acid, and from 6.8 +/- 0.7 to 3.2 +/- 0.4 by 66 ug/ml BRB extract. The inhibitions were statistically significant. In contrast, no significant effect PA on mutagenesis by 140 nM DBPDE was observed, with mutant fractions similar to that of DBPDE (3.2 +/- 0.45 mutants/10E5 pfu) being obtained over a range of 0.1 - 1 uM PA. Similarly 10-50 ug/ml BRB extract had no effect on DBPDE-induced mutagenesis. Consistent with these results, levels of total DBPDE adducts were unchanged over a range of PA concentrations, with the high dose of 1uM PA yielding, 4.6 +/- 0.7 adducts/10E5 nucleotides and 4.5 +/- 0.9 without PA. Presumably the short halflife of DBPDE precludes detoxification, or scavenging of DBPDE. The effects of PA and BRB extract on oxidative stress in a human oral cancer cell line (SCC 1483, retromolar trigone) were also examined. The redox sensitive fluorescent probe, dihydrodichlorofluorescein diacetate (DHDCF) was added to the cells at 5uM and BRB (0.5-5 ug/ml) or PA (0.02-0.2 uM) was added shortly afterwards. 2 hr later all doses of BRB extract and PA inhibited the oxidation of DHDCF to its fluorescent product, in a dose-dependent manner. At 5ug/ml BRB extract the increase in fluorescence was reduced from 304 +/- 3.3 to 177 +/- 7.1 relative fluorescence units (RFU) and at 0.2 uM PA the increase was reduced to 224 +/- 25 RFU. In summary, BRB extract and PA inhibited mutagenesis and DNA adduct formation by DBP-dihydrodiol, but not DBPDE. BRB extract and PA were also effective inhibitors of oxidative stress. These mechanisms may a play a role in the protective effects of BRB on oral and other cancers in experimental animals. Supported by NIH grant #CA173465. Citation Format: Joseph B. Guttenplan, Seungjin Kim, Ying Zhou, Wieslawa Kosinska, Ana-Vera Golgotiu, Shantu Amin, Gary D. Stoner, Yuan-Wan Sun, Kun-Ming Chen, Karam El-Bayoumy. Effects of black raspberry extract (BRB) and related compounds on mutagenesis induced by metabolites of the tobacco carcinogen, dibenzo(a,l)pyrene in cultured rat oral fibroblasts; and on oxidative stress in human oral cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 915. doi:10.1158/1538-7445.AM2015-915

Synthesis and evaluation of antioxidant activity of new quinoline-2-carbaldehyde hydrazone derivatives: bioisosteric melatonin analogues

Overproduction of reactive oxygen species results in oxidative stress that can cause fatal damage to vital cell structures. It is known that the use of antioxidants could be beneficial in the prevention or delay of numerous diseases associated with oxidative stress. Melatonin (MLT) is known as a powerful free-radical scavenger and antioxidant. It was found that indole ring of MLT can be employed by bioisosteric replacement by other aromatic rings. Quinoline derivatives constitute an important class of compounds for new drug development. Owing to quinoline and hydrazones appealing physiological properties and are mostly found in numerous biologically active compounds a series of quinoline-2-carbaldehyde hydrazone derivatives were synthesized as bioisosteric analogues of MLT, characterized and in vitro antioxidant activity was investigated by evaluating their reducing effect against oxidation of a redox-sensitive fluorescent probe. Cytotoxicity potential of all compounds was investigated both by lactate dehydrogenase leakage assay and by MTT assay.

Redox-based selective fluorometric detection of homocysteine.

Plasma homocysteine (Hcy) is an important risk factor for various diseases. A novel redox-sensitive fluorescent probe is developed for the selective detection of Hcy. A linear calibration curve has been obtained in buffer and plasma for the quantitative determination of Hcy in such media.

Lipopolysaccharides elicit an oxidative burst as a component of the innate immune system in the seagrass Thalassia testudinum

This study represents the first report characterizing the biological effects of a lipopolysaccharide (LPS) immune modulator on a marine vascular plant. LPS was shown to serve as a strong elicitor of the early defense response in the subtropical seagrass Thalassia testudinum Banks ex König and was capable of inducing an oxidative burst identified at the single cell level. The formation of reactive oxygen species (ROS), detected by a redox-sensitive fluorescent probe and luminol-based chemiluminescence, included a diphenyleneiodonium sensitive response, suggesting the involvement of an NADPH oxidase. A 900bp cDNA fragment coding for this enzyme was sequenced and found to encode a NAD binding pocket domain with extensive homology to the Arabidopsis thaliana rbohF (respiratory burst oxidase homolog) gene. The triggered release of ROS occurred at 20min post-elicitation and was dose-dependent, requiring a minimal threshold of 50μg/mL LPS. Pharmacological dissection of the early events preceding ROS emission indicated that the signal transduction chain of events involved extracellular alkalinization, G-proteins, phospholipase A2, as well as K(+), Ca(2+), and anion channels. Despite exclusively thriving in a marine environment, seagrasses contain ROS-generating machinery and signal transduction components that appear to be evolutionarily conserved with the well-characterized defense response systems found in terrestrial plants.

Kaempferol Inhibits P. intermedia Lipopolysaccharide‐Induced Production of Nitric Oxide Through Translational Regulation in Murine Macrophages: Critical Role of Heme Oxygenase‐1‐Mediated ROS Reduction

Nitric oxide (NO) could be a potential target for the development of new therapeutic approaches to the treatment of periodontal disease because this molecule plays a significant role in the tissue destruction observed in periodontitis. In this study, the authors investigate the effect of kaempferol on the production of NO by murine macrophage-like RAW264.7 cells stimulated with lipopolysaccharide (LPS) from Prevotella intermedia, a pathogen implicated in periodontal disease, and try to determine the underlying mechanisms of action. NO production was assayed by measuring the accumulation of nitrite in culture supernatants. Real-time polymerase chain reaction was performed to quantify inducible NO synthase (iNOS) and heme oxygenase-1 (HO-1) mRNA expression. iNOS and HO-1 protein expression and phosphorylation of c-Jun N-terminal kinase and p38 were characterized via immunoblot analysis. Reactive oxygen species (ROS) production was measured using the redox-sensitive fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate. Kaempferol significantly inhibited NO production and expression of iNOS protein in P. intermedia LPS-stimulated RAW246.7 cells without affecting iNOS mRNA expression. Kaempferol upregulated HO-1 expression in LPS-activated cells. Inhibition of HO-1 activity by tin protoporphyrin IX (SnPP) abolished the suppressive effect of kaempferol on NO production. In addition, kaempferol significantly attenuated P. intermedia LPS-induced increase of intracellular ROS, and SnPP blocked this reduction. Treatment with antioxidants downregulated the production of LPS-induced NO. Kaempferol inhibits NO production and iNOS protein expression in P. intermedia LPS-stimulated RAW264.7 cells at the translational level via HO-1-mediated ROS reduction and could be an efficient modulator of host response in the treatment of periodontal disease.

Antioxidant activity of indole-based melatonin analogues in erythrocytes and their voltammetric characterization

Melatonin (MLT) is a strong free-radical scavenger, which protects the body from the effects of oxidants. In recent years, MLT have been described resulting in much attention in the development of synthetic compounds possessing. As a part of our ongoing study a series of indole-based MLT analogue hydrazide/hydrazone derivatives were synthesized, characterized and in vitro antioxidant activity was investigated by evaluating their reducing effect against oxidation of a redox sensitive fluorescent probe. Membrane stabilizing effect of all compounds was also investigated by lactate dehydrogenase leakage assay. Furthermore voltammetric methods have been applied to the synthesized compounds to characterize oxidation potentials to get insight into their metabolism owing to the oxidation mechanisms taking place at the electrode and in the body share similar principles.

Oxidative burst inPorphyra haitanensis(Rhodophyta)

Oligosaccharides could induce some physiological responses in algae to defend attacks from pathogens. One of the earliest responses induced by oligosaccharides was so-called oxidative burst. This study has surveyed the recognition and oxidative burst response of Porphyra haitanenesis to agaro-oligosaccharide. Oxygen consumption was detected by an oxygen electrode. H2O2 concentrations in the medium were determined by measuring the dimerization of (p-hydroxyphenyl) acetic acid. The elicited oxidative burst was observed by loading the P. haitanenesis with redox-sensitive fluorescent probe dichlorohydrofluorescin (DCFH-DA). Gene expression level of NADPH oxidase (named Phrboh) was detected by real-time PCR. 2-AMAC labeled agaro-oligosaccharides were used to research the binding of agaro-oligosaccharide on algae. Results showed that two respiration peaks were observed after addition of 100 μg/mL agaro-oligosaccharide in P. haitanensis. Fourfold increase occurred in the first respiration peak 4 minutes after the addition, and fourteen times higher in the second response 10 minutes after the exposure to agaro-oligosaccharide. The accumulation of H2O2 in the culture medium was detected 5 minutes after challenged with agaro-oligosaccharide. The concentration of H2O2 reached its peak after 15 minutes, which was nearly ten times higher than control. The gene expression level of NADPH oxidase in P. haitanensis was up-regulated 3 minutes after the addition of agaro-oligosaccharide, suggesting that the synthesis of H2O2 was closely related to the activation of Phrboh. Based on the analyses of the fluorescence of redox-sensitive dye, activated oxygen species mainly accumulated around the plasma membrane. Under the fluorescent microscope, specific binding of 2-AMAC labeled agaro-oligosaccharide on cell plasma membrane was also observed, which indicated that there was recognition site of agaro-oligosaccharide on P. haitanensis plasma membrane. In summary, P. haitanensis could recognize agaro-oligosaccharide and respond with oxidative burst which was associated with the activation of NADPH oxidase.

Mitochondrial fragmentation causes cellular acidification in the nematode C. elegans

Mitochondrial structural dynamics (MSD) occur through the opposing processes of membrane fission and fusion. Diseases such as autosomal dominant optic atrophy and Charcot‐Marie‐Tooth syndrome Type 2A result from haploinsufficiency at genetic loci whose gene products regulate MSD and null mutations in these genes are lethal in mice. However, the nematode C. elegans tolerates a complete loss of fission and fusion proteins, and the physiologic consequences of MSD loss‐of‐function (lf) can be studied in a whole animal genetic model. Dynamic fluorescent imaging of genetically‐encoded biosensors in live transgenic worms indicates that mitochondrial fragmentation caused by mutation of the profusion gene eat‐3 (Opa1 in man) induces cellular acidification in multiple tissues. Epistasis analysis further suggests that fragmentation itself rather than an unrelated protein function is causative. The results of dichloroacetate supplementation experiments demonstrate that it is unlikely that the acidosis is caused by lactate accumulation, while AMP/ATP ratios suggest that basal energy balance is conserved in MSD mutants. However, using a redox‐sensitive fluorescent probe we have found that the mitochondrial matrix in eat‐3lf mutants is significantly oxidized compared to that of wildtype worms. We are currently exploring the hypothesis that reactive oxygen species play a role in the acidification. Support: USPHS F31GM084506 (DWJ).

Novel Indole-Based Analogs of Melatonin: Synthesis and in Vitro Antioxidant Activity Studies

The aim of this study was to synthesize and examine possible in vitro antioxidant effects of indole-based melatonin analogue compounds. As a part of our ongoing study nineteen indole hydrazide/hydrazone derivatives were synthesized, characterized and their in vitro antioxidant activity was investigated by three different assays: by evaluating their reducing effect against oxidation of a redox sensitive fluorescent probe, by examining their protective effect against H2O2-induced membrane lipid peroxidation and by determining their inhibitory effect on AAPH–induced hemolysis of human erythrocytes. The results indicated significant strong antioxidant activity for most of the compounds, when compared to melatonin.