Induction of extracellular ATP mediates increase in intracellular thioredoxin in RAW264.7 cells exposed to low-dose γ-rays.

PubMed

Ohshima, Yasuhiro; Kitami, Akihiro; Kawano, Ayumi; Tsukimoto, Mitsutoshi; Kojima, Shuji

2011-09-15

We previously showed that low doses (0.25-0.5 Gy) of γ-rays elevated thioredoxin (Trx-1) in various organs of mice after whole-body irradiation. Also, it is reported that extracellular ATP, which is released in response to various stresses, regulates the expression of intracellular antioxidants through activation of P2 receptors. We have recently found that low-dose γ-rays induce ATP release from the exposed cells. However, it is not yet clear whether the radiation-induced extracellular ATP modulates the cellular redox balance. Here, we investigated whether γ-ray irradiation-induced release of extracellular ATP contributes to the induction of the cellular antioxidant Trx-1, using mouse macrophage-like RAW264.7 cells. Irradiation with γ-rays or exogenously added ATP increased the expression of Trx-1, and in both cases the increase was blocked by pretreatment with an ectonucleotidase, apyrase. Then, the involvement of ATP-dependent reactive oxygen species (ROS) generation in the increase in antioxidant capacity was examined. ATP stimulation promoted the generation of intracellular ROS and also increased Trx-1 expression. The increase in Trx-1 expression was significantly suppressed by pretreatment of the cells with antioxidants. In conclusion, the γ-ray irradiation-induced release of extracellular ATP may, at least in part, contribute to the production of ROS via purinergic signaling, leading to promotion of intracellular antioxidants as an adaptive response to an oxidative stress. Copyright © 2011 Elsevier Inc. All rights reserved.

Hydrogen peroxide generated by xanthine/xanthine oxidase system represses the proliferation of colorectal cancer cell line Caco-2.

PubMed

Sakuma, Satoru; Abe, Muneyuki; Kohda, Tetsuya; Fujimoto, Yohko

2015-01-01

The twin character of reactive oxygen species is substantiated by a growing body of evidence that reactive oxygen species within cells act as inducers and accelerators of the oncogenic phenotype of cancer cells, while reactive oxygen species can also induce cancer cell death and can therefore function as anti-tumorigenic species. The aim of this study was to assess a possible influence of xanthine/xanthine oxidase on the proliferation of colorectal cancer cell line Caco-2. xanthine/xanthine oxidase (2.5 µM/0.25 mU/ml-25 µM/2.5 mU/ml) dose-dependently inhibited the proliferation of Caco-2 cells. Experiments utilizing reactive oxygen species scavengers (superoxide dismutase, catalase and mannitol) and exogenous hydrogen peroxide revealed a major role of hydrogen peroxide in the xanthine/xanthine oxidase effect. Investigations utilizing annexin V-fluorescein/PI assay using flow cytometry, and the lactate dehydrogenase extracellular release assay indicated that hydrogen peroxide induced necrosis, but not apoptosis, in Caco-2 cells. These results suggest that hydrogen peroxide generated by xanthine/xanthine oxidase has the potential to suppress colorectal cancer cell proliferation.

Benzo(a)pyrene quinones increase cell proliferation, generate reactive oxygen species, and transactivate the epidermal growth factor receptor in breast epithelial cells.

PubMed

Burdick, Andrew D; Davis, John W; Liu, Ke Jian; Hudson, Laurie G; Shi, Honglian; Monske, Michael L; Burchiel, Scott W

2003-11-15

Polycyclic aromatic hydrocarbons, such as benzo(a)pyrene (BaP), are known mammary carcinogens in rodents and may be involved in human breast cancer. We have reported previously that BaP can mimic growth factor signaling and increase cell proliferation in primary human mammary epithelial cells and the human mammary epithelial cell line MCF-10A. BaP-quinones (BPQs) are important metabolites of BaP that have been associated with the production of reactive oxygen species. Using a model of epidermal growth factor (EGF) withdrawal in MCF-10A, we hypothesized that production of reactive oxygen species by BPQs could lead to the activation of the EGF receptor (EGFR). Here, we demonstrate through electron paramagnetic resonance spectroscopy and flow cytometry that 1,6-BPQ and 3,6-BPQ produce superoxide anion and hydrogen peroxide in MCF-10A cells. Furthermore, we show that BPQs increase EGFR, Akt, and extracellular signal-regulated kinase activity, leading to increased cell number in the absence of EGF. The BPQ-induced EGFR activity and associated cell proliferation were attenuated by the EGFR inhibitor AG1478, as well as by the antioxidant N-acetyl cysteine. Overexpression of catalase, but not Cu/Zn superoxide dismutase, reduced the extent of BPQ-dependent increased cell number and EGFR pathway activation. Moreover, the direct treatment of MCF-10A cells with hydrogen peroxide enhanced EGFR, Akt, and extracellular-regulated kinase phosphorylation that could be similarly inhibited by AG1478, N-acetyl cysteine, and catalase. Taken together, these data indicate that BPQs, through the generation of hydrogen peroxide, activate the EGFR in MCF-10A cells, leading to increased cell number under EGF-deficient conditions.

Extracellular calcium triggers unique transcriptional programs and modulates staurosporine-induced cell death in Neurospora crassa

PubMed Central

Gonçalves, A. P.; Monteiro, João; Lucchi, Chiara; Kowbel, David J.; Cordeiro, J. M.; Correia-de-Sá, Paulo; Rigden, Daniel J.; Glass, N. L.; Videira, Arnaldo

2014-01-01

Alterations in the intracellular levels of calcium are a common response to cell death stimuli in animals and fungi and, particularly, in the Neurospora crassa response to staurosporine. We highlight the importance of the extracellular availability of Ca2+ for this response. Limitation of the ion in the culture medium further sensitizes cells to the drug and results in increased accumulation of reactive oxygen species (ROS). Conversely, an approximately 30-fold excess of external Ca2+ leads to increased drug tolerance and lower ROS generation. In line with this, distinct staurosporine-induced cytosolic Ca2+ signaling profiles were observed in the absence or presence of excessive external Ca2+. High-throughput RNA sequencing revealed that different concentrations of extracellular Ca2+ define distinct transcriptional programs. Our transcriptional profiling also pointed to two putative novel Ca2+-binding proteins, encoded by the NCU08524 and NCU06607 genes, and provides a reference dataset for future investigations on the role of Ca2+ in fungal biology. PMID:28357255

Hypochlorous acid regulates neutrophil extracellular trap release in humans

PubMed Central

Palmer, L J; Cooper, P R; Ling, M R; Wright, H J; Huissoon, A; Chapple, I L C

2012-01-01

Neutrophil extracellular traps (NETs) comprise extracellular chromatin and granule protein complexes that immobilize and kill bacteria. NET release represents a recently discovered, novel anti-microbial strategy regulated non-exclusively by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase generation of reactive oxygen intermediates (ROIs), particularly hydrogen peroxide. This study aimed to characterize the role of ROIs in the process of NET release and to identify the dominant ROI trigger. We employed various enzymes, inhibitors and ROIs to record their effect fluorometrically on in vitro NET release by human peripheral blood neutrophils. Treatment with exogenous superoxide dismutase (SOD) supported the established link between hydrogen peroxide and NET production. However, treatment with myeloperoxidase inhibitors and direct addition of hypochlorous acid (HOCl; generated in situ from sodium hypochlorite) established that HOCl was a necessary and sufficient ROI for NET release. This was confirmed by the ability of HOCl to stimulate NET release in chronic granulomatous disease (CGD) patient neutrophils which, due to the lack of a functional NADPH oxidase, also lack the capacity for NET release in response to classical stimuli. Moreover, the exogenous addition of taurine, abundantly present within the neutrophil cytosol, abrogated NET production stimulated by phorbol myristate acetate (PMA) and HOCl, providing a novel mode of cytoprotection by taurine against oxidative stress by taurine. PMID:22236002

Tangeretin reduces ultraviolet B (UVB)-induced cyclooxygenase-2 expression in mouse epidermal cells by blocking mitogen-activated protein kinase (MAPK) activation and reactive oxygen species (ROS) generation.

PubMed

Yoon, Ji Hye; Lim, Tae-Gyu; Lee, Kyung Mi; Jeon, Ae Ji; Kim, Su Yeon; Lee, Ki Won

2011-01-12

The present study examined the effects of tangeretin, a polymethoxylated flavonone present in citrus fruits, on ultraviolet B (UVB)-induced cyclooxygenase-2 (COX-2) expression in JB6 P+ mouse skin epidermal cells. Tangeretin suppressed UVB-induced COX-2 expression and transactivation of nuclear factor-κB and activator protein-1 in JB6 P+ cells. Moreover, tangeretin blocked UVB-induced phosphorylation of Akt and mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated protein kinase, c-Jun N-terminal kinase, and p38, and attenuated the phosphorylation of MAPK kinases 1/2, 3/6, and 4. Tangeretin also limited the endogenous generation of reactive oxygen species (ROS), thereby protecting the cells against oxidative stress. However, tangeretin did not scavenge the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and influence the nicotinamide adenine dinucleotide phosphate oxidase activity. These results suggest that the anti-inflammatory effects of tangeretin stem from its modulation of cell signaling and suppression of intracellular ROS generation. Tangeretin may have a potent chemopreventive effect in skin cancer.

Impaired neutrophil extracellular trap formation: a novel defect in the innate immune system of aged individuals.

PubMed

Hazeldine, Jon; Harris, Phillipa; Chapple, Iain L; Grant, Melissa; Greenwood, Hannah; Livesey, Amy; Sapey, Elizabeth; Lord, Janet M

2014-08-01

Neutrophil extracellular traps (NETs) are a recently discovered addition to the defensive armamentarium of neutrophils, assisting in the immune response against rapidly dividing bacteria. Although older adults are more susceptible to such infections, no study has examined whether aging in humans influences NET formation. We report that TNF-α-primed neutrophils generate significantly more NETs than unprimed neutrophils and that lipopolysaccharide (LPS)- and interleukin-8 (IL-8)-induced NET formation exhibits a significant age-related decline. NET formation requires generation of reactive oxygen species (ROS), and this was also reduced in neutrophils from older donors identifying a mechanism for reduced NET formation. Expression of IL-8 receptors (CXCR1 and CXCR2) and the LPS receptor TLR4 was similar on neutrophils from young and old subjects, and neutrophils challenged with phorbol-12-myristate-13-acetate (PMA) showed no age-associated differences in ROS or NET production. Taken together, these data suggest a defect in proximal signalling underlies the age-related decline in NET and ROS generation. TNF-α priming involves signalling through p38 MAP kinase, but activation kinetics were comparable in neutrophils from young and old donors. In a clinical setting, we assessed the capacity of neutrophils from young and older patients with chronic periodontitis to generate NETs in response to PMA and hypochlorous acid (HOCL). Neutrophil extracellular trap generation to HOCL, but not PMA, was lower in older periodontitis patients but not in comparison with age-matched controls. Impaired NET formation is thus a novel defect of innate immunity in older adults but does not appear to contribute to the increased incidence of periodontitis in older adults. © 2014 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

Impaired neutrophil extracellular trap formation: a novel defect in the innate immune system of aged individuals

PubMed Central

Hazeldine, Jon; Harris, Phillipa; Chapple, Iain L; Grant, Melissa; Greenwood, Hannah; Livesey, Amy; Sapey, Elizabeth; Lord, Janet M

2014-01-01

Neutrophil extracellular traps (NETs) are a recently discovered addition to the defensive armamentarium of neutrophils, assisting in the immune response against rapidly dividing bacteria. Although older adults are more susceptible to such infections, no study has examined whether aging in humans influences NET formation. We report that TNF-α-primed neutrophils generate significantly more NETs than unprimed neutrophils and that lipopolysaccharide (LPS)- and interleukin-8 (IL-8)-induced NET formation exhibits a significant age-related decline. NET formation requires generation of reactive oxygen species (ROS), and this was also reduced in neutrophils from older donors identifying a mechanism for reduced NET formation. Expression of IL-8 receptors (CXCR1 and CXCR2) and the LPS receptor TLR4 was similar on neutrophils from young and old subjects, and neutrophils challenged with phorbol-12-myristate-13-acetate (PMA) showed no age-associated differences in ROS or NET production. Taken together, these data suggest a defect in proximal signalling underlies the age-related decline in NET and ROS generation. TNF-α priming involves signalling through p38 MAP kinase, but activation kinetics were comparable in neutrophils from young and old donors. In a clinical setting, we assessed the capacity of neutrophils from young and older patients with chronic periodontitis to generate NETs in response to PMA and hypochlorous acid (HOCL). Neutrophil extracellular trap generation to HOCL, but not PMA, was lower in older periodontitis patients but not in comparison with age-matched controls. Impaired NET formation is thus a novel defect of innate immunity in older adults but does not appear to contribute to the increased incidence of periodontitis in older adults. PMID:24779584

Quantifying intracellular hydrogen peroxide perturbations in terms of concentration

PubMed Central

Huang, Beijing K.; Sikes, Hadley D.

2014-01-01

Molecular level, mechanistic understanding of the roles of reactive oxygen species (ROS) in a variety of pathological conditions is hindered by the difficulties associated with determining the concentration of various ROS species. Here, we present an approach that converts fold-change in the signal from an intracellular sensor of hydrogen peroxide into changes in absolute concentration. The method uses extracellular additions of peroxide and an improved biochemical measurement of the gradient between extracellular and intracellular peroxide concentrations to calibrate the intracellular sensor. By measuring peroxiredoxin activity, we found that this gradient is 650-fold rather than the 7–10-fold that is widely cited. The resulting calibration is important for understanding the mass-action kinetics of complex networks of redox reactions, and it enables meaningful characterization and comparison of outputs from endogenous peroxide generating tools and therapeutics across studies. PMID:25460730

The biofilm matrix polysaccharides cellulose and alginate both protect Pseudomonas putida mt-2 against reactive oxygen species generated under matric stress and copper exposure.

PubMed

Svenningsen, Nanna B; Martínez-García, Esteban; Nicolaisen, Mette H; de Lorenzo, Victor; Nybroe, Ole

2018-06-01

In natural environments most bacteria live in biofilms embedded in complex matrices of extracellular polymeric substances (EPS). This lifestyle is known to increase protection against environmental stress. Pseudomonas putida mt-2 harbours genes for the production of at least four different EPS polysaccharides, including alginate and cellulose. Little is known about the functional properties of cellulose, while alginate attenuates the accumulation of reactive oxygen species (ROS) caused by matric stress. By using mutants that are deficient in either alginate or cellulose production we show that even cellulose attenuates the accumulation of matric stress-induced ROS for cells in biofilms. Further, both cellulose and alginate attenuate ROS generated through exposure to copper. Interestingly, the two EPS polysaccharides protect cells in both liquid culture and in biofilms against ROS caused by matric stress, indicating that cellulose and alginate do not need to be produced as an integral part of the biofilm lifestyle to provide tolerance towards environmental stressors.

The role of extracellular conductivity profiles in compartmental models for neurons: particulars for layer 5 pyramidal cells.

PubMed

Wang, Kai; Riera, Jorge; Enjieu-Kadji, Herve; Kawashima, Ryuta

2013-07-01

With the rapid increase in the number of technologies aimed at observing electric activity inside the brain, scientists have felt the urge to create proper links between intracellular- and extracellular-based experimental approaches. Biophysical models at both physical scales have been formalized under assumptions that impede the creation of such links. In this work, we address this issue by proposing a multicompartment model that allows the introduction of complex extracellular and intracellular resistivity profiles. This model accounts for the geometrical and electrotonic properties of any type of neuron through the combination of four devices: the integrator, the propagator, the 3D connector, and the collector. In particular, we applied this framework to model the tufted pyramidal cells of layer 5 (PCL5) in the neocortex. Our model was able to reproduce the decay and delay curves of backpropagating action potentials (APs) in this type of cell with better agreement with experimental data. We used the voltage drops of the extracellular resistances at each compartment to approximate the local field potentials generated by a PCL5 located in close proximity to linear microelectrode arrays. Based on the voltage drops produced by backpropagating APs, we were able to estimate the current multipolar moments generated by a PCL5. By adding external current sources in parallel to the extracellular resistances, we were able to create a sensitivity profile of PCL5 to electric current injections from nearby microelectrodes. In our model for PCL5, the kinetics and spatial profile of each ionic current were determined based on a literature survey, and the geometrical properties of these cells were evaluated experimentally. We concluded that the inclusion of the extracellular space in the compartmental models of neurons as an extra electrotonic medium is crucial for the accurate simulation of both the propagation of the electric potentials along the neuronal dendrites and the neuronal reactivity to an electrical stimulation using external microelectrodes.

[Cell surface peroxidase--generator of superoxide anion in wheat root cells under wound stress].

PubMed

Chasov, A V; Gordon, L Kh; Kolesnikov, O P; Minibaeva, F V

2002-01-01

Development of wound stress in excised wheat roots is known to be accompanied with an increase in reactive oxygen species (ROS) production, fall of membrane potential, release of K+ from cells, alkalization of extracellular solution, changes in respiration and metabolism of structural lipids. Dynamics of superoxide release correlates with changes in other physiological parameters, indicating the cross-reaction of these processes. Activity of peroxidase in extracellular solution after a 1 h incubation and removal of roots was shown to be stimulated by the range of organic acids, detergents, metals, and to be inhibited by cyanide. Superoxide production was sensitive to the addition of Mn2+ and H2O2. Increase in superoxide production correlates with the enhancement of peroxidase activity at the application of organic acids and detergents. The results obtained indicate that cell surface peroxidase is one of the main generators of superoxide in wounded wheat root cells. Different ways of stimulation of the ROS producing activity in root cells is supposed. By controlling superoxide and hydrogen peroxide formation, the cell surface peroxidase can control the adaptation processes in stressed plant cells.

Glycoconjugates and methods

DOEpatents

Bertozzi, Carolyn C [Albany, CA; Yarema, Kevin J [Albany, CA; Mahal, Lara K [Berkeley, CA

2008-04-01

Methods for making the functionalized glycoconjugates include (a) contacting a cell with a first monosaccharide, and (b) incubating the cell under conditions whereby the cell (i) internalizes the first monosaccharide, (ii) biochemically processes the first monosaccharide into a second saccharide, (iii) conjugates the saccharide to a carrier to form a glycoconjugate, and (iv) extracellularly expresses the glycoconjugate to form an extracellular glycoconjugate comprising a selectively reactive functional group. Methods for forming products at a cell further comprise contacting the functional group of the extracellularly expressed glycoconjugate with an agent which selectively reacts with the functional group to form a product. Subject compositions include cyto-compatible monosaccharides comprising a nitrogen or ether linked functional group selectively reactive at a cell surface and compositions and cells comprising such saccharides.

siRNA-based Analysis of the Abrogation of the Protective Function of Membrane-associated Catalase of Tumor Cells.

PubMed

Bauer, Georg

2017-02-01

Tumor cells, in contrast to non-malignant cells, show sustained expression of membrane-associated NADPH oxidase-1 and therefore generate extracellular superoxide anions and their dismutation product H 2 O 2 In order to prevent intercellular reactive oxygen species/reactive nitrogen species (ROS/RNS)-dependent apoptosis-inducing signaling, tumor cells need to express membrane-associated catalase that interferes with HOCl and nitric oxide/peroxynitrite signaling. Catalase is attached to tumor cells through the activity of transglutaminase-2 and is prevented from superoxide anion-dependent inhibition through coexpression of membrane-associated superoxide dismutase. Therefore, specific inhibition of membrane-associated catalase should reactivate intercellular ROS/RNS-dependent apoptosis-inducing signaling. These processes are analyzed here through small interfering RNA-mediated knockdown of essential signaling compounds. This allows to establish a rather comprehensive picture of intercellular ROS/RNS signaling that may be instrumental for future therapeutic approaches. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Effect of single oral dose of azithromycin, clarithromycin, and roxithromycin on polymorphonuclear leukocyte function assessed ex vivo by flow cytometry.

PubMed Central

Wenisch, C; Parschalk, B; Zedtwitz-Liebenstein, K; Weihs, A; el Menyawi, I; Graninger, W

1996-01-01

Azithromycin was given as a single oral dose (20 mg/kg of body weight) to 12 volunteers in a crossover study with roxithromycin (8 to 12 mg/kg) and clarithromycin (8 to 12 mg/kg). Flow cytometry was used to study the phagocytic functions and the release of reactive oxygen products following phagocytosis by neutrophil granulocytes prior to administration of the three drugs, 16 h after azithromycin administration, and 3 h after clarithromycin and roxithromycin administration. Phagocytic capacity was assessed by measuring the uptake of fluorescein isothiocyanate-labeled bacteria. Reactive oxygen generation after phagocytosis of unlabeled bacteria was estimated by the amount of dihydrorhodamine 123 converted to rhodamine 123 intracellularly. Azithromycin resulted in decreased capacities of the cells to phagocytize Escherichia coli (median [range], 62% [27 to 91%] of the control values; P < 0.01) and generate reactive oxygen products (75% [34 to 26%] of the control values; P < 0.01). Clarithromycin resulted in reduced phagocytosis (82% [75 to 98%] of control values; P < 0.01) but did not alter reactive oxygen production (84% [63 to 113%] of the control values; P > 0.05). Roxithromycin treatment did not affect granulocyte phagocytosis (92% [62 to 118%] of the control values; P > 0.05) or reactive oxygen production (94% [66 to 128%] of the control value; P > 0.05). No relation between intra- and/or extracellular concentrations of azithromycin and/or roxithromycin and the polymorphonuclear phagocyte function and/or reactive oxygen production existed (P > 0.05 for all comparisons). These results demonstrate that the accumulation of macrolides in neutrophils can suppress the response of phagocytic cells to bacterial pathogens after a therapeutic dose. PMID:8878577

Critical role for the NLRP3 inflammasome during acute lung injury1

PubMed Central

Grailer, Jamison J.; Canning, Bethany A.; Kalbitz, Miriam; Haggadone, Mikel D.; Dhond, Rasika M.; Andjelkovic, Anuska V.; Zetoune, Firas S.; Ward, Peter A.

2014-01-01

The inflammasome is a key factor in innate immunity and senses soluble pathogen and danger associated molecular patterns as well as biological crystals (urate, cholesterol, etc.), resulting in expression of IL-1β and IL-18. Using a standard model of acute lung injury (ALI) in mice featuring airway instillation of LPS, ALI was dependent on availability of NLRP3 as well as caspase-1, which are known features of the NLRP3 inflammasome. The appearance of IL-1β, a product of NLRP3 inflammasome activation, was detected in bronchoalveolar lavage fluids (BALF) in a macrophage- and neutrophil-dependent manner. Neutrophil-derived extracellular histones appeared in the BALF during ALI and directly activated the NLRP3 inflammasome. Antibody-mediated neutralization of histones significantly reduced IL-1β levels in BALF during ALI. Inflammasome activation by extracellular histones in LPS-primed macrophages required NLRP3 and caspase-1 as well as extrusion of K+, increased [Ca+2]i, and generation of reactive oxygen species. NLRP3 and caspase-1 were also required for full extracellular histone presence during ALI, suggesting a positive feedback mechanism. Extracellular histone and IL-1β levels in BALF were also elevated in C5a-induced and IgG immune complex ALI models suggesting a common inflammatory mechanism. These data indicate an interaction between extracellular histones and the NLRP3 inflammasome, resulting in ALI. Such findings suggest novel targets for treatment of ALI, for which there is currently no known efficacious drug. PMID:24795455

Ex vivo effects of an Oenothera paradoxa extract on the reactive oxygen species generation and neutral endopeptidase activity in neutrophils from patients after acute myocardial infarction.

PubMed

Kiss, Anna K; Kapłon-Cieślicka, Agnieszka; Filipiak, Krzysztof J; Opolski, Grzegorz; Naruszewicz, Marek

2012-04-01

Oxidative stress induced by reactive oxygen species (ROS) is considered to play an important part in the aetiology of coronary heart disease. Apart from ROS, neutrophils are a source of neutral endopeptidase (NEP) that inactivates protective natriuretic peptides. The aim of the present study was to evaluate the in vitro ROS generation and inhibition of NEP activity in neutrophils obtained from healthy volunteers and from patients after acute myocardial infarction (AMI) by an aqueous extract of Oenothera paradoxa. Neutrophils isolated from AMI patients showed two-fold higher ROS generation compared with cells from healthy donors, especially in the lucigenin-enhanced luminescence model, which suggests intensive O₂⁻ generation. The addition of O. paradoxa extract at concentrations of 0.2, 2 and 20 µg/mL resulted in a significant reduction in ROS generation. The extracellular NEP activity was higher in patients after AMI compared with healthy individuals (15.0 ± 0.9 versus 10.3 ± 0.5 nmol AMC/10(6) cells/60 min; p = 0.001). The addition of O. paradoxa extract at concentrations of 20, 50 and 100 µg/mL resulted in a significant reduction in NEP activity in both groups. O. paradoxa extract appears to be an interesting candidate for supplementation in the prevention of cardiovascular diseases. Copyright © 2011 John Wiley & Sons, Ltd.

Prenatal Nicotine Increases Matrix Metalloproteinase 2 (MMP-2) Expression in Fetal Guinea Pig Hearts

PubMed Central

Thompson, Loren P.; Liu, Hongshan; Evans, LaShauna; Mong, Jessica A.

2011-01-01

This study tested the hypothesis that maternal nicotine ingestion increases matrix metalloproteinase (MMP) expression in fetal hearts, which is mediated by the generation of reactive oxygen species. Timed pregnant guinea pigs were administered either water alone, nicotine (200 μg/mL), N-acetylcysteine (NAC), or nicotine plus NAC in their drinking water for 10 days at 52-day gestation (term = 65 days). Near-term (62 days), anesthetized fetuses were extracted, hearts were excised, and left cardiac ventricles snap frozen for analysis of MMP-2/-9/-13 protein and activity levels. Interstitial collagens were identified by Picrosirius red stain to assess changes in the extracellular matrix. Prenatal nicotine increased active MMP-2 forms and interstitial collagen but had no effect on either pro- or active MMP-9 or MMP-13 forms. In the presence of nicotine, NAC decreased active MMP-2 protein levels and reversed the nicotine-induced increase in collagen staining. We conclude that prenatal nicotine alters MMP-2 expression in fetal hearts that may be mediated by reactive oxygen species generation. PMID:21775771

Xanthohumol induces generation of reactive oxygen species and triggers apoptosis through inhibition of mitochondrial electron transfer chain complex I.

PubMed

Zhang, Bo; Chu, Wei; Wei, Peng; Liu, Ying; Wei, Taotao

2015-12-01

Xanthohumol is a prenylflavonoid extracted from hops (Humulus lupulus). It possesses anti-cancer and anti-inflammatory activities in vitro and in vivo, and offers therapeutic benefits for treatment of metabolic syndromes. However, the precise mechanisms underlying its pharmacological effects remain to be elucidated, together with its cellular target. Here, we provide evidence that xanthohumol directly interacts with the mitochondrial electron transfer chain complex I (NADH dehydrogenase), inhibits the oxidative phosphorylation, triggers the production of reactive oxygen species, and induces apoptosis. In addition, we show that as a result of the inhibition of the mitochondrial oxidative phosphorylation, xanthohumol exposure causes a rapid decrease of mitochondrial transmembrane potential. Furthermore, we showed that xanthohumol up-regulates the glycolytic capacity in cells, and thus compensates cellular ATP generation. Dissection of the multiple steps of aerobic respiration by extracellular flux assays revealed that xanthohumol specifically inhibits the activity of mitochondrial complex I, but had little effect on that of complex II, III and IV. Inhibition of complex I by xanthohumol caused the overproduction of reactive oxygen species, which are responsible for the induction of apoptosis in cancer cells. We also found that isoxanthohumol, the structural isomer of xanthohumol, is inactive to cells, suggesting that the reactive 2-hydroxyl group of xanthohumol is crucial for its targeting to the mitochondrial complex I. Together, the remodeling of cell metabolism revealed here has therapeutic potential for the use of xanthohumol. Copyright © 2015 Elsevier Inc. All rights reserved.

Classical ROS-dependent and early/rapid ROS-independent release of Neutrophil Extracellular Traps triggered by Leishmania parasites.

PubMed

Rochael, Natalia C; Guimarães-Costa, Anderson B; Nascimento, Michelle T C; DeSouza-Vieira, Thiago S; Oliveira, Matheus P; Garcia e Souza, Luiz F; Oliveira, Marcus F; Saraiva, Elvira M

2015-12-17

Neutrophil extracellular traps (NETs) extruded from neutrophils upon activation are composed of chromatin associated with cytosolic and granular proteins, which ensnare and kill microorganisms. This microbicidal mechanism named classical netosis has been shown to dependent on reactive oxygen species (ROS) generation by NADPH oxidase and also chromatin decondensation dependent upon the enzymes (PAD4), neutrophil elastase (NE) and myeloperoxidase (MPO). NET release also occurs through an early/rapid ROS-independent mechanism, named early/rapid vital netosis. Here we analyze the role of ROS, NE, MPO and PAD4 in the netosis stimulated by Leishmania amazonensis promastigotes in human neutrophils. We demonstrate that promastigotes induce a classical netosis, dependent on the cellular redox imbalance, as well as by a chloroamidine sensitive and elastase activity mechanism. Additionally, Leishmania also induces the early/rapid NET release occurring only 10 minutes after neutrophil-parasite interaction. We demonstrate here, that this early/rapid mechanism is dependent on elastase activity, but independent of ROS generation and chloroamidine. A better understanding of both mechanisms of NET release, and the NETs effects on the host immune system modulation, could support the development of new potential therapeutic strategies for leishmaniasis.

Influence of extracellular zinc on M1 microglial activation.

PubMed

Higashi, Youichirou; Aratake, Takaaki; Shimizu, Shogo; Shimizu, Takahiro; Nakamura, Kumiko; Tsuda, Masayuki; Yawata, Toshio; Ueba, Tetuya; Saito, Motoaki

2017-02-27

Extracellular zinc, which is released from hippocampal neurons in response to brain ischaemia, triggers morphological changes in microglia. Under ischaemic conditions, microglia exhibit two opposite activation states (M1 and M2 activation), which may be further regulated by the microenvironment. We examined the role of extracellular zinc on M1 activation of microglia. Pre-treatment of microglia with 30-60 μM ZnCl 2 resulted in dose-dependent increases in interleukin-1 beta (IL-1β), interleukin-6 (IL-6), and tumour necrosis factor-alpha (TNFα) secretion when M1 activation was induced by lipopolysaccharide administration. In contrast, the cell-permeable zinc chelator TPEN, the radical scavenger Trolox, and the P2X7 receptor antagonist A438079 suppressed the effects of zinc pre-treatment on microglia. Furthermore, endogenous zinc release was induced by cerebral ischaemia-reperfusion, resulting in increased expression of IL-1β, IL-6, TNFα, and the microglial M1 surface marker CD16/32, without hippocampal neuronal cell loss, in addition to impairments in object recognition memory. However, these effects were suppressed by the zinc chelator CaEDTA. These findings suggest that extracellular zinc may prime microglia to enhance production of pro-inflammatory cytokines via P2X7 receptor activation followed by reactive oxygen species generation in response to stimuli that trigger M1 activation, and that these inflammatory processes may result in deficits in object recognition memory.

Cystic fibrosis transmembrane conductance regulator: temperature-dependent cysteine reactivity suggests different stable conformers of the conduction pathway.

PubMed

Liu, Xuehong; Dawson, David C

2011-11-29

Cysteine scanning has been widely used to identify pore-lining residues in mammalian ion channels, including the cystic fibrosis transmembrane conductance regulator (CFTR). These studies, however, have been typically conducted at room temperature rather than human body temperature. Reports of substantial effects of temperature on gating and anion conduction in CFTR channels as well as an unexpected pattern of cysteine reactivity in the sixth transmembrane segment (TM6) prompted us to investigate the effect of temperature on the reactivity of cysteines engineered into TM6 of CFTR. We compared reaction rates at temperatures ranging from 22 to 37 °C for cysteines placed on either side of an apparent size-selective accessibility barrier previously defined by comparing reactivity toward channel-permeant and channel-impermeant, thiol-directed reagents. The results indicate that the reactivity of cysteines at three positions extracellular to the position of the accessibility barrier, 334, 336, and 337, is highly temperature-dependent. At 37 °C, cysteines at these positions were highly reactive toward MTSES(-), whereas at 22 °C, the reaction rates were 2-6-fold slower to undetectable. An activation energy of 157 kJ/mol for the reaction at position 337 is consistent with the hypothesis that, at physiological temperature, the extracellular portion of the CFTR pore can adopt conformations that differ significantly from those that can be accessed at room temperature. However, the position of the accessibility barrier defined empirically by applying channel-permeant and channel-impermeant reagents to the extracellular aspect of the pore is not altered. The results illuminate previous scanning results and indicate that the assay temperature is a critical variable in studies designed to use chemical modification to test structural models for the CFTR anion conduction pathway.

Candida albicans Biofilms Do Not Trigger Reactive Oxygen Species and Evade Neutrophil Killing

PubMed Central

Xie, Zhihong; Thompson, Angela; Sobue, Takanori; Kashleva, Helena; Xu, Hongbin; Vasilakos, John; Dongari-Bagtzoglou, Anna

2012-01-01

Neutrophils are found within Candida albicans biofilms in vivo and could play a crucial role in clearing the pathogen from biofilms forming on catheters and mucosal surfaces. Our goal was to compare the antimicrobial activity of neutrophils against developing and mature C. albicans biofilms and identify biofilm-specific properties mediating resistance to immune cells. Antibiofilm activity was measured with the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)2H-tetrazolium-5-carboxanilide assay and a molecular Candida viability assay. Reactive oxygen species generation was assessed by measuring fluorescence of 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester in preloaded neutrophils. We found that mature biofilms were resistant to leukocytic killing and did not trigger reactive oxygen species, even though neutrophils retained their viability and functional activation potential. Beta-glucans found in the extracellular matrix negatively affected antibiofilm activities. We conclude that these polymers act as a decoy mechanism to prevent neutrophil activation and that this represents an important innate immune evasion mechanism of C. albicans biofilms. PMID:23033146

Hydrogen peroxide generation by the pepper extracellular peroxidase CaPO2 activates local and systemic cell death and defense response to bacterial pathogens.

PubMed

Choi, Hyong Woo; Kim, Young Jin; Lee, Sung Chul; Hong, Jeum Kyu; Hwang, Byung Kook

2007-11-01

Reactive oxygen species (ROS) are responsible for mediating cellular defense responses in plants. Controversy has existed over the origin of ROS in plant defense. We have isolated a novel extracellular peroxidase gene, CaPO2, from pepper (Capsicum annuum). Local or systemic expression of CaPO2 is induced in pepper by avirulent Xanthomonas campestris pv vesicatoria (Xcv) infection. We examined the function of the CaPO2 gene in plant defense using the virus-induced gene silencing technique and gain-of-function transgenic plants. CaPO2-silenced pepper plants were highly susceptible to Xcv infection. Virus-induced gene silencing of the CaPO2 gene also compromised hydrogen peroxide (H(2)O(2)) accumulation and hypersensitive cell death in leaves, both locally and systemically, during avirulent Xcv infection. In contrast, overexpression of CaPO2 in Arabidopsis (Arabidopsis thaliana) conferred enhanced disease resistance accompanied by cell death, H(2)O(2) accumulation, and PR gene induction. In CaPO2-overexpression Arabidopsis leaves infected by Pseudomonas syringae pv tomato, H(2)O(2) generation was sensitive to potassium cyanide (a peroxidase inhibitor) but insensitive to diphenylene iodonium (an NADPH oxidase inhibitor), suggesting that H(2)O(2) generation depends on peroxidase in Arabidopsis. Together, these results indicate that the CaPO2 peroxidase is involved in ROS generation, both locally and systemically, to activate cell death and PR gene induction during the defense response to pathogen invasion.

Oxidative stress and myocardial injury in the diabetic heart

PubMed Central

Ansley, David M.; Wang, Baohua

2013-01-01

Reactive oxygen or nitrogen species play an integral role in both myocardial injury and repair. This dichotomy is differentiated at the level of species type, amount, duration of free radical generated. Homeostatic mechanisms designed to prevent free radical generation in the first instance, scavenge, or enzymatically convert them to less toxic forms and water, play crucial roles in maintenance of cellular structure and function. The outcome between functional recovery and dysfunction is dependent upon the inherent ability of these homeostatic antioxidant defenses to withstand acute free radical generation, in the order of seconds to minutes. Alternatively, pre-existent antioxidant capacity (from intracellular and extracellular sources) may regulate the degree of free radical generation. This converts reactive oxygen and nitrogen species to the role of second messenger involved in cell signalling. The adaptive capacity of the cell is altered by the balance between death or survival signal converging at the level of the mitochondria, with distinct pathophysiologic consequences that extends the period of injury from hours to days and weeks. Hyperglycemia, hyperlipidemia, and insulin resistance enhance oxidative stress in diabetic myocardium that cannot adapt to ischemia reperfusion. Altered glucose flux, mitochondrial derangements and nitric oxide synthase uncoupling in the presence of decreased antioxidant defense and impaired prosurvival cell signalling may render the diabetic myocardium more vulnerable to injury, remodelling and heart failure. PMID:23011912

Oxidative Stress Facilitates IFN-γ-Induced Mimic Extracellular Trap Cell Death in A549 Lung Epithelial Cancer Cells.

PubMed

Lin, Chiou-Feng; Chen, Chia-Ling; Chien, Shun-Yi; Tseng, Po-Chun; Wang, Yu-Chih; Tsai, Tsung-Ting

2016-01-01

We previously demonstrated that IFN-γ induces an autophagy-regulated mimic extracellular trap cell death (ETosis) in A549 human lung cancer cells. Regarding reactive oxygen species (ROS) are involved in ETosis, this study investigated the role of oxidative stress. After IFN-γ stimulation, a necrosis-like cell death mimic ETosis occurred accompanied by the inhibition of cell growth, aberrant nuclear staining, and nucleosome release. ROS were generated in a time-dependent manner with an increase in NADPH oxidase component protein expression. STAT1-mediated IFN regulatory factor-1 activation was essential for upregulating ROS production. By genetically silencing p47phox, IFN-γ-induced ROS and mimic ETosis were significantly attenuated. This mechanistic study indicated that ROS may mediate DNA damage followed by histone H3 citrullination. Furthermore, ROS promoted IFN-γ-induced mimic ETosis in cooperation with autophagy. These findings further demonstrate that ROS regulates IFN-γ-induced mimic ETosis in lung epithelial malignancy.

Oxidative Stress Facilitates IFN-γ-Induced Mimic Extracellular Trap Cell Death in A549 Lung Epithelial Cancer Cells

PubMed Central

Lin, Chiou-Feng; Chen, Chia-Ling; Chien, Shun-Yi; Tseng, Po-Chun; Wang, Yu-Chih; Tsai, Tsung-Ting

2016-01-01

We previously demonstrated that IFN-γ induces an autophagy-regulated mimic extracellular trap cell death (ETosis) in A549 human lung cancer cells. Regarding reactive oxygen species (ROS) are involved in ETosis, this study investigated the role of oxidative stress. After IFN-γ stimulation, a necrosis-like cell death mimic ETosis occurred accompanied by the inhibition of cell growth, aberrant nuclear staining, and nucleosome release. ROS were generated in a time-dependent manner with an increase in NADPH oxidase component protein expression. STAT1-mediated IFN regulatory factor-1 activation was essential for upregulating ROS production. By genetically silencing p47phox, IFN-γ-induced ROS and mimic ETosis were significantly attenuated. This mechanistic study indicated that ROS may mediate DNA damage followed by histone H3 citrullination. Furthermore, ROS promoted IFN-γ-induced mimic ETosis in cooperation with autophagy. These findings further demonstrate that ROS regulates IFN-γ-induced mimic ETosis in lung epithelial malignancy. PMID:27575372

Redox Signaling in Diabetic Wound Healing Regulates Extracellular Matrix Deposition.

PubMed

Kunkemoeller, Britta; Kyriakides, Themis R

2017-10-20

Impaired wound healing is a major complication of diabetes, and can lead to development of chronic foot ulcers in a significant number of patients. Despite the danger posed by poor healing, very few specific therapies exist, leaving patients at risk of hospitalization, amputation, and further decline in overall health. Recent Advances: Redox signaling is a key regulator of wound healing, especially through its influence on the extracellular matrix (ECM). Normal redox signaling is disrupted in diabetes leading to several pathological mechanisms that alter the balance between reactive oxygen species (ROS) generation and scavenging. Importantly, pathological oxidative stress can alter ECM structure and function. There is limited understanding of the specific role of altered redox signaling in the diabetic wound, although there is evidence that ROS are involved in the underlying pathology. Preclinical studies of antioxidant-based therapies for diabetic wound healing have yielded promising results. Redox-based therapeutics constitute a novel approach for the treatment of wounds in diabetes patients that deserve further investigation. Antioxid. Redox Signal. 27, 823-838.

Sulforaphane Restores Cellular Glutathione Levels and Reduces Chronic Periodontitis Neutrophil Hyperactivity In Vitro

PubMed Central

Dias, Irundika H. K.; Chapple, Ian L. C.; Milward, Mike; Grant, Melissa M.; Hill, Eric; Brown, James; Griffiths, Helen R.

2013-01-01

The production of high levels of reactive oxygen species by neutrophils is associated with the local and systemic destructive phenotype found in the chronic inflammatory disease periodontitis. In the present study, we investigated the ability of sulforaphane (SFN) to restore cellular glutathione levels and reduce the hyperactivity of circulating neutrophils associated with chronic periodontitis. Using differentiated HL60 cells as a neutrophil model, here we show that generation of extracellular O2 . - by the nicotinamide adenine dinucleotide (NADPH) oxidase complex is increased by intracellular glutathione depletion. This may be attributed to the upregulation of thiol regulated acid sphingomyelinase driven lipid raft formation. Intracellular glutathione was also lower in primary neutrophils from periodontitis patients and, consistent with our previous findings, patients neutrophils were hyper-reactive to stimuli. The activity of nuclear factor erythroid-2-related factor 2 (Nrf2), a master regulator of the antioxidant response, is impaired in circulating neutrophils from chronic periodontitis patients. Although patients’ neutrophils exhibit a low reduced glutathione (GSH)/oxidised glutathione (GSSG) ratio and a higher total Nrf2 level, the DNA-binding activity of nuclear Nrf2 remained unchanged relative to healthy controls and had reduced expression of glutamate cysteine ligase catalytic (GCLC), and modifier (GCLM) subunit mRNAs, compared to periodontally healthy subjects neutrophils. Pre-treatment with SFN increased expression of GCLC and GCM, improved intracellular GSH/GSSG ratios and reduced agonist-activated extracellular O2 . - production in both dHL60 and primary neutrophils from patients with periodontitis and controls. These findings suggest that a deficiency in Nrf2-dependent pathways may underpin susceptibility to hyper-reactivity in circulating primary neutrophils during chronic periodontitis. PMID:23826097

Sulforaphane restores cellular glutathione levels and reduces chronic periodontitis neutrophil hyperactivity in vitro.

PubMed

Dias, Irundika H K; Chapple, Ian L C; Milward, Mike; Grant, Melissa M; Hill, Eric; Brown, James; Griffiths, Helen R

2013-01-01

The production of high levels of reactive oxygen species by neutrophils is associated with the local and systemic destructive phenotype found in the chronic inflammatory disease periodontitis. In the present study, we investigated the ability of sulforaphane (SFN) to restore cellular glutathione levels and reduce the hyperactivity of circulating neutrophils associated with chronic periodontitis. Using differentiated HL60 cells as a neutrophil model, here we show that generation of extracellular O2 (. -) by the nicotinamide adenine dinucleotide (NADPH) oxidase complex is increased by intracellular glutathione depletion. This may be attributed to the upregulation of thiol regulated acid sphingomyelinase driven lipid raft formation. Intracellular glutathione was also lower in primary neutrophils from periodontitis patients and, consistent with our previous findings, patients neutrophils were hyper-reactive to stimuli. The activity of nuclear factor erythroid-2-related factor 2 (Nrf2), a master regulator of the antioxidant response, is impaired in circulating neutrophils from chronic periodontitis patients. Although patients' neutrophils exhibit a low reduced glutathione (GSH)/oxidised glutathione (GSSG) ratio and a higher total Nrf2 level, the DNA-binding activity of nuclear Nrf2 remained unchanged relative to healthy controls and had reduced expression of glutamate cysteine ligase catalytic (GCLC), and modifier (GCLM) subunit mRNAs, compared to periodontally healthy subjects neutrophils. Pre-treatment with SFN increased expression of GCLC and GCM, improved intracellular GSH/GSSG ratios and reduced agonist-activated extracellular O2 (. -) production in both dHL60 and primary neutrophils from patients with periodontitis and controls. These findings suggest that a deficiency in Nrf2-dependent pathways may underpin susceptibility to hyper-reactivity in circulating primary neutrophils during chronic periodontitis.

Up-regulation of A1M/α1-microglobulin in skin by heme and reactive oxygen species gives protection from oxidative damage.

PubMed

Olsson, Magnus G; Allhorn, Maria; Larsson, Jörgen; Cederlund, Martin; Lundqvist, Katarina; Schmidtchen, Artur; Sørensen, Ole E; Mörgelin, Matthias; Akerström, Bo

2011-01-01

During bleeding the skin is subjected to oxidative insults from free heme and radicals, generated from extracellular hemoglobin. The lipocalin α(1)-microglobulin (A1M) was recently shown to have reductase properties, reducing heme-proteins and other substrates, and to scavenge heme and radicals. We investigated the expression and localization of A1M in skin and the possible role of A1M in the protection of skin tissue from damage induced by heme and reactive oxygen species. Skin explants, keratinocyte cultures and purified collagen I were exposed to heme, reactive oxygen species, and/or A1M and investigated by biochemical methods and electron microscopy. The results demonstrate that A1M is localized ubiquitously in the dermal and epidermal layers, and that the A1M-gene is expressed in keratinocytes and up-regulated after exposure to heme and reactive oxygen species. A1M inhibited the heme- and reactive oxygen species-induced ultrastructural damage, up-regulation of antioxidation and cell cycle regulatory genes, and protein carbonyl formation in skin and keratinocytes. Finally, A1M bound to purified collagen I (K(d) = 0.96×10(-6) M) and could inhibit and repair the destruction of collagen fibrils by heme and reactive oxygen species. The results suggest that A1M may have a physiological role in protection of skin cells and matrix against oxidative damage following bleeding.

plasmatis Center for Innovation Competence: Controlling reactive component output of atmospheric pressure plasmas in plasma medicine

NASA Astrophysics Data System (ADS)

Reuter, Stephan

2012-10-01

The novel approach of using plasmas in order to alter the local chemistry of cells and cell environment presents a significant development in biomedical applications. The plasmatis center for innovation competence at the INP Greifswald e.V. performs fundamental research in plasma medicine in two interdisciplinary research groups. The aim of our plasma physics research group ``Extracellular Effects'' is (a) quantitative space and time resolved diagnostics and modelling of plasmas and liquids to determine distribution and composition of reactive species (b) to control the plasma and apply differing plasma source concepts in order to produce a tailored output of reactive components and design the chemical composition of the liquids/cellular environment and (c) to identify and understand the interaction mechanisms of plasmas with liquids and biological systems. Methods to characterize the plasma generated reactive species from plasma-, gas- and liquid phase and their biological effects will be presented. The diagnostic spectrum ranges from absorption/emission/laser spectroscopy and molecular beam mass spectrometry to electron paramagnetic resonance spectroscopy and cell biological diagnostic techniques. Concluding, a presentation will be given of the comprehensive approach to plasma medicine in Greifswald where the applied and clinical research of the Campus PlasmaMed association is combined with the fundamental research at plasmatis center.

Influence of extracellular zinc on M1 microglial activation

PubMed Central

Higashi, Youichirou; Aratake, Takaaki; Shimizu, Shogo; Shimizu, Takahiro; Nakamura, Kumiko; Tsuda, Masayuki; Yawata, Toshio; Ueba, Tetuya; Saito, Motoaki

2017-01-01

Extracellular zinc, which is released from hippocampal neurons in response to brain ischaemia, triggers morphological changes in microglia. Under ischaemic conditions, microglia exhibit two opposite activation states (M1 and M2 activation), which may be further regulated by the microenvironment. We examined the role of extracellular zinc on M1 activation of microglia. Pre-treatment of microglia with 30–60 μM ZnCl2 resulted in dose-dependent increases in interleukin-1 beta (IL-1β), interleukin-6 (IL-6), and tumour necrosis factor-alpha (TNFα) secretion when M1 activation was induced by lipopolysaccharide administration. In contrast, the cell-permeable zinc chelator TPEN, the radical scavenger Trolox, and the P2X7 receptor antagonist A438079 suppressed the effects of zinc pre-treatment on microglia. Furthermore, endogenous zinc release was induced by cerebral ischaemia–reperfusion, resulting in increased expression of IL-1β, IL-6, TNFα, and the microglial M1 surface marker CD16/32, without hippocampal neuronal cell loss, in addition to impairments in object recognition memory. However, these effects were suppressed by the zinc chelator CaEDTA. These findings suggest that extracellular zinc may prime microglia to enhance production of pro-inflammatory cytokines via P2X7 receptor activation followed by reactive oxygen species generation in response to stimuli that trigger M1 activation, and that these inflammatory processes may result in deficits in object recognition memory. PMID:28240322

Neither eosinophils nor neutrophils require ATG5-dependent autophagy for extracellular DNA trap formation.

PubMed

Germic, Nina; Stojkov, Darko; Oberson, Kevin; Yousefi, Shida; Simon, Hans-Uwe

2017-11-01

The importance of extracellular traps (ETs) in innate immunity is well established, but the molecular mechanisms responsible for their formation remain unclear and in scientific dispute. ETs have been defined as extracellular DNA scaffolds associated with the granule proteins of eosinophils or neutrophils. They are capable of killing bacteria extracellularly. Based mainly on results with phosphoinositide 3-kinase (PI3K) inhibitors such as 3-methyladenine (3-MA) and wortmannin, which are commonly used to inhibit autophagy, several groups have reported that autophagy is required for neutrophil extracellular trap (NET) formation. We decided to investigate this apparent dependence on autophagy for ET release and generated genetically modified mice that lack, specifically in eosinophils or neutrophils, autophagy-related 5 (Atg5), a gene encoding a protein essential for autophagosome formation. Interestingly, neither eosinophils nor neutrophils from Atg5-deficient mice exhibited abnormalities in ET formation upon physiological activation or exposure to low concentrations of PMA, although we could confirm that human and mouse eosinophils and neutrophils, after pre-treatment with inhibitors of class III PI3K, show a block both in reactive oxygen species (ROS) production and in ET formation. The so-called late autophagy inhibitors bafilomycin A1 and chloroquine, on the other hand, were without effect. These data indicate that ET formation occurs independently of autophagy and that the inhibition of ROS production and ET formation in the presence of 3-MA and wortmannin is probably owing to their additional ability to block the class I PI3Ks, which are involved in signalling cascades initiated by triggers of ET formation. © 2017 John Wiley & Sons Ltd.

Redox linked flavin sites in extracellular decaheme proteins involved in microbe-mineral electron transfer.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Edwards, Marcus J.; White, Gaye F.; Norman, Michael

2015-07-01

Extracellular microbe-mineral electron transfer is a major driving force for the oxidation of organic carbon in many subsurface environments. Extracellular multi-heme cytochromes of the Shewenella genus play a major role in this process but the mechanism of electron exchange at the interface between cytochrome and acceptor is widely debated. The 1.8 Å x-ray crystal structure of the decaheme MtrC revealed a highly conserved CX₈C disulfide that, when substituted for AX₈A, severely compromised the ability of S. oneidensis to grow under aerobic conditions. Reductive cleavage of the disulfide in the presence of flavin mononucleotide (FMN) resulted in the reversible formation ofmore » a stable flavocytochrome. Similar results were also observed with other decaheme cytochromes, OmcA, MtrF and UndA. The data suggest that these decaheme cytochromes can transition between highly reactive flavocytochromes or less reactive cytochromes, and that this transition is controlled by a redox active disulfide that responds to the presence of oxygen.« less

Atrial fibrillation in the elderly: the potential contribution of reactive oxygen species

PubMed Central

Schillinger, Kurt J.; Patel, Vickas V.

2012-01-01

Atrial fibrillation (AF) is the most commonly encountered cardiac arrhythmia, and is a significant source of healthcare expenditures throughout the world. It is an arrhythmia with a very clearly defined predisposition for individuals of advanced age, and this fact has led to intense study of the mechanistic links between aging and AF. By promoting oxidative damage to multiple subcellular and cellular structures, reactive oxygen species (ROS) have been shown to induce the intra- and extra-cellular changes necessary to promote the pathogenesis of AF. In addition, the generation and accumulation of ROS have been intimately linked to the cellular processes which underlie aging. This review begins with an overview of AF pathophysiology, and introduces the critical structures which, when damaged, predispose an otherwise healthy atrium to AF. The available evidence that ROS can lead to damage of these critical structures is then reviewed. Finally, the evidence linking the process of aging to the pathogenesis of AF is discussed. PMID:23341843

Generation and characterization of anti-MUC4 monoclonal antibodies reactive with normal and cancer cells in humans.

PubMed

Moniaux, Nicolas; Varshney, Grish Chandra; Chauhan, Subhash Chand; Copin, Marie Christine; Jain, Maneesh; Wittel, Uwe A; Andrianifahanana, Mahefatiana; Aubert, Jean-Pierre; Batra, Surinder Kumar

2004-02-01

We have previously cloned the full-length cDNA (approximately 28 Kb) and established the complete genomic organization (25 exons/introns over 100 kb) of the human MUC4 mucin. This large molecule is predicted to protrude over 2 microm above the cell surface, in which MUC4alpha is an extracellular mucin-type glycoprotein subunit and MUC4beta is the transmembrane subunit. Over two thirds of the encoded protein sequence consists of 16-amino-acid tandem repeats (TR), which are flanked by unique sequences. In this study we generated and characterized monoclonal antibodies (MAbs) directed against the TR region of MUC4. Mice were immunized with a KLH-conjugated MUC4 TR peptide, STGDTTPLPVTDTSSV. Several clones were purified by three rounds of limited dilutions and stable clones presenting a sustained antibody production were selected for subsequent characterization. Antibodies were tested for their reactivity and specificity to recognize the MUC4 peptide and further screened by enzyme-linked immunosorbent assay (ELISA) and Western blotting analyses. One of the MAbs (8G7) was strongly reactive against the MUC4 peptide and with native MUC4 from human tissues or pancreatic cancer cells in Western blotting, immunohistochemistry, and confocal analysis. Anti-MUC4 MAb may represent a powerful tool for the study of MUC4 function under normal and pathological conditions and for diagnosis of solid tumors including those in the breast, pancreas, lungs, and ovaries.

Measurement of Reactive Oxygen Species in the Culture Media Using Acridan Lumigen PS-3 Assay

PubMed Central

Uy, Benedict; McGlashan, Susan R.; Shaikh, Shamim B.

2011-01-01

Reactive oxygen species (ROS) are generated continuously during aerobic metabolism. ROS are highly reactive molecules and in excessive amounts, can lead to protein and DNA oxidation, protein cross-linking, and cell death. Cell-culture models provide a valuable tool in understanding the mechanisms that lead to cell death. Accumulation of ROS within cells and/or their release into the culture media are highly cell type-specific. The ability to estimate ROS levels in the culture media is an important step in understanding the mechanisms contributing to disease processes. In this paper, we describe the optimization of a simple method to estimate ROS levels in the culture media using the Acridan Lumigen PS-3 reagent provided in the Amersham ECL Plus kit (GE Healthcare, UK). We have shown that the Acridan Lumigen PS-3 assay generates ROS-specific chemiluminescence in fresh as well as media stored at −20°C, in as little as 10–20 μl of samples. The method was able to detect the dose (of stimulants)- and time (acute and chronic)-dependent changes in ROS levels in media collected from various cell types. Our results suggest that the kit reagents, PBS buffer, and various media did not contribute significantly to the overall chemiluminescence generated in the assay; however, we suggest that the unused medium specific for each cell type should be used as blanks and final readings of test samples normalized against these readings. As this method uses commonly available laboratory equipment and commercially available reagents, we believe this assay is convenient, economical, and specific in estimating ROS released extracellularly into the culture media. PMID:21966257

Measurement of reactive oxygen species in the culture media using Acridan Lumigen PS-3 assay.

PubMed

Uy, Benedict; McGlashan, Susan R; Shaikh, Shamim B

2011-09-01

Reactive oxygen species (ROS) are generated continuously during aerobic metabolism. ROS are highly reactive molecules and in excessive amounts, can lead to protein and DNA oxidation, protein cross-linking, and cell death. Cell-culture models provide a valuable tool in understanding the mechanisms that lead to cell death. Accumulation of ROS within cells and/or their release into the culture media are highly cell type-specific. The ability to estimate ROS levels in the culture media is an important step in understanding the mechanisms contributing to disease processes. In this paper, we describe the optimization of a simple method to estimate ROS levels in the culture media using the Acridan Lumigen PS-3 reagent provided in the Amersham ECL Plus kit (GE Healthcare, UK). We have shown that the Acridan Lumigen PS-3 assay generates ROS-specific chemiluminescence in fresh as well as media stored at -20°C, in as little as 10-20 μl of samples. The method was able to detect the dose (of stimulants)- and time (acute and chronic)-dependent changes in ROS levels in media collected from various cell types. Our results suggest that the kit reagents, PBS buffer, and various media did not contribute significantly to the overall chemiluminescence generated in the assay; however, we suggest that the unused medium specific for each cell type should be used as blanks and final readings of test samples normalized against these readings. As this method uses commonly available laboratory equipment and commercially available reagents, we believe this assay is convenient, economical, and specific in estimating ROS released extracellularly into the culture media.

The Antitumor Effect of Singlet Oxygen.

PubMed

Bauer, Georg

2016-11-01

Tumor cells are protected against intercellular apoptosis-inducing signaling through expression of membrane-associated catalase and superoxide dismutase. Exogenous singlet oxygen derived from activated photosensitizers or from cold atmospheric plasma causes local inactivation of protective catalase which is followed by the generation of secondary extracellular singlet oxygen. This process is specific for tumor cells and is driven by a complex interaction between H 2 O 2 and peroxynitrite. Secondary singlet oxygen has the potential for autoamplification of its generation, resulting in optimal inactivation of protective catalase and reactivation of intercellular apoptosis-inducing signaling. An increase in the endogenous NO concentration also causes inactivation of catalase and autoamplificatory generation of secondary singlet oxygen. This principle is essential for the antitumor activity of secondary plant products, such as cyanidins and other inhibitors of NO dioxygenase. It seems that the action of the established chemotherapeutic taxol and the recently established antitumor effect of certain azoles are based on the same principles. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

The role of oxidative stress in the metabolic syndrome.

PubMed

Whaley-Connell, Adam; McCullough, Peter A; Sowers, James R

2011-01-01

Loss of reduction-oxidation (redox) homeostasis and generation of excess free oxygen radicals play an important role in the pathogenesis of diabetes, hypertension, and consequent cardiovascular disease. Reactive oxygen species are integral in routine in physiologic mechanisms. However, loss of redox homeostasis contributes to proinflammatory and profibrotic pathways that promote impairments in insulin metabolic signaling, reduced endothelial-mediated vasorelaxation, and associated cardiovascular and renal structural and functional abnormalities. Redox control of metabolic function is a dynamic process with reversible pro- and anti-free radical processes. Labile iron is necessary for the catalysis of superoxide anion, hydrogen peroxide, and the generation of the damaging hydroxyl radical. Acute hypoxia and cellular damage in cardiovascular tissue liberate larger amounts of cytosolic and extracellular iron that is poorly liganded; thus, large increases in the generation of oxygen free radicals are possible, causing tissue damage. The understanding of iron and the imbalance of redox homeostasis within the vasculature is integral in hypertension and progression of metabolic dysregulation that contributes to insulin resistance, endothelial dysfunction, and cardiovascular and kidney disease.

Algal extracellular release in river-floodplain dissolved organic matter: response of extracellular enzymatic activity during a post-flood period

PubMed Central

Sieczko, Anna; Maschek, Maria; Peduzzi, Peter

2015-01-01

River-floodplain systems are susceptible to rapid hydrological events. Changing hydrological connectivity of the floodplain generates a broad range of conditions, from lentic to lotic. This creates a mixture of allochthonously and autochthonously derived dissolved organic matter (DOM). Autochthonous DOM, including photosynthetic extracellular release (PER), is an important source supporting bacterial secondary production (BSP). Nonetheless, no details are available regarding microbial extracellular enzymatic activity (EEA) as a response to PER under variable hydrological settings in river-floodplain systems. To investigate the relationship between bacterial and phytoplankton components, we therefore used EEA as a tool to track the microbial response to non-chromophoric, but reactive and ecologically important DOM. The study was conducted in three floodplain subsystems with distinct hydrological regimes (Danube Floodplain National Park, Austria). The focus was on the post-flood period. Enhanced %PER (up to 48% of primary production) in a hydrologically isolated subsystem was strongly correlated with β-glucosidase, which was related to BSP. This shows that—in disconnected floodplain backwaters with high terrestrial input—BSP can also be driven by autochthonous carbon sources (PER). In a semi-isolated section, in the presence of fresh labile material from primary producers, enhanced activity of phenol oxidase was observed. In frequently flooded river-floodplain systems, BSP was mainly driven by enzymatic degradation of particulate primary production. Our research demonstrates that EEA measurements are an excellent tool to describe the coupling between bacteria and phytoplankton, which cannot be deciphered when focusing solely on chromophoric DOM. PMID:25741326

Extracellular Production of Reactive Oxygen Species by Marine Microbiota

NASA Astrophysics Data System (ADS)

Schneider, R. J.; Roe, K. L.; Voelker, B. M.; Hansel, C. M.

2016-02-01

The reactive oxygen species (ROS) superoxide (O2-) and hydrogen peroxide (H2O2) are important to the cycling of trace metals and carbon in marine systems. Previous studies have shown that biological ROS production in the ocean may be significant. We examined the ability of five common species of diatoms to produce and break down ROS in the presence and absence of light by suspending cells on filters and measuring downstream ROS concentrations using chemiluminescence probes. Results show a wide range of rates (undetectable to 7.3 x 10-16 mol cell-1 hr-1) and suggest that extracellular ROS production occurs through a variety of pathways. H2O2 decay appears to be mediated primarily by active cell processes, while O2- appears to occur through a combination of active and passive cell processes. Extracellular H2O2 production and decay were also determined for twelve species of heterotrophic bacteria using two different methodologies. Measured decay rates were consistent despite methodological differences. By contrast, large variability of production rates was observed could vary significantly even among between replicates of the same species measured using the same methodology. Although production rates cannot be stated with certainty, it is likely that extracellular H2O2 production occurs in most bacterial species.

Can injured adult CNS axons regenerate by recapitulating development?

PubMed

Hilton, Brett J; Bradke, Frank

2017-10-01

In the adult mammalian central nervous system (CNS), neurons typically fail to regenerate their axons after injury. During development, by contrast, neurons extend axons effectively. A variety of intracellular mechanisms mediate this difference, including changes in gene expression, the ability to form a growth cone, differences in mitochondrial function/axonal transport and the efficacy of synaptic transmission. In turn, these intracellular processes are linked to extracellular differences between the developing and adult CNS. During development, the extracellular environment directs axon growth and circuit formation. In adulthood, by contrast, extracellular factors, such as myelin and the extracellular matrix, restrict axon growth. Here, we discuss whether the reactivation of developmental processes can elicit axon regeneration in the injured CNS. © 2017. Published by The Company of Biologists Ltd.

Depletion of intracellular zinc from neurons by use of an extracellular chelator in vivo and in vitro.

PubMed

Frederickson, Christopher J; Suh, Sang W; Koh, Jae-Young; Cha, Yoo K; Thompson, Richard B; LaBuda, Christopher J; Balaji, Rengarajan V; Cuajungco, Math P

2002-12-01

The membrane-impermeable chelator CaEDTA was introduced extracellularly among neurons in vivo and in vitro for the purpose of chelating extracellular Zn(2+). Unexpectedly, this treatment caused histochemically reactive Zn(2+) in intracellular compartments to drop rapidly. The same general result was seen with intravesicular Zn(2+), which fell after CaEDTA infusion into the lateral ventricle of the brain, with perikaryal Zn(2+) in Purkinje neurons (in vivo) and with cortical neurons (in vitro). These findings suggest either that the volume of zinc ion efflux and reuptake is higher than previously suspected or that EDTA can enter cells and vesicles. Caution is therefore warranted in attempting to manipulate extracellular or intracellular Zn(2+) selectively.

Arabidopsis annexin1 mediates the radical-activated plasma membrane Ca²+- and K+-permeable conductance in root cells.

PubMed

Laohavisit, Anuphon; Shang, Zhonglin; Rubio, Lourdes; Cuin, Tracey A; Véry, Anne-Aliénor; Wang, Aihua; Mortimer, Jennifer C; Macpherson, Neil; Coxon, Katy M; Battey, Nicholas H; Brownlee, Colin; Park, Ohkmae K; Sentenac, Hervé; Shabala, Sergey; Webb, Alex A R; Davies, Julia M

2012-04-01

Plant cell growth and stress signaling require Ca²⁺ influx through plasma membrane transport proteins that are regulated by reactive oxygen species. In root cell growth, adaptation to salinity stress, and stomatal closure, such proteins operate downstream of the plasma membrane NADPH oxidases that produce extracellular superoxide anion, a reactive oxygen species that is readily converted to extracellular hydrogen peroxide and hydroxyl radicals, OH•. In root cells, extracellular OH• activates a plasma membrane Ca²⁺-permeable conductance that permits Ca²⁺ influx. In Arabidopsis thaliana, distribution of this conductance resembles that of annexin1 (ANN1). Annexins are membrane binding proteins that can form Ca²⁺-permeable conductances in vitro. Here, the Arabidopsis loss-of-function mutant for annexin1 (Atann1) was found to lack the root hair and epidermal OH•-activated Ca²⁺- and K⁺-permeable conductance. This manifests in both impaired root cell growth and ability to elevate root cell cytosolic free Ca²⁺ in response to OH•. An OH•-activated Ca²⁺ conductance is reconstituted by recombinant ANN1 in planar lipid bilayers. ANN1 therefore presents as a novel Ca²⁺-permeable transporter providing a molecular link between reactive oxygen species and cytosolic Ca²⁺ in plants.

Engineering Synthetic Proteins to Generate Ca2+ Signals in Mammalian Cells.

PubMed

Qudrat, Anam; Truong, Kevin

2017-03-17

The versatility of Ca 2+ signals allows it to regulate diverse cellular processes such as migration, apoptosis, motility and exocytosis. In some receptors (e.g., VEGFR2), Ca 2+ signals are generated upon binding their ligand(s) (e.g., VEGF-A). Here, we employed a design strategy to engineer proteins that generate a Ca 2+ signal upon binding various extracellular stimuli by creating fusions of protein domains that oligomerize to the transmembrane domain and the cytoplasmic tail of the VEGFR2. To test the strategy, we created chimeric proteins that generate Ca 2+ signals upon stimulation with various extracellular stimuli (e.g., rapamycin, EDTA or extracellular free Ca 2+ ). By coupling these chimeric proteins that generate Ca 2+ signals with proteins that respond to Ca 2+ signals, we rewired, for example, dynamic cellular blebbing to increases in extracellular free Ca 2+ . Thus, using this design strategy, it is possible to engineer proteins to generate a Ca 2+ signal to rewire a wide range of extracellular stimuli to a wide range of Ca 2+ -activated processes.

An ethanol extract of Piper betle Linn. mediates its anti-inflammatory activity via down-regulation of nitric oxide.

PubMed

Ganguly, Sudipto; Mula, Soumyaditya; Chattopadhyay, Subrata; Chatterjee, Mitali

2007-05-01

The leaves of Piper betle (locally known as Paan) have long been in use in the Indian indigenous system of medicine for the relief of pain; however, the underlying molecular mechanisms of this effect have not been elucidated. The anti-inflammatory and immunomodulatory effects of an ethanolic extract of the leaves of P. betle (100 mg kg(-1); PB) were demonstrated in a complete Freund's adjuvant-induced model of arthritis in rats with dexamethasone (0.1 mg kg(-1)) as the positive control. At non-toxic concentrations of PB (5-25 microg mL(-1)), a dose-dependent decrease in extracellular production of nitric oxide in murine peritoneal macrophages was measured by the Griess assay and corroborated by flow cytometry using the nitric oxide specific probe, 4,5-diaminofluorescein-2 diacetate. This decreased generation of reactive nitrogen species was mediated by PB progressively down-regulating transcription of inducible nitric oxide synthase in macrophages, and concomitantly causing a dose-dependent decrease in the expression of interleukin-12 p40, indicating the ability of PB to down-regulate T-helper 1 pro-inflammatory responses. Taken together, the anti-inflammatory and anti-arthrotic activity of PB is attributable to its ability to down-regulate the generation of reactive nitrogen species, thus meriting further pharmacological investigation.

Dietary cladode powder from wild type and domesticated Opuntia species reduces atherogenesis in apoE knock-out mice.

PubMed

Garoby-Salom, Sandra; Guéraud, Françoise; Camaré, Caroline; de la Rosa, Ana-Paulina Barba; Rossignol, Michel; Santos Díaz, María del Socorro; Salvayre, Robert; Negre-Salvayre, Anne

2016-03-01

Dietary intake of Opuntia species may prevent the development of cardiovascular diseases. The present study was designed to characterize the biological antioxidant and anti-inflammatory properties of Opuntia species and to investigate whether Opuntia cladodes prevent the development of atherosclerosis in vivo, in apoE(-)KO mice. The effects of the two Opuntia species, the wild Opuntia streptacantha and the domesticated Opuntia ficus-indica, were tested on the generation of intra- and extracellular reactive oxygen species (ROS) production and kinetics of the LDL oxidation by murine CRL2181 endothelial cells and on the subsequent inflammatory signaling leading to the adhesion of monocytes on the activated endothelium and the formation of foam cells. Opuntia species blocked the extracellular ROS (superoxide anion) generation and LDL oxidation by CRL2181, as well as the intracellular ROS rise and signaling evoked by the oxidized LDL, including the nuclear translocation of the transcription factor NFκB, the expression of ICAM-1 and VCAM-1 adhesion molecules, and the adhesion of monocytes to CRL2181. In vivo, Opuntia significantly reduced the formation of atherosclerotic lesions and the accumulation of 4-hydroxynonenal adducts in the vascular wall of apoE-KO mice, indicating that Opuntia cladodes prevent lipid oxidation in the vascular wall. In conclusion, wild and domesticated Opuntia species exhibit antioxidant, anti-inflammatory, and antiatherogenic properties which emphasize their nutritional benefit for preventing cardiovascular diseases.

Pathophysiology of neutrophil-mediated extracellular redox reactions.

PubMed

Jaganjac, Morana; Cipak, Ana; Schaur, Rudolf Joerg; Zarkovic, Neven

2016-01-01

Neutrophil granulocyte leukocytes (neutrophils) play fundamental role in the innate immune response. In the presence of adequate stimuli, neutrophils release excessive amount of reactive oxygen species (ROS) that may induce cell and tissue injury. Oxidative burst of neutrophils acts as a double-edged sword. It may contribute to the pathology of atherosclerosis and brain injury but is also necessary in resolving infections. Moreover, neutrophil-derived ROS may also have both a tumor promoting and tumor suppressing role. ROS have a specific activities and diffusion distance, which is related to their short lifetime. Therefore, the manner in which ROS will act depends on the cells targeted and the intra- and extracellular levels of individual ROS, which can further cause production of reactive aldehydes like 4-hydroxynonenal (HNE) that act as a second messengers of ROS. In this review we discuss the influence of neutrophil mediated extracellular redox reactions in ischemia reperfusion injury, transplant rejection and chronic diseases (atherosclerosis, inflammatory bowel diseases and cancer). At the end a brief overview of cellular mechanisms to maintain ROS homeostasis is given.

Enhanced migration of murine fibroblast-like 3T3-L1 preadipocytes on type I collagen-coated dish is reversed by silibinin treatment.

PubMed

Liu, Xiaoling; Xu, Qian; Liu, Weiwei; Yao, Guodong; Zhao, Yeli; Xu, Fanxing; Hayashi, Toshihiko; Fujisaki, Hitomi; Hattori, Shunji; Tashiro, Shin-Ichi; Onodera, Satoshi; Yamato, Masayuki; Ikejima, Takashi

2018-04-01

Migration of fibroblast-like preadipocytes is important for the development of adipose tissue, whereas excessive migration is often responsible for impaired adipose tissue related with obesity and fibrotic diseases. Type I collagen (collagen I) is the most abundant component of extracellular matrix and has been shown to regulate fibroblast migration in vitro, but its role in adipose tissue is not known. Silibinin is a bioactive natural flavonoid with antioxidant and antimetastasis activities. In this study, we found that type I collagen coating promoted the proliferation and migration of murine 3T3-L1 preadipocytes in a dose-dependent manner, implying that collagen I could be an extracellular signal. Regarding the mechanisms of collagen I-stimulated 3T3-L1 migration, we found that NF-κB p65 is activated, including the increased nuclear translocation of NF-κB p65 as well as the upregulation of NF-κB p65 phosphorylation and acetylation, accompanied by the increased expressions of proinflammatory factors and the generation of reactive oxygen species (ROS). Reduction of collagen I-enhanced migration of cells by treatment with silibinin was associated with suppression of NF-κB p65 activity and ROS generation, and negatively correlated with the increasing sirt1 expression. Taken together, the enhanced migration of 3T3-L1 cells induced on collagen I-coated dish is mediated by the activation of NF-κB p65 function and ROS generation that can be alleviated with silibinin by upregulation of sirt1, leading to the repression of NF-κB p65 function and ROS generation.

Revealing mechanisms of selective, concentration-dependent potentials of 4-hydroxy-2-nonenal to induce apoptosis in cancer cells through inactivation of membrane-associated catalase.

PubMed

Bauer, Georg; Zarkovic, Neven

2015-04-01

Tumor cells generate extracellular superoxide anions and are protected against superoxide anion-mediated intercellular apoptosis-inducing signaling by the expression of membrane-associated catalase. 4-Hydroxy-2-nonenal (4-HNE), a versatile second messenger generated during lipid peroxidation, has been shown to induce apoptosis selectively in malignant cells. The findings described in this paper reveal the strong, concentration-dependent potential of 4-HNE to specifically inactivate extracellular catalase of tumor cells both indirectly and directly and to consequently trigger apoptosis in malignant cells through superoxide anion-mediated intercellular apoptosis-inducing signaling. Namely, 4-HNE caused apoptosis selectively in NOX1-expressing tumor cells through inactivation of their membrane-associated catalase, thus reactivating subsequent intercellular signaling through the NO/peroxynitrite and HOCl pathways, followed by the mitochondrial pathway of apoptosis. Concentrations of 4-HNE of 1.2 µM and higher directly inactivated membrane-associated catalase of tumor cells, whereas at lower concentrations, 4-HNE triggered a complex amplificatory pathway based on initial singlet oxygen formation through H2O2 and peroxynitrite interaction. Singlet-oxygen-dependent activation of the FAS receptor and caspase-8 increased superoxide anion generation by NOX1 and amplification of singlet oxygen generation, which allowed singlet-oxygen-dependent inactivation of catalase. 4-HNE and singlet oxygen cooperate in complex autoamplificatory loops during this process. The finding of these novel anticancer pathways may be useful for understanding the role of 4-HNE in the control of malignant cells and for the optimization of ROS-dependent therapeutic approaches including antioxidant treatments. Copyright © 2015 Elsevier Inc. All rights reserved.

Neutrophils in chronic and aggressive periodontitis in interaction with Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans.

PubMed

Guentsch, A; Puklo, M; Preshaw, P M; Glockmann, E; Pfister, W; Potempa, J; Eick, S

2009-06-01

This study analyzed the interaction of Porphyromonas gingivalis ATCC 33277 and Aggregatibacter actinomycetemcomitans Y4 with peripheral blood polymorphonuclear neutrophils taken from patients with aggressive periodontitis and chronic periodontitis. Peripheral blood polymorphonuclear neutrophils obtained from 12 patients with chronic periodontitis, six patients with aggressive periodontitis and 12 healthy controls were exposed to P. gingivalis and A. actinomycetemcomitans following opsonization of the bacteria using the patient's own serum. Serum immunoglobulin G (IgG) levels against both periodontopathogens were measured. Phagocytosis and killing of the bacteria, as well as the extracellular human neutrophil elastase activity, were quantified. The total amount and the extracellular release of reactive oxygen species were measured using luminol-dependent and isoluminol-dependent chemiluminescence. Polymorphonuclear neutrophils from patients with chronic (62.16 +/- 19.39%) and aggressive (43.26 +/- 26.63%) periodontitis phagocytosed more P. gingivalis than the healthy controls (24.43 +/- 19.87%) at the 30-min time point after exposure to the bacteria (p < 0.05). High serum IgG levels against P. gingivalis and A. actinomycetemcomitans were detected in subjects with periodontitis. Polymorphonuclear neutrophils from subjects with chronic and aggressive periodontitis released significantly more reactive oxygen species and demonstrated greater human neutrophil elastase activity in the absence of any stimulus than polymorphonuclear neutrophils from healthy controls (p < 0.05). Polymorphonuclear neutrophils in chronic periodontitis released significantly more reactive oxygen species when exposed to P. gingivalis and A. actinomycetemcomitans than polymorphonuclear neutrophils in aggressive periodontitis. High serum IgG levels against P. gingivalis and A. actinomycetemcomitans promote phagocytosis in periodontitis. The extracellular release of reactive oxygen species and neutrophil elastase by polymorphonuclear neutrophils may also contribute to damage of the surrounding periodontal tissues.

Effects of coarse chalk dust particles (2.5-10 μm) on respiratory burst and oxidative stress in alveolar macrophages.

PubMed

Zhang, Yuexia; Yang, Zhenhua; Feng, Yan; Li, Ruijin; Zhang, Quanxi; Geng, Hong; Dong, Chuan

2015-08-01

The main aim of the present study was to examine in vitro responses of rat alveolar macrophages (AMs) exposed to coarse chalk dust particles (particulate matter in the size range 2.5-10 μm, PM(coarse)) by respiratory burst and oxidative stress. Chalk PM(coarse)-induced respiratory burst in AMs was measured by using a luminol-dependent chemiluminescence (CL) method. Also, the cell viability; lactate dehydrogenase (LDH) release; levels of cellular superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), malondialdehyde (MDA), and acid phosphatase (ACP); plasma membrane ATPase; and extracellular nitric oxide (NO) level were determined 4 h following the treatment with the different dosages of chalk PM(coarse). The results showed that chalk PM(coarse) initiated the respiratory burst of AMs as indicated by strong CL, which was inhibited by diphenyleneiodonium chloride and L-N-nitro-L-arginine methyl ester hydrochloride. It suggested that chalk PM(coarse) induced the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in AMs. This hypothesis was confirmed by the fact that chalk PM(coarse) resulted in a significant decrease of intracellular SOD, GSH, ACP, and ATPase levels and a notable increase of intracellular CAT, MDA content, and extracellular NO level, consequently leading to a decrease of the cell viability and a increase of LDH release. It was concluded that AMs exposed to chalk PM(coarse) can suffer from cytotoxicity which may be mediated by generation of excessive ROS/RNS. Graphical Abstract The possible mechanism of coarse chalk particles-induced adverse effects in AMs.

5-Hydroxytryptamine1A receptor/Gibetagamma stimulates mitogen-activated protein kinase via NAD(P)H oxidase and reactive oxygen species upstream of src in chinese hamster ovary fibroblasts.

PubMed Central

Mukhin, Y V; Garnovskaya, M N; Collinsworth, G; Grewal, J S; Pendergrass, D; Nagai, T; Pinckney, S; Greene, E L; Raymond, J R

2000-01-01

The hypothesis of this work is that the 'serotonin' or 5-hydroxytryptamine (5-HT)(1A) receptor, which activates the extracellular signal-regulated kinase (ERK) through a G(i)betagamma-mediated pathway, does so through the intermediate actions of reactive oxygen species (ROS). Five criteria were shown to support a key role for ROS in the activation of ERK by the 5-HT(1A) receptor. (1) Antioxidants inhibit activation of ERK by 5-HT. (2) Application of cysteine-reactive oxidant molecules activates ERK. (3) The 5-HT(1A) receptor alters cellular redox properties, and generates both superoxide and hydrogen peroxide. (4) A specific ROS-producing enzyme [NAD(P)H oxidase] is involved in the activation of ERK. (5) There is specificity both in the effects of various chemical oxidizers, and in the putative location of the ROS in the ERK activation pathway. We propose that NAD(P)H oxidase is located in the ERK activation pathway stimulated by the transfected 5-HT(1A) receptor in Chinese hamster ovary (CHO) cells downstream of G(i)betagamma subunits and upstream of or at the level of the non-receptor tyrosine kinase, Src. Moreover, these experiments provide confirmation that the transfected human 5-HT(1A) receptor induces the production of ROS (superoxide and hydrogen peroxide) in CHO cells, and support the possibility that an NAD(P)H oxidase-like enzyme might be involved in the 5-HT-mediated generation of both superoxide and hydrogen peroxide. PMID:10727402

Real-time electrochemical detection of extracellular nitric oxide in tobacco cells exposed to cryptogein, an elicitor of defence responses

PubMed Central

Besson-Bard, Angélique; Griveau, Sophie; Bedioui, Fethi; Wendehenne, David

2008-01-01

It was previously reported that cryptogein, an elicitor of defence responses, induces an intracellular production of nitric oxide (NO) in tobacco. Here, the possibility was explored that cryptogein might also trigger an increase of NO extracellular content through two distinct approaches, an indirect method using the NO probe 4,5-diaminofluorescein (DAF-2) and an electrochemical method involving a chemically modified microelectrode probing free NO in biological media. While the chemical nature of DAF-2-reactive compound(s) is still uncertain, the electrochemical modified microelectrodes provide real-time evidence that cryptogein induces an increase of extracellular NO. Direct measurement of free extracellular NO might offer important new insights into its role in plants challenged by biotic stresses. PMID:18653691

Increasing the endogenous NO level causes catalase inactivation and reactivation of intercellular apoptosis signaling specifically in tumor cells

PubMed Central

Bauer, Georg

2015-01-01

Tumor cells generate extracellular superoxide anions and are protected against intercellular apoptosis-inducing HOCl- and NO/peroxynitrite signaling through the expression of membrane-associated catalase. This enzyme decomposes H2O2 and thus prevents HOCl synthesis. It efficiently interferes with NO/peroxynitrite signaling through oxidation of NO and decomposition of peroxynitrite. The regulatory potential of catalase at the crosspoint of ROS and RNS chemical biology, as well as its high local concentration on the outside of the cell membrane of tumor cells, establish tight control of intercellular signaling and thus prevent tumor cell apoptosis. Therefore, inhibition of catalase or its inactivation by singlet oxygen reactivate intercellular apoptosis-inducing signaling. Nitric oxide and peroxynitrite are connected with catalase in multiple and meaningful ways, as (i) NO can be oxidated by compound I of catalase, (ii) NO can reversibly inhibit catalase, (iii) peroxynitrite can be decomposed by catalase and (iv) the interaction between peroxynitrite and H2O2 leads to the generation of singlet oxygen that inactivates catalase. Therefore, modulation of the concentration of free NO through addition of arginine, inhibition of arginase, induction of NOS expression or inhibition of NO dioxygenase triggers an autoamplificatory biochemical cascade that is based on initial formation of singlet oxygen, amplification of superoxide anion/H2O2 and NO generation through singlet oxygen dependent stimulation of the FAS receptor and caspase-8. Finally, singlet oxygen is generated at sufficiently high concentration to inactivate protective catalase and to reactivate intercellular apoptosis-inducing ROS signaling. This regulatory network allows to establish several pathways for synergistic interactions, like the combination of modulators of NO metabolism with enhancers of superoxide anion generation, modulators of NO metabolism that act at different targets and between modulators of NO metabolism and direct catalase inhibitors. The latter aspect is explicitely studied for the interaction between catalase inhibiting acetylsalicylic acid and an NO donor. It is also shown that hybrid molecules like NO-aspirin utilize this synergistic potential. Our data open novel approaches for rational tumor therapy based on specific ROS signaling and its control in tumor cells. PMID:26342455

Convalescent Plasmodium falciparum-specific seroreactivity does not correlate with paediatric malaria severity or Plasmodium antigen exposure.

PubMed

Kessler, Anne; Campo, Joseph J; Harawa, Visopo; Mandala, Wilson L; Rogerson, Stephen J; Mowrey, Wenzhu B; Seydel, Karl B; Kim, Kami

2018-04-25

Antibody immunity is thought to be essential to prevent severe Plasmodium falciparum infection, but the exact correlates of protection are unknown. Over time, children in endemic areas acquire non-sterile immunity to malaria that correlates with development of antibodies to merozoite invasion proteins and parasite proteins expressed on the surface of infected erythrocytes. A 1000 feature P. falciparum 3D7 protein microarray was used to compare P. falciparum-specific seroreactivity during acute infection and 30 days after infection in 23 children with uncomplicated malaria (UM) and 25 children with retinopathy-positive cerebral malaria (CM). All children had broad P. falciparum antibody reactivity during acute disease. IgM reactivity decreased and IgG reactivity increased in convalescence. Antibody reactivity to CIDR domains of "virulent" PfEMP1 proteins was low with robust reactivity to the highly conserved, intracellular ATS domain of PfEMP1 in both groups. Although children with UM and CM differed markedly in parasite burden and PfEMP1 exposure during acute disease, neither acute nor convalescent PfEMP1 seroreactivity differed between groups. Greater seroprevalence to a conserved Group A-associated ICAM binding extracellular domain was observed relative to linked extracellular CIDRα1 domains in both case groups. Pooled immune IgG from Malawian adults revealed greater reactivity to PfEMP1 than observed in children. Children with uncomplicated and cerebral malaria have similar breadth and magnitude of P. falciparum antibody reactivity. The utility of protein microarrays to measure serological recognition of polymorphic PfEMP1 antigens needs to be studied further, but the study findings support the hypothesis that conserved domains of PfEMP1 are more prominent targets of cross reactive antibodies than variable domains in children with symptomatic malaria. Protein microarrays represent an additional tool to identify cross-reactive Plasmodium antigens including PfEMP1 domains that can be investigated as strain-transcendent vaccine candidates.

Glutamine activates STAT3 to control cancer cell proliferation independently of glutamine metabolism

PubMed Central

Vazeille, Thibaut; Sonveaux, Pierre

2016-01-01

Cancer cells can use a variety of metabolic substrates to fulfill the bioenergetic and biosynthetic needs of their oncogenic program. Besides bioenergetics, cancer cell metabolism also directly influences genetic, epigenetic and signaling events associated with tumor progression. Many cancer cells are addicted to glutamine, and this addiction is observed in oxidative as well as in glycolytic cells. While both oxidative and bioreductive glutamine metabolism can contribute to cancer progression and glutamine can further serve to generate peptides (including glutathione) and proteins, we report that glutamine promotes the proliferation of cancer cells independently of its use as a metabolic fuel or as a precursor of glutathione. Extracellular glutamine activates transcription factor STAT3, which is necessary and sufficient to mediate the proliferative effects of glutamine in glycolytic and in oxidative cancer cells. Glutamine also activates transcription factors HIF-1, mTOR and c-Myc, but these factors do not mediate the effects of glutamine on cancer cell proliferation. Our findings shed a new light on the anticancer effects of L-asparaginase that possesses glutaminase activity and converts glutamine into glutamate extracellularly. Conversely, cancer resistance to treatments that block glutamine metabolism could arise from glutamine-independent STAT3 re-activation. PMID:27748760

Reduction of inflammatory responses and enhancement of extracellular matrix formation by vanillin-incorporated poly(lactic-co-glycolic acid) scaffolds.

PubMed

Lee, Yujung; Kwon, Jeongil; Khang, Gilson; Lee, Dongwon

2012-10-01

Vanillin is one of the major components of vanilla, a commonly used flavoring agent and preservative and is known to exert potent antioxidant and anti-inflammatory activities. In this work, vanillin-incorporated poly(lactic-co-glycolic acid) (PLGA) films and scaffolds were fabricated to evaluate the effects of vanillin on the inflammatory responses and extracellular matrix (ECM) formation in vitro and in vivo. The incorporation of vanillin to PLGA films induced hydrophilic nature, resulting in the higher cell attachment and proliferation than the pure PLGA film. Vanillin also reduced the generation of reactive oxygen species (ROS) in cells cultured on the pure PLGA film and significantly inhibited the PLGA-induced inflammatory responses in vivo, evidenced by the reduced accumulation of inflammatory cells and thinner fibrous capsules. The effects of vanillin on the ECM formation were evaluated using annulus fibrous (AF) cell-seeded porous PLGA/vanillin scaffolds. PLGA/vanillin scaffolds elicited the more production of glycosaminoglycan and collagen than the pure PLGA scaffold, in a concentration-dependent manner. Based on the low level of inflammatory responses and enhanced ECM formation, vanillin-incorporated PLGA constructs make them promising candidates in the future biomedical applications.

Protein cross-linking by chlorinated polyamines and transglutamylation stabilizes neutrophil extracellular traps.

PubMed

Csomós, Krisztián; Kristóf, Endre; Jakob, Bernadett; Csomós, István; Kovács, György; Rotem, Omri; Hodrea, Judit; Bagoly, Zsuzsa; Muszbek, Laszlo; Balajthy, Zoltán; Csősz, Éva; Fésüs, László

2016-08-11

Neutrophil extracellular trap (NET) ejected from activated dying neutrophils is a highly ordered structure of DNA and selected proteins capable to eliminate pathogenic microorganisms. Biochemical determinants of the non-randomly formed stable NETs have not been revealed so far. Studying the formation of human NETs we have observed that polyamines were incorporated into the NET. Inhibition of myeloperoxidase, which is essential for NET formation and can generate reactive chlorinated polyamines through hypochlorous acid, decreased polyamine incorporation. Addition of exogenous primary amines that similarly to polyamines inhibit reactions catalyzed by the protein cross-linker transglutaminases (TGases) has similar effect. Proteomic analysis of the highly reproducible pattern of NET components revealed cross-linking of NET proteins through chlorinated polyamines and ɛ(γ-glutamyl)lysine as well as bis-γ-glutamyl polyamine bonds catalyzed by the TGases detected in neutrophils. Competitive inhibition of protein cross-linking by monoamines disturbed the cross-linking pattern of NET proteins, which resulted in the loss of the ordered structure of the NET and significantly reduced capacity to trap bacteria. Our findings provide explanation of how NETs are formed in a reproducible and ordered manner to efficiently neutralize microorganisms at the first defense line of the innate immune system.

Protein cross-linking by chlorinated polyamines and transglutamylation stabilizes neutrophil extracellular traps

PubMed Central

Csomós, Krisztián; Kristóf, Endre; Jakob, Bernadett; Csomós, István; Kovács, György; Rotem, Omri; Hodrea, Judit; Bagoly, Zsuzsa; Muszbek, Laszlo; Balajthy, Zoltán; Csősz, Éva; Fésüs, László

2016-01-01

Neutrophil extracellular trap (NET) ejected from activated dying neutrophils is a highly ordered structure of DNA and selected proteins capable to eliminate pathogenic microorganisms. Biochemical determinants of the non-randomly formed stable NETs have not been revealed so far. Studying the formation of human NETs we have observed that polyamines were incorporated into the NET. Inhibition of myeloperoxidase, which is essential for NET formation and can generate reactive chlorinated polyamines through hypochlorous acid, decreased polyamine incorporation. Addition of exogenous primary amines that similarly to polyamines inhibit reactions catalyzed by the protein cross-linker transglutaminases (TGases) has similar effect. Proteomic analysis of the highly reproducible pattern of NET components revealed cross-linking of NET proteins through chlorinated polyamines and ɛ(γ-glutamyl)lysine as well as bis-γ-glutamyl polyamine bonds catalyzed by the TGases detected in neutrophils. Competitive inhibition of protein cross-linking by monoamines disturbed the cross-linking pattern of NET proteins, which resulted in the loss of the ordered structure of the NET and significantly reduced capacity to trap bacteria. Our findings provide explanation of how NETs are formed in a reproducible and ordered manner to efficiently neutralize microorganisms at the first defense line of the innate immune system. PMID:27512953

Quercetin attenuates mitochondrial dysfunction and biogenesis via upregulated AMPK/SIRT1 signaling pathway in OA rats.

PubMed

Qiu, Linan; Luo, Yuju; Chen, Xiaojuan

2018-07-01

Despite the severity of osteoarthritis (OA), current medical therapy strategies for OA aim at symptom control and pain reduction, as there is no ideal drug for effective OA treatment. OA rat model was used to explore the therapeutic function of quercetin on remission of OA, by determining the reactive oxygen species (ROS) levels, mitochondrial function and extracellular matrix integrity. Quercetin could attenuate ROS generation and augment the glutathione (GSH) and glutathione peroxidase (GPx) expression levels in OA rat. Quercetin not only enhanced mitochondrial membrane potential, oxygen consumption, adenosine triphosphate (ATP) levels in mitochondria, but also increased the mitochondrial copy number. Furthermore, the interlukin (IL)-1β-induced accumulation of nitric oxide (NO), matrixmetalloproteinase (MMP)-3) and MMP-13 could be suppressed by quercetin. Finally, we confirmed that the therapeutic properties of quercetin on OA might function through the adenosine monophosphate-activated protein kinase/sirtuin 1 (AMPK/SIRT1) signaling pathway. In summary, quercetin could alleviate OA through attenuating the ROS levels, reversing the mitochondrial dysfunction and keeping the integrality of extracellular matrix of joint cartilage. The underlying mechanism might involve the regulation of AMPK/SIRT1 signaling pathway. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Concentration-dependent protein loading of extracellular vesicles released by Histoplasma capsulatum after antibody treatment and its modulatory action upon macrophages

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baltazar, Ludmila Matos; Zamith-Miranda, Daniel; Burnet, Meagan C.

Here, diverse pathogenic fungi secrete extracellular vesicles (EV) that contain macromolecules, including virulence factors that can modulate the host immune response. We recently demonstrated that the binding of monoclonal antibodies (mAb) modulates how Histoplasma capsulatum load and releases its extracellular vesicles (EV). In the present paper, we addressed a concentration-dependent impact on the fungus’ EV loading and release with different mAb, as well as the pathophysiological role of these EV during the host-pathogen interaction. We found that the mAbs differentially regulate EV content in concentration-dependent and independent manners. Enzymatic assays demonstrated that laccase activity in EV from H. capsulatum opsonizedmore » with 6B7 was reduced, but urease activity was not altered. The uptake of H. capsulatum by macrophages pre-treated with EV, presented an antibody concentration-dependent phenotype. The intracellular killing of yeast cells was potently inhibited in macrophages pre-treated with EV from 7B6 (non-protective) mAb-opsonized H. capsulatum and this inhibition was associated with a decrease in the reactive-oxygen species generated by these macrophages. In summary, our findings show that opsonization quantitatively and qualitatively modifies H. capsulatum EV load and secretion leading to distinct effects on the host’s immune effector mechanisms, supporting the hypothesis that EV sorting and secretion are dynamic mechanisms for a fine-tuned response by fungal cells.« less

Concentration-dependent protein loading of extracellular vesicles released by Histoplasma capsulatum after antibody treatment and its modulatory action upon macrophages

DOE PAGES

Baltazar, Ludmila Matos; Zamith-Miranda, Daniel; Burnet, Meagan C.; ...

2018-05-23

Here, diverse pathogenic fungi secrete extracellular vesicles (EV) that contain macromolecules, including virulence factors that can modulate the host immune response. We recently demonstrated that the binding of monoclonal antibodies (mAb) modulates how Histoplasma capsulatum load and releases its extracellular vesicles (EV). In the present paper, we addressed a concentration-dependent impact on the fungus’ EV loading and release with different mAb, as well as the pathophysiological role of these EV during the host-pathogen interaction. We found that the mAbs differentially regulate EV content in concentration-dependent and independent manners. Enzymatic assays demonstrated that laccase activity in EV from H. capsulatum opsonizedmore » with 6B7 was reduced, but urease activity was not altered. The uptake of H. capsulatum by macrophages pre-treated with EV, presented an antibody concentration-dependent phenotype. The intracellular killing of yeast cells was potently inhibited in macrophages pre-treated with EV from 7B6 (non-protective) mAb-opsonized H. capsulatum and this inhibition was associated with a decrease in the reactive-oxygen species generated by these macrophages. In summary, our findings show that opsonization quantitatively and qualitatively modifies H. capsulatum EV load and secretion leading to distinct effects on the host’s immune effector mechanisms, supporting the hypothesis that EV sorting and secretion are dynamic mechanisms for a fine-tuned response by fungal cells.« less

Grape seed extract triggers apoptosis in Caco-2 human colon cancer cells through reactive oxygen species and calcium increase: extracellular signal-regulated kinase involvement.

PubMed

Dinicola, Simona; Mariggiò, Maria Addolorata; Morabito, Caterina; Guarnieri, Simone; Cucina, Alessandra; Pasqualato, Alessia; D'Anselmi, Fabrizio; Proietti, Sara; Coluccia, Pierpaolo; Bizzarri, Mariano

2013-09-14

Grape seed extract (GSE) from Italia, Palieri and Red Globe cultivars inhibits cell growth and induces apoptosis in Caco-2 human colon cancer cells in a dose-dependent manner. In order to investigate the mechanism(s) supporting the apoptotic process, we analysed reactive oxygen species (ROS) production, intracellular Ca2+ handling and extracellular signal-regulated kinase (ERK) activation. Upon exposure to GSE, ROS and intracellular Ca2+ levels increased in Caco-2 cells, concomitantly with ERK inactivation. As ERK activity is thought to be essential for promoting survival pathways, inhibition of this kinase is likely to play a relevant role in GSE-mediated anticancer effects. Indeed, pretreatment with N-acetyl cysteine, a ROS scavenger, reversed GSE-induced apoptosis, and promoted ERK phosphorylation. This effect was strengthened by ethylene glycol tetraacetic acid-mediated inhibition of extracellular Ca2+ influx. ROS and Ca2+ influx inhibition, in turn, increased ERK phosphorylation, and hence almost entirely suppressed GSE-mediated apoptosis. These data suggested that GSE triggers a previously unrecognised ERK-based mechanism, involving both ROS production and intracellular Ca2+ increase, eventually leading to apoptosis in cancer cells.

Fosfomycin enhances phagocyte-mediated killing of Staphylococcus aureus by extracellular traps and reactive oxygen species.

PubMed

Shen, Fengge; Tang, Xudong; Cheng, Wei; Wang, Yang; Wang, Chao; Shi, Xiaochen; An, Yanan; Zhang, Qiaoli; Liu, Mingyuan; Liu, Bo; Yu, Lu

2016-01-18

The successful treatment of bacterial infections is the achievement of a synergy between the host's immune defences and antibiotics. Here, we examined whether fosfomycin (FOM) could improve the bactericidal effect of phagocytes, and investigated the potential mechanisms. FOM enhanced the phagocytosis and extra- or intracellular killing of S. aureus by phagocytes. And FOM enhanced the extracellular killing of S. aureus in macrophage (MФ) and in neutrophils mediated by extracellular traps (ETs). ET production was related to NADPH oxidase-dependent reactive oxygen species (ROS). Additionally, FOM increased the intracellular killing of S. aureus in phagocytes, which was mediated by ROS through the oxidative burst process. Our results also showed that FOM alone induced S. aureus producing hydroxyl radicals in order to kill the bacterial cells in vitro. In a mouse peritonitis model, FOM treatment increased the bactericidal extra- and intracellular activity in vivo, and FOM strengthened ROS and ET production from peritoneal lavage fluid ex vivo. An IVIS imaging system assay further verified the observed in vivo bactericidal effect of the FOM treatment. This work may provide a deeper understanding of the role of the host's immune defences and antibiotic interactions in microbial infections.

Fosfomycin enhances phagocyte-mediated killing of Staphylococcus aureus by extracellular traps and reactive oxygen species

PubMed Central

Shen, Fengge; Tang, Xudong; Cheng, Wei; Wang, Yang; Wang, Chao; Shi, Xiaochen; An, Yanan; Zhang, Qiaoli; Liu, Mingyuan; Liu, Bo; Yu, Lu

2016-01-01

The successful treatment of bacterial infections is the achievement of a synergy between the host’s immune defences and antibiotics. Here, we examined whether fosfomycin (FOM) could improve the bactericidal effect of phagocytes, and investigated the potential mechanisms. FOM enhanced the phagocytosis and extra- or intracellular killing of S. aureus by phagocytes. And FOM enhanced the extracellular killing of S. aureus in macrophage (MФ) and in neutrophils mediated by extracellular traps (ETs). ET production was related to NADPH oxidase-dependent reactive oxygen species (ROS). Additionally, FOM increased the intracellular killing of S. aureus in phagocytes, which was mediated by ROS through the oxidative burst process. Our results also showed that FOM alone induced S. aureus producing hydroxyl radicals in order to kill the bacterial cells in vitro. In a mouse peritonitis model, FOM treatment increased the bactericidal extra- and intracellular activity in vivo, and FOM strengthened ROS and ET production from peritoneal lavage fluid ex vivo. An IVIS imaging system assay further verified the observed in vivo bactericidal effect of the FOM treatment. This work may provide a deeper understanding of the role of the host’s immune defences and antibiotic interactions in microbial infections. PMID:26778774

RP-1551s, a family of azaphilones produced by Penicillium sp., inhibit the binding of PDGF to the extracellular domain of its receptor.

PubMed

Toki, S; Tanaka, T; Uosaki, Y; Yoshida, M; Suzuki, Y; Kita, K; Mihara, A; Ando, K; Lokker, N A; Giese, N A; Matsuda, Y

1999-03-01

Nine azaphilones designated RP-1551-1, -2, -3, -4, -5, -6, -7, -M1, and -M2 were isolated from the culture broth of Penicillium sp. SPC-21609 as inhibitors of PDGF binding to its receptor. RP-1551s inhibit the binding of PDGF AA to the extracellular domain of PDGF alpha-receptor with IC50 values ranging from 0.1 to 2 microM without affecting PDGF BB binding to the extracellular domain of PDGF beta-receptor. PDGF binding was not restored after the PDGF alpha-receptor extracellular domain was washed in an attempt to remove the RP-1551-1 bound to the receptor. This result suggests that RP-1551-1 may irreversibly interact with the PDGF alpha-receptor. Since many azaphilone compounds possess high reactivity with an amino group, RP-1551-1 may prevent PDGF AA binding by reacting with amino groups on the alpha-receptor extracellular domain.

Reactive Power Compensation Method Considering Minimum Effective Reactive Power Reserve

NASA Astrophysics Data System (ADS)

Gong, Yiyu; Zhang, Kai; Pu, Zhang; Li, Xuenan; Zuo, Xianghong; Zhen, Jiao; Sudan, Teng

2017-05-01

According to the calculation model of minimum generator reactive power reserve of power system voltage stability under the premise of the guarantee, the reactive power management system with reactive power compensation combined generator, the formation of a multi-objective optimization problem, propose a reactive power reserve is considered the minimum generator reactive power compensation optimization method. This method through the improvement of the objective function and constraint conditions, when the system load growth, relying solely on reactive power generation system can not meet the requirement of safe operation, increase the reactive power reserve to solve the problem of minimum generator reactive power compensation in the case of load node.

A perspective on reagent diversity and non-covalent binding of reactive carbonyl species (RCS) and effector reagents in nonenzymatic glycation (NEG): Mechanistic considerations and implications for future research

NASA Astrophysics Data System (ADS)

Rodnick, Kenneth J.; Holman, R. W.; Ropski, Pamela S.; Huang, Mingdong; Swislocki, Arthur L. M.

2017-06-01

This perspective focuses on illustrating the underappreciated connections between reactive carbonyl species (RCS), initial binding in the nonenzymatic glycation (NEG) process, and nonenzymatic covalent protein modification (here termed NECPM). While glucose is the central species involved in NEG, recent studies indicate that the initially-bound glucose species in the NEG of human hemoglobin (HbA) and human serum albumin (HSA) are non-RCS ring-closed isomers. The ring-opened glucose, an RCS structure that reacts in the NEG process, is most likely generated from previously-bound ring-closed isomers undergoing concerted acid/base reactions while bound to protein. The generation of the glucose RCS can involve concomitantly-bound physiological species (e.g., inorganic phosphate, water, etc.); here termed effector reagents. Extant NEG schemes do not account for these recent findings. In addition, effector reagent reactions with glucose in the serum and erythrocyte cytosol can generate RCS (e.g., glyoxal, glyceraldehyde, etc.). Recent research has shown that these RCS covalently modify proteins in vivo via NECPM mechanisms. A general scheme that reflects both the reagent and mechanistic diversity that can lead to NEG and NECPM is presented here. A perspective that accounts for the relationships between RCS, NEG, and NECPM can facilitate the understanding of site selectivity, may help explain overall glycation rates, and may have implications for the clinical assessment/control of diabetes mellitus. In view of this perspective, concentrations of ribose, fructose, Pi, bicarbonate, counter ions, and the resulting RCS generated within intracellular and extracellular compartments may be of importance and of clinical relevance. Future research is also proposed.

Regulating NETosis: Increasing pH Promotes NADPH Oxidase-Dependent NETosis

PubMed Central

Khan, Meraj A.; Philip, Lijy M.; Cheung, Guillaume; Vadakepeedika, Shawn; Grasemann, Hartmut; Sweezey, Neil; Palaniyar, Nades

2018-01-01

Neutrophils migrating from the blood (pH 7.35–7.45) into the surrounding tissues encounter changes in extracellular pH (pHe) conditions. Upon activation of NADPH oxidase 2 (Nox), neutrophils generate large amounts of H+ ions reducing the intracellular pH (pHi). Nevertheless, how extracellular pH regulates neutrophil extracellular trap (NET) formation (NETosis) is not clearly established. We hypothesized that increasing pH increases Nox-mediated production of reactive oxygen species (ROS) and neutrophil protease activity, stimulating NETosis. Here, we found that raising pHe (ranging from 6.6 to 7.8; every 0.2 units) increased pHi of both activated and resting neutrophils within 10–20 min (Seminaphtharhodafluor dual fluorescence measurements). Since Nox activity generates H+ ions, pHi is lower in neutrophils that are activated compared to resting. We also found that higher pH stimulated Nox-dependent ROS production (R123 generation; flow cytometry, plate reader assay, and imaging) during spontaneous and phorbol myristate acetate-induced NETosis (Sytox Green assays, immunoconfocal microscopy, and quantifying NETs). In neutrophils that are activated and not resting, higher pH stimulated histone H4 cleavage (Western blots) and NETosis. Raising pH increased Escherichia coli lipopolysaccharide-, Pseudomonas aeruginosa (Gram-negative)-, and Staphylococcus aureus (Gram-positive)-induced NETosis. Thus, higher pHe promoted Nox-dependent ROS production, protease activity, and NETosis; lower pH has the opposite effect. These studies provided mechanistic steps of pHe-mediated regulation of Nox-dependent NETosis. Raising pH either by sodium bicarbonate or Tris base (clinically known as Tris hydroxymethyl aminomethane, tromethamine, or THAM) increases NETosis. Each Tris molecule can bind 3H+ ions, whereas each bicarbonate HCO3− ion binds 1H+ ion. Therefore, the amount of Tris solution required to cause the same increase in pH level is less than that of equimolar bicarbonate solution. For that reason, regulating NETosis by pH with specific buffers such as THAM could be more effective than bicarbonate in managing NET-related diseases. PMID:29487850

Regulating NETosis: Increasing pH Promotes NADPH Oxidase-Dependent NETosis.

PubMed

Khan, Meraj A; Philip, Lijy M; Cheung, Guillaume; Vadakepeedika, Shawn; Grasemann, Hartmut; Sweezey, Neil; Palaniyar, Nades

2018-01-01

Neutrophils migrating from the blood (pH 7.35-7.45) into the surrounding tissues encounter changes in extracellular pH (pH e ) conditions. Upon activation of NADPH oxidase 2 (Nox), neutrophils generate large amounts of H + ions reducing the intracellular pH (pH i ). Nevertheless, how extracellular pH regulates neutrophil extracellular trap (NET) formation (NETosis) is not clearly established. We hypothesized that increasing pH increases Nox-mediated production of reactive oxygen species (ROS) and neutrophil protease activity, stimulating NETosis. Here, we found that raising pH e (ranging from 6.6 to 7.8; every 0.2 units) increased pH i of both activated and resting neutrophils within 10-20 min (Seminaphtharhodafluor dual fluorescence measurements). Since Nox activity generates H + ions, pH i is lower in neutrophils that are activated compared to resting. We also found that higher pH stimulated Nox-dependent ROS production (R123 generation; flow cytometry, plate reader assay, and imaging) during spontaneous and phorbol myristate acetate-induced NETosis (Sytox Green assays, immunoconfocal microscopy, and quantifying NETs). In neutrophils that are activated and not resting, higher pH stimulated histone H4 cleavage (Western blots) and NETosis. Raising pH increased Escherichia coli lipopolysaccharide-, Pseudomonas aeruginosa (Gram-negative)-, and Staphylococcus aureus (Gram-positive)-induced NETosis. Thus, higher pH e promoted Nox-dependent ROS production, protease activity, and NETosis; lower pH has the opposite effect. These studies provided mechanistic steps of pH e -mediated regulation of Nox-dependent NETosis. Raising pH either by sodium bicarbonate or Tris base (clinically known as Tris hydroxymethyl aminomethane, tromethamine, or THAM) increases NETosis. Each Tris molecule can bind 3H + ions, whereas each bicarbonate HCO3 - ion binds 1H + ion. Therefore, the amount of Tris solution required to cause the same increase in pH level is less than that of equimolar bicarbonate solution. For that reason, regulating NETosis by pH with specific buffers such as THAM could be more effective than bicarbonate in managing NET-related diseases.

Role of redoximiRs in fibrogenesis.

PubMed

Fierro-Fernández, Marta; Miguel, Verónica; Lamas, Santiago

2016-04-01

Fibrosis can be defined as an excessive accumulation of extracellular matrix (ECM) components, ultimately leading to stiffness, scarring and devitalized tissue. MicroRNAs (miRNAs) are short, 19-25 nucleotides (nt), non-coding RNAs involved in the post-transcriptional regulation of gene expression. Recently, miRNAs have also emerged as powerful regulators of fibrotic processes and have been termed "fibromiRs". Oxidative stress represents a self-perpetuating mechanism in fibrogenesis. MiRNAs can also influence the expression of genes responsible for the generation of reactive oxygen species (ROS) and antioxidant defence and are termed "redoximiRs". Here, we review the current knowledge of mechanisms by which "redoximiRs" regulate fibrogenesis. This new set of miRNAs may be called "redoxifibromiRs". Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Antioxidant effect of thiazolidine molecules in cell culture media improves stability and performance.

PubMed

Kuschelewski, Jennifer; Schnellbaecher, Alisa; Pering, Sascha; Wehsling, Maria; Zimmer, Aline

2017-05-01

The ability of cell culture media components to generate reactive species as well as their sensitivity to oxidative degradation, affects the overall stability of media and the behavior of cells cultured in vitro. This study investigates the influence of thiazolidine molecules, formed from the condensation between cysteine and alpha-ketoacids, on the stability of these complex mixtures and on the performance of cell culture processes aiming to produce therapeutically relevant monoclonal antibodies. Results presented in this study indicate that 2-methyl-1,3-thiazolidine-2,4-dicarboxylic acid and 2-(2-carboxyethyl)-1,3-thiazolidine-2,4-dicarboxylic acid, obtained by condensation of cysteine with pyruvate or alpha-ketoglutarate, respectively, are able to stabilize cell culture media formulations, in particular redox sensitive molecules like folic acid, thiamine, l-methionine (met) and l-tryptophan (trp). The use of thiazolidine containing feeds in Chinese hamster ovary fed-batch processes showed prolonged culture duration and increased productivity. This enhanced performance was correlated with lower reactive species generation, extracellularly and intracellularly. Moreover, an anti-oxidative response was triggered via the induction of superoxide dismutase and an increase in the total glutathione pool, the major intracellular antioxidant. In total, the results confirm that cells in vitro are not cultured in an oxidant-free environment, a concept that has to be considered when studying the influence of reactive species in human diseases. Furthermore, this study indicates that thiazolidines are an interesting class of antioxidant molecules, capable of increasing cell culture media stability and process performance. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:759-770, 2017. © 2017 American Institute of Chemical Engineers.

Endocrine Imbalance Associated With Proteome Changes in Diabetes.

PubMed

Khairallah, Ahmed; Farag, Abo-Alela; Johar, Dina; Bernstein, Larry

2017-11-01

The dynamics of cellular metabolism involves rapid interactions between proteins and nucleic acids, proteins and proteins, and signaling. These involve the interactions with respect to the sulfur bond, noncovalent electrostatic interactions, protein structure stabilization and protein-ligand binding, weak electrostatic interactions in proteins, oxygen radicals that initiate a change in conformation and a chain of events. We review a development in molecular medicine that is a very promising work in progress. We also review the current and future research methods involving mitochondria. Long-term effects of diabetes include glycation of proteins, for example, glycohemoglobin (HbA1c), increased risk of cardiovascular diseases, atherosclerosis, retinopathy, nephropathy, and neurological dysfunctions. Tissues are exposed to significant quantities of highly reactive chemical species including nitric oxide • NO and reactive oxygen species ROS over months to years, to an extent generated by mitochondrial activities. The reactions of • NO can be broadly discussed with reference to three main processes which control their fate in biological systems: (1) diffusion and intra-cellular consumption; (2) autooxidation to form nitrous anhydride N 2 O 3 ; and (3) reaction with superoxide O2 • - to form peroxynitrite ONOO-. Reactive nitrogen species produced by macrophages and neutrophils in the interstitial space, with emphasis on • NO, N 2 O 3 , ONOO-, and nitrogen dioxide radicals • NO 2 generate protein and DNA damage. Serum thiol (-SH) groups act as an important extracellular scavenger of peroxides and are therefore helpful in protecting the surrounding tissues. The events described here are a homeostatic endocrine imbalance that is associated with proteostasis. The advances we have seen in untangling this web of interactions are sure to continue at a breathtaking pace. J. Cell. Biochem. 118: 3569-3576, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Human neutrophils in auto-immunity.

PubMed

Thieblemont, Nathalie; Wright, Helen L; Edwards, Steven W; Witko-Sarsat, Véronique

2016-04-01

Human neutrophils have great capacity to cause tissue damage in inflammatory diseases via their inappropriate activation to release reactive oxygen species (ROS), proteases and other tissue-damaging molecules. Furthermore, activated neutrophils can release a wide variety of cytokines and chemokines that can regulate almost every element of the immune system. In addition to these important immuno-regulatory processes, activated neutrophils can also release, expose or generate neoepitopes that have the potential to break immune tolerance and result in the generation of autoantibodies, that characterise a number of human auto-immune diseases. For example, in vasculitis, anti-neutrophil cytoplasmic antibodies (ANCA) that are directed against proteinase 3 or myeloperoxidase are neutrophil-derived autoantigens and activated neutrophils are the main effector cells of vascular damage. In other auto-immune diseases, these neutrophil-derived neoepitopes may arise from a number of processes that include release of granule enzymes and ROS, changes in the properties of components of their plasma membrane as a result of activation or apoptosis, and via the release of Neutrophil Extracellular Traps (NETs). NETs are extracellular structures that contain chromatin that is decorated with granule enzymes (including citrullinated proteins) that can act as neo-epitopes to generate auto-immunity. This review therefore describes the processes that can result in neutrophil-mediated auto-immunity, and the role of neutrophils in the molecular pathologies of auto-immune diseases such as vasculitis, rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). We discuss the potential role of NETs in these processes and some of the debate in the literature regarding the role of this phenomenon in microbial killing, cell death and auto-immunity. Copyright © 2016 Elsevier Ltd. All rights reserved.

Intramitochondrial Zn2+ accumulation via the Ca2+ uniporter contributes to acute ischemic neurodegeneration

PubMed Central

Medvedeva, Yuliya V.; Weiss, John H.

2014-01-01

Ca2+ and Zn2+ have both been implicated in the induction of acute ischemic neurodegeneration. We recently examined changes in intracellular Zn2+ and Ca2+ in CA1 pyramidal neurons subjected to oxygen glucose deprivation (OGD), and found that Zn2+ rises precede and contribute to the onset of terminal Ca2+ rises (“Ca2+ deregulation”), which are causatively linked to a lethal loss of membrane integrity. The present study seeks to examine the specific role of intramitochondrial Zn2+ accumulation in ischemic injury, using blockers of the mitochondrial Ca2+ uniporter (MCU), through which both Zn2+ and Ca2+ appear able to enter the mitochondrial matrix. In physiological extracellular Ca2+, treatment with the MCU blocker, Ruthenium Red (RR), accelerated the Ca2+ deregulation, most likely by disrupting mitochondrial Ca2+ buffering and thus accelerating the lethal cytosolic Ca2+ overload. However, when intracellular Ca2+ overload was slowed, either by adding blockers of major Ca2+ entry channels or by lowering the concentration of Ca2+ in the extracellular buffer, Ca2+ deregulation was delayed, and under these conditions either Zn2+ chelation or MCU blockade resulted in similar further delays of the Ca2+ deregulation. In parallel studies using the reactive oxygen species (ROS) indicator, hydroethidine, lowering Ca2+ surprisingly accelerated OGD induced ROS generation, and in these low Ca2+ conditions, either Zn2+ chelation or MCU block slowed the ROS generation. These studies suggest that, during acute ischemia, Zn2+ entry into mitochondria via the MCU induces mitochondrial dysfunction (including ROS generation) that occurs upstream of, and contributes to the terminal Ca2+ deregulation. PMID:24787898

Gp120 binding with DC-SIGN induces reactivation of HIV-1 provirus via the NF-κB signaling pathway

PubMed Central

Jin, Changzhong; Li, Jie; Cheng, Linfang; Liu, Fumin; Wu, Nanping

2016-01-01

The reactivation mechanism of latent human immunodeficiency virus type 1 (HIV-1) infection is unclear, especially in dendritic cells (DC). DC-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) binds with HIV-1 and other pathogens to activate the extracellular regulated protein kinase (ERK) and nuclear factor-kappa B (NF-κB) pathways and regulate cytokine expression. We hypothesized that DC-SIGN-induced signaling pathways may activate HIV-1 provirus. To investigate this hypothesis, we generated a model by transfecting 293T cells with a DC-SIGN expression plasmid and an HIV-1 5′ long terminal repeat (LTR) reporter plasmid, and then stimulated the 293T cells with HIV-1 gp120 protein, wild-type HIV-1 or VSV-G-pNL4.3 pseudotype virus (without gp120 protein). It was found that the HIV-1 5′LTR was reactivated by HIV-1 gp120 in DC-SIGN-expressing 293T cells. Then the HIV-1 chronically infected CEM-Bru cells were transfected with DC-SIGN expression plasmid and stimulated by HIV-1 gp120 protein. It was found that early and late HIV-1 provirus replication was reactivated by the HIV-1 gp120/DC-SIGN stimulation. We then investigated the involvement of the ERK, p38 mitogen-activated protein kinases and NF-κB signaling pathways in HIV-1 gp120/DC-SIGN-induced activation of HIV-1 provirus by inhibiting the pathways specifically. Our results indicated that HIV-1 gp120/DC-SIGN stimulation reactivates latent HIV-1 provirus via the NF-κB signal pathway. PMID:26837416

Growth of Acidithiobacillus Ferrooxidans ATCC 23270 in Thiosulfate Under Oxygen-Limiting Conditions Generates Extracellular Sulfur Globules by Means of a Secreted Tetrathionate Hydrolase

PubMed Central

Beard, Simón; Paradela, Alberto; Albar, Juan P.; Jerez, Carlos A.

2011-01-01

Production of sulfur globules during sulfide or thiosulfate oxidation is a characteristic feature of some sulfur bacteria. Although their generation has been reported in Acidithiobacillus ferrooxidans, its mechanism of formation and deposition, as well as the physiological significance of these globules during sulfur compounds oxidation, are currently unknown. Under oxygen-sufficient conditions (OSC), A. ferrooxidans oxidizes thiosulfate to tetrathionate, which accumulates in the culture medium. Tetrathionate is then oxidized by a tetrathionate hydrolase (TTH) generating thiosulfate, elemental sulfur, and sulfate as final products. We report here a massive production of extracellular conspicuous sulfur globules in thiosulfate-grown A. ferrooxidans cultures shifted to oxygen-limiting conditions (OLC). Concomitantly with sulfur globule deposition, the extracellular concentration of tetrathionate greatly diminished and sulfite accumulated in the culture supernatant. A. ferrooxidans cellular TTH activity was negligible in OLC-incubated cells, indicating that this enzymatic activity was not responsible for tetrathionate disappearance. On the other hand, supernatants from both OSC- and OLC-incubated cells showed extracellular TTH activity, which most likely accounted for tetrathionate consumption in the culture medium. The extracellular TTH activity described here: (i) gives experimental support to the TTH-driven model for hydrophilic sulfur globule generation, (ii) explains the extracellular location of A. ferrooxidans sulfur deposits, and (iii) strongly suggests that the generation of sulfur globules in A. ferrooxidans corresponds to an early step during its adaptation to an anaerobic lifestyle. PMID:21833324

Mn(II) oxidation by an ascomycete fungus is linked to superoxide production during asexual reproduction

PubMed Central

Hansel, Colleen M.; Zeiner, Carolyn A.; Santelli, Cara M.; Webb, Samuel M.

2012-01-01

Manganese (Mn) oxides are among the most reactive minerals within the environment, where they control the bioavailability of carbon, nutrients, and numerous metals. Although the ability of microorganisms to oxidize Mn(II) to Mn(III/IV) oxides is scattered throughout the bacterial and fungal domains of life, the mechanism and physiological basis for Mn(II) oxidation remains an enigma. Here, we use a combination of compound-specific chemical assays, microspectroscopy, and electron microscopy to show that a common Ascomycete filamentous fungus, Stilbella aciculosa, oxidizes Mn(II) to Mn oxides by producing extracellular superoxide during cell differentiation. The reactive Mn oxide phase birnessite and the reactive oxygen species superoxide and hydrogen peroxide are colocalized at the base of asexual reproductive structures. Mn oxide formation is not observed in the presence of superoxide scavengers (e.g., Cu) and inhibitors of NADPH oxidases (e.g., diphenylene iodonium chloride), enzymes responsible for superoxide production and cell differentiation in fungi. Considering the recent identification of Mn(II) oxidation by NADH oxidase-based superoxide production by a common marine bacterium (Roseobacter sp.), these results introduce a surprising homology between some prokaryotic and eukaryotic organisms in the mechanisms responsible for Mn(II) oxidation, where oxidation appears to be a side reaction of extracellular superoxide production. Given the versatility of superoxide as a redox reactant and the widespread ability of fungi to produce superoxide, this microbial extracellular superoxide production may play a central role in the cycling and bioavailability of metals (e.g., Hg, Fe, Mn) and carbon in natural systems. PMID:22802654

Mn(II) oxidation by an ascomycete fungus is linked to superoxide production during asexual reproduction.

PubMed

Hansel, Colleen M; Zeiner, Carolyn A; Santelli, Cara M; Webb, Samuel M

2012-07-31

Manganese (Mn) oxides are among the most reactive minerals within the environment, where they control the bioavailability of carbon, nutrients, and numerous metals. Although the ability of microorganisms to oxidize Mn(II) to Mn(III/IV) oxides is scattered throughout the bacterial and fungal domains of life, the mechanism and physiological basis for Mn(II) oxidation remains an enigma. Here, we use a combination of compound-specific chemical assays, microspectroscopy, and electron microscopy to show that a common Ascomycete filamentous fungus, Stilbella aciculosa, oxidizes Mn(II) to Mn oxides by producing extracellular superoxide during cell differentiation. The reactive Mn oxide phase birnessite and the reactive oxygen species superoxide and hydrogen peroxide are colocalized at the base of asexual reproductive structures. Mn oxide formation is not observed in the presence of superoxide scavengers (e.g., Cu) and inhibitors of NADPH oxidases (e.g., diphenylene iodonium chloride), enzymes responsible for superoxide production and cell differentiation in fungi. Considering the recent identification of Mn(II) oxidation by NADH oxidase-based superoxide production by a common marine bacterium (Roseobacter sp.), these results introduce a surprising homology between some prokaryotic and eukaryotic organisms in the mechanisms responsible for Mn(II) oxidation, where oxidation appears to be a side reaction of extracellular superoxide production. Given the versatility of superoxide as a redox reactant and the widespread ability of fungi to produce superoxide, this microbial extracellular superoxide production may play a central role in the cycling and bioavailability of metals (e.g., Hg, Fe, Mn) and carbon in natural systems.

A comparison of antibacterial and antibiofilm efficacy of phenothiazinium dyes between Gram positive and Gram negative bacterial biofilm.

PubMed

Misba, Lama; Zaidi, Sahar; Khan, Asad U

2017-06-01

Antimicrobial photodynamic therapy (APDT) is a process that generates reactive oxygen species (ROS) in presence of photosensitizer, visible light and oxygen which destroys the bacterial cells. We investigated the photoinactivation efficiency of phenothiazinium dyes and the effect of ROS generation on Gram positive and Gram negative bacterial cell as well as on biofilm. Enterococcus faecalis and Klebsiella pneumonia were incubated with all the three phenothiazinium dyes and exposed to 630nm of light. After PDT, colony forming unit (CFU) were performed to estimate the cell survival fraction. Intracellular reactive oxygen species (ROS) was detected by DCFH-DA. Crystal violet (CV) assay and extracellular polysaccharides (EPS) reduction assay were performed to analyze antibiofilm effect. Confocal laser electron microscope (CLSM) scanning electron microscope (SEM) was performed to assess the disruption of biofilm. 8log 10 reduction in bacterial count was observed in Enterococcus faecalis while 3log 10 in Klebsiella pneumoniae. CV and EPS reduction assay revealed that photodynamic inhibition was more pronounced in Enterococcus faecalis. In addition to this CLSM and SEM study showed an increase in cell permeability of propidium iodide and leakage of cellular constituents in treated preformed biofilm which reflects the antibiofilm action of photodynamic therapy. We conclude that Gram-positive bacteria (Enterococcus faecalis) are more susceptible to APDT due to increased level of ROS generation inside the cell, higher photosensitizer binding efficiency and DNA degradation. Phenothiazinium dyes are proved to be highly efficient against both planktonic and biofilm state of cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Induction of autoimmune response to the extracellular loop of the HERG channel pore induces QTc prolongation in guinea‐pigs

PubMed Central

Fabris, Frank; Yue, Yuankun; Qu, Yongxia; Chahine, Mohamed; Sobie, Eric; Lee, Peng; Wieczorek, Rosemary; Jiang, Xian‐Cheng; Capecchi, Pier‐Leopoldo; Laghi‐Pasini, Franco; Lazzerini, Pietro‐Enea

2016-01-01

Key points Channelopathies of autoimmune origin are novel and are associated with corrected QT (QTc) prolongation and complex ventricular arrhythmias.We have recently demonstrated that anti‐SSA/Ro antibodies from patients with autoimmune diseases and with QTc prolongation on the ECG target the human ether‐à‐go‐go‐related gene (HERG) K+ channel by inhibiting the corresponding current, I Kr, at the pore region.Immunization of guinea‐pigs with a peptide (E‐pore peptide) corresponding to the extracellular loop region connecting the S5 and S6 segments of the HERG channel induces high titres of antibodies that inhibit I Kr, lengthen the action potential and cause QTc prolongation on the surface ECG. In addition, anti‐SSA/Ro‐positive sera from patients with connective tissue diseases showed high reactivity to the E‐pore peptide.The translational impact is the development of a peptide‐based approach for the diagnosis and treatment of autoimmune‐associated long QT syndrome. Abstract We recently demonstrated that anti‐SSA/52 kDa Ro antibodies (Abs) from patients with autoimmune diseases and corrected QT (QTc) prolongation directly target and inhibit the human ether‐à‐go‐go‐related gene (HERG) K+ channel at the extracellular pore (E‐pore) region, where homology with SSA/52 kDa Ro antigen was demonstrated. We tested the hypothesis that immunization of guinea‐pigs with a peptide corresponding to the E‐pore region (E‐pore peptide) will generate pathogenic inhibitory Abs and cause QTc prolongation. Guinea‐pigs were immunized with a 31‐amino‐acid peptide corresponding to the E‐pore region of HERG. On days 10–62 after immunization, ECGs were recorded and blood was sampled for the detection of E‐pore peptide Abs. Serum samples from patients with autoimmune diseases were evaluated for reactivity to E‐pore peptide by enzyme‐linked immunosorbent assay (ELISA), and histology was performed on hearts using Masson's Trichrome. Inhibition of the HERG channel was assessed by electrophysiology and by computational modelling of the human ventricular action potential. The ELISA results revealed the presence of high titres of E‐pore peptide Abs and significant QTc prolongation after immunization. High reactivity to E‐pore peptide was found using anti‐SSA/Ro Ab‐positive sera from patients with QTc prolongation. Histological data showed no evidence of fibrosis in immunized hearts. Simulations of simultaneous inhibition of repolarizing currents by anti‐SSA/Ro Ab‐positive sera showed the predominance of the HERG channel in controlling action potential duration and the QT interval. These results are the first to demonstrate that inhibitory Abs to the HERG E‐pore region induce QTc prolongation in immunized guinea‐pigs by targeting the HERG channel independently from fibrosis. The reactivity of anti‐SSA/Ro Ab‐positive sera from patients with connective tissue diseases with the E‐pore peptide opens novel pharmacotherapeutic avenues in the diagnosis and management of autoimmune‐associated QTc prolongation. PMID:27296897

Bio-inspired benchmark generator for extracellular multi-unit recordings

PubMed Central

Mondragón-González, Sirenia Lizbeth; Burguière, Eric

2017-01-01

The analysis of multi-unit extracellular recordings of brain activity has led to the development of numerous tools, ranging from signal processing algorithms to electronic devices and applications. Currently, the evaluation and optimisation of these tools are hampered by the lack of ground-truth databases of neural signals. These databases must be parameterisable, easy to generate and bio-inspired, i.e. containing features encountered in real electrophysiological recording sessions. Towards that end, this article introduces an original computational approach to create fully annotated and parameterised benchmark datasets, generated from the summation of three components: neural signals from compartmental models and recorded extracellular spikes, non-stationary slow oscillations, and a variety of different types of artefacts. We present three application examples. (1) We reproduced in-vivo extracellular hippocampal multi-unit recordings from either tetrode or polytrode designs. (2) We simulated recordings in two different experimental conditions: anaesthetised and awake subjects. (3) Last, we also conducted a series of simulations to study the impact of different level of artefacts on extracellular recordings and their influence in the frequency domain. Beyond the results presented here, such a benchmark dataset generator has many applications such as calibration, evaluation and development of both hardware and software architectures. PMID:28233819

Pseudomonas aeruginosa inactivation mechanism is affected by capsular extracellular polymeric substances reactivity with chlorine and monochloramine.

PubMed

Xue, Zheng; Hessler, Christopher M; Panmanee, Warunya; Hassett, Daniel J; Seo, Youngwoo

2013-01-01

The reactivity of capsular extracellular polymeric substances (EPS) to chlorine and monochloramine was assessed and compared in this study. The impact of capsular EPS on Gram-negative bacteria Pseudomonas aeruginosa inactivation mechanisms was investigated both qualitatively and quantitatively using a combination of batch experiments, viability tests with LIVE/DEAD staining, and Fourier transform infrared spectroscopy (FTIR). Both wild-type and isogenic mutant strains with different alginate EPS production capabilities were used to evaluate their susceptibility to chlorine and monochloramine. The mucA22 mutant strain, which overproduces the EPS composed largely of acidic polysaccharide alginate, exhibited high resistance and prolonged inactivation time to both chlorine and monochloramine relative to PAO1 (wild-type) and algT(U) mutant strains (alginate EPS deficient). Multiple analyses were combined to better understand the mechanistic role of EPS against chlorine-based disinfectants. The extracted EPS exhibited high reactivity with chlorine and very low reactivity with monochloramine, suggesting different mechanism of protection against disinfectants. Moreover, capsular EPS on cell membrane appeared to reduce membrane permeabilization by disinfectants as suggested by deformation of key functional groups in EPS and cell membrane (the C-O-C stretching of carbohydrate and the C=O stretching of ester group). The combined results supported that capsular EPS, acting either as a disinfectant consumer (for chlorine inactivation) or limiting access to reactive sites on cell membrane (for monochloramine inactivation), provide a protective role for bacterial cells against regulatory residual disinfectants by reducing membrane permeabilization. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Adaptive response of Yersinia pestis to extracellular effectors of innate immunity during bubonic plague.

PubMed

Sebbane, Florent; Lemaître, Nadine; Sturdevant, Daniel E; Rebeil, Roberto; Virtaneva, Kimmo; Porcella, Stephen F; Hinnebusch, B Joseph

2006-08-01

Yersinia pestis causes bubonic plague, characterized by an enlarged, painful lymph node, termed a bubo, that develops after bacterial dissemination from a fleabite site. In susceptible animals, the bacteria rapidly escape containment in the lymph node, spread systemically through the blood, and produce fatal sepsis. The fulminant progression of disease has been largely ascribed to the ability of Y. pestis to avoid phagocytosis and exposure to antimicrobial effectors of innate immunity. In vivo microarray analysis of Y. pestis gene expression, however, revealed an adaptive response to nitric oxide (NO)-derived reactive nitrogen species and to iron limitation in the extracellular environment of the bubo. Polymorphonuclear neutrophils recruited to the infected lymph node expressed abundant inducible NO synthase, and several Y. pestis homologs of genes involved in the protective response to reactive nitrogen species were up-regulated in the bubo. Mutation of one of these genes, which encodes the Hmp flavohemoglobin that detoxifies NO, attenuated virulence. Thus, the ability of Y. pestis to destroy immune cells and remain extracellular in the bubo appears to limit exposure to some but not all innate immune effectors. High NO levels induced during plague may also influence the developing adaptive immune response and contribute to septic shock.

Rapid polyether cleavage via extracellular one-electron oxidation by a brown-rot basidiomycete.

PubMed

Kerem, Z; Bao, W; Hammel, K E

1998-09-01

Fungi that cause brown rot of wood are essential biomass recyclers and also the principal agents of decay in wooden structures, but the extracellular mechanisms by which they degrade lignocellulose remain unknown. To test the hypothesis that brown-rot fungi use extracellular free radical oxidants as biodegradative tools, Gloeophyllum trabeum was examined for its ability to depolymerize an environmentally recalcitrant polyether, poly(ethylene oxide) (PEO), that cannot penetrate cell membranes. Analyses of degraded PEOs by gel permeation chromatography showed that the fungus cleaved PEO rapidly by an endo route. 13C NMR analyses of unlabeled and perdeuterated PEOs recovered from G. trabeum cultures showed that a major route for depolymerization was oxidative C---C bond cleavage, a reaction diagnostic for hydrogen abstraction from a PEO methylene group by a radical oxidant. Fenton reagent (Fe(II)/H2O2) oxidized PEO by the same route in vitro and therefore might account for PEO biodegradation if it is produced by the fungus, but the data do not rule out involvement of less reactive radicals. The reactivity and extrahyphal location of this PEO-degrading system suggest that its natural function is to participate in the brown rot of wood and that it may enable brown-rot fungi to degrade recalcitrant organopollutants.

ERK pathway inhibitors: how low should we go?

PubMed

Nissan, Moriah H; Rosen, Neal; Solit, David B

2013-07-01

Resistance to RAF inhibitors is generally accompanied by reactivation of extracellular signal-regulated kinase (ERK) signaling. SCH772984, a selective, ATP-competitive inhibitor of ERK1 and ERK2, is effective in BRAF-mutant models in which resistance is the result of ERK reactivation. SCH772984 may also have a role in the treatment of tumors in which ERK is dysregulated by mutant RAS, NF1, or activated receptor tyrosine kinases, settings in which current RAF inhibitors are ineffective. ©2013 AACR.

Biomechanical cell regulatory networks as complex adaptive systems in relation to cancer.

PubMed

Feller, Liviu; Khammissa, Razia Abdool Gafaar; Lemmer, Johan

2017-01-01

Physiological structure and function of cells are maintained by ongoing complex dynamic adaptive processes in the intracellular molecular pathways controlling the overall profile of gene expression, and by genes in cellular gene regulatory circuits. Cytogenetic mutations and non-genetic factors such as chronic inflammation or repetitive trauma, intrinsic mechanical stresses within extracellular matrix may induce redirection of gene regulatory circuits with abnormal reactivation of embryonic developmental programmes which can now drive cell transformation and cancer initiation, and later cancer progression and metastasis. Some of the non-genetic factors that may also favour cancerization are dysregulation in epithelial-mesenchymal interactions, in cell-to-cell communication, in extracellular matrix turnover, in extracellular matrix-to-cell interactions and in mechanotransduction pathways. Persistent increase in extracellular matrix stiffness, for whatever reason, has been shown to play an important role in cell transformation, and later in cancer cell invasion. In this article we review certain cell regulatory networks driving carcinogenesis, focussing on the role of mechanical stresses modulating structure and function of cells and their extracellular matrices.

Extracellular RNA is transported from one generation to the next in Caenorhabditis elegans

PubMed Central

Marré, Julia; Traver, Edward C.

2016-01-01

Experiences during the lifetime of an animal have been proposed to have consequences for subsequent generations. Although it is unclear how such intergenerational transfer of information occurs, RNAs found extracellularly in animals are candidate molecules that can transfer gene-specific regulatory information from one generation to the next because they can enter cells and regulate gene expression. In support of this idea, when double-stranded RNA (dsRNA) is introduced into some animals, the dsRNA can silence genes of matching sequence and the silencing can persist in progeny. Such persistent gene silencing is thought to result from sequence-specific interaction of the RNA within parents to generate chromatin modifications, DNA methylation, and/or secondary RNAs, which are then inherited by progeny. Here, we show that dsRNA can be directly transferred between generations in the worm Caenorhabditis elegans. Intergenerational transfer of dsRNA occurs even in animals that lack any DNA of matching sequence, and dsRNA that reaches progeny can spread between cells to cause gene silencing. Surprisingly, extracellular dsRNA can also reach progeny without entry into the cytosol, presumably within intracellular vesicles. Fluorescently labeled dsRNA is imported from extracellular space into oocytes along with yolk and accumulates in punctate structures within embryos. Subsequent entry into the cytosol of early embryos causes gene silencing in progeny. These results demonstrate the transport of extracellular RNA from one generation to the next to regulate gene expression in an animal and thus suggest a mechanism for the transmission of experience-dependent effects between generations. PMID:27791108

Quantitative iTRAQ-based secretome analysis reveals species-specific and temporal shifts in carbon utilization strategies among manganese(II)-oxidizing Ascomycete fungi.

PubMed

Zeiner, Carolyn A; Purvine, Samuel O; Zink, Erika M; Paša-Tolić, Ljiljana; Chaput, Dominique L; Wu, Si; Santelli, Cara M; Hansel, Colleen M

2017-09-01

Fungi generate a wide range of extracellular hydrolytic and oxidative enzymes and reactive metabolites, collectively known as the secretome, that synergistically drive plant litter decomposition in the environment. While secretome studies of model organisms have greatly expanded our knowledge of these enzymes, few have extended secretome characterization to environmental isolates, particularly filamentous Ascomycetes, or directly compared temporal patterns of enzyme utilization among diverse species. Thus, the mechanisms of carbon (C) degradation by many ubiquitous soil fungi remain poorly understood. Here we use a combination of iTRAQ proteomics and extracellular enzyme activity assays to compare the protein composition of the secretomes of four manganese(II)-oxidizing Ascomycete fungi over a three-week time course. We demonstrate that the fungi exhibit striking differences in the regulation of extracellular lignocellulose-degrading enzymes among species and over time, revealing species-specific and temporal shifts in C utilization strategies as they degrade the same substrate. Specifically, our findings suggest that Alternaria alternata SRC1lrK2f and Paraconiothyrium sporulosum AP3s5-JAC2a employ sequential enzyme secretion patterns concomitant with decreasing resource availability. Stagonospora sp. SRC1lsM3a preferentially degrades proteinaceous substrate before switching to carbohydrates, and Pyrenochaeta sp. DS3sAY3a utilizes primarily peptidases to aggressively attack carbon sources in a concentrated burst. This work highlights the diversity of operative metabolic strategies among understudied yet ubiquitous cellulose-degrading Ascomycetes, enhancing our understanding of their contribution to C turnover in the environment. Copyright © 2017. Published by Elsevier Inc.

Male genital tract antioxidant enzymes--their ability to preserve sperm DNA integrity.

PubMed

O, Wai-Sum; Chen, H; Chow, P H

2006-05-16

Male germ cells are unique because they lose a bulk of their cytoplasm as cytoplasmic droplets when they develop, leading to a decrease in endogenous antioxidant and hence a dependence on extracellular antioxidant system to overcome oxidative stress. Spermatozoa are particularly vulnerable to oxidative stress because their plasma membrane is rich in polyunsaturated fatty acids and membrane-bound NADPH oxidase. To protect spermatozoa from oxidative attack, an optimal amount of reactive oxygen species is maintained by balancing the reactive oxygen species generated during sperm maturation in the epididymidis and antioxidants in secretions of the male reproductive tract. The male accessory sex glands secretions have been shown to be the major source of antioxidant enzymes in the ejaculate and have the important function of preserving sperm DNA integrity from oxidative stress experienced in the uterine environment. In our in vivo golden hamster model, ablation of the five major male accessory sex glands, namely the ampullary glands, coagulating glands, dorsolateral prostate, ventral prostate and seminal vesicle, was found to cause higher incidence and greater degree of DNA damage in spermatozoa. These damaged sperm are able to undergo fertilization at the same rate as intact ones; however, the outcome of embryos sired is seriously affected.

Fractionation and Characterization of Biologically-active Polysaccharides from Artemisia tripartita

PubMed Central

Xie, Gang; Schepetkin, Igor A.; Siemsen, Daniel W.; Kirpotina, Liliya N.; Wiley, James A.; Quinn, Mark T.

2008-01-01

The leaves of Artemisia species have been traditionally used for prevention and treatment of a number of diseases. In this study, five polysaccharide fractions (designated A-I to A-V) were isolated from the leaves of Artemisia tripartita Rydb. by the sequential use of hot-water extraction, ethanol precipitation, ultra-filtration, and chromatography. The homogeneity and average molecular weight of each fraction were determined by high performance size-exclusion chromatography. Sugar composition analysis revealed that Artemisia polysaccharides consisted primarily of xylose, glucose, arabinose, galactose, and galactosamine. Moreover, all fractions contained at least 3.4% sulfate, and fractions A-II through A-V contained an arabinogalactan type II structure. All fractions exhibited macrophage-activating activity, enhancing production of intracellular reactive oxygen species and release of nitric oxide, interleukin 6, interleukin 10, tumor necrosis factor α, and monocyte chemotactic protein-1. In addition, all fractions exhibited scavenging activity for reactive oxygen species generated enzymatically or produced extracellularly by human neutrophils. Finally, fractions A-I and A-V exhibited complement-fixing activity. Taken together, our results provide a molecular basis to explain at least part of the beneficial therapeutic effects of Artemisia extracts, and suggest the possibility of using Artemisia polysaccharides as an immunotherapeutic adjuvant. PMID:18325553

Pigment epithelium-derived factor stimulates skeletal muscle glycolytic activity through NADPH oxidase-dependent reactive oxygen species production.

PubMed

Carnagarin, Revathy; Carlessi, Rodrigo; Newsholme, Philip; Dharmarajan, Arun M; Dass, Crispin R

2016-09-01

Pigment epithelium-derived factor is a multifunctional serpin implicated in insulin resistance in metabolic disorders. Recent evidence suggests that exposure of peripheral tissues such as skeletal muscle to PEDF has profound metabolic consequences with predisposition towards chronic conditions such as obesity, type 2 diabetes, metabolic syndrome and polycystic ovarian syndrome. Chronic inflammation shifts muscle metabolism towards increased glycolysis and decreased oxidative metabolism. In the present study, we demonstrate a novel effect of PEDF on cellular metabolism in mouse cell line (C2C12) and human primary skeletal muscle cells. PEDF addition to skeletal muscle cells induced enhanced phospholipase A2 activity. This was accompanied with increased production of reactive oxygen species in a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent manner that triggered a shift towards a more glycolytic phenotype. Extracellular flux analysis and glucose consumption assays demonstrated that PEDF treatment resulted in enhanced glycolysis but did not change mitochondrial respiration. Our results demonstrate that skeletal muscle cells express a PEDF-inducible oxidant generating system that enhances glycolysis but is sensitive to antioxidants and NADPH oxidase inhibition. Copyright © 2016 Elsevier Ltd. All rights reserved.

Identification of Manganese Superoxide Dismutase from Sphingobacterium sp. T2 as a Novel Bacterial Enzyme for Lignin Oxidation.

PubMed

Rashid, Goran M M; Taylor, Charles R; Liu, Yangqingxue; Zhang, Xiaoyang; Rea, Dean; Fülöp, Vilmos; Bugg, Timothy D H

2015-10-16

The valorization of aromatic heteropolymer lignin is an important unsolved problem in the development of a biomass-based biorefinery, for which novel high-activity biocatalysts are needed. Sequencing of the genomic DNA of lignin-degrading bacterial strain Sphingobacterium sp. T2 revealed no matches to known lignin-degrading genes. Proteomic matches for two manganese superoxide dismutase proteins were found in partially purified extracellular fractions. Recombinant MnSOD1 and MnSOD2 were both found to show high activity for oxidation of Organosolv and Kraft lignin, and lignin model compounds, generating multiple oxidation products. Structure determination revealed that the products result from aryl-Cα and Cα-Cβ bond oxidative cleavage and O-demethylation. The crystal structure of MnSOD1 was determined to 1.35 Å resolution, revealing a typical MnSOD homodimer harboring a five-coordinate trigonal bipyramidal Mn(II) center ligated by three His, one Asp, and a water/hydroxide in each active site. We propose that the lignin oxidation reactivity of these enzymes is due to the production of a hydroxyl radical, a highly reactive oxidant. This is the first demonstration that MnSOD is a microbial lignin-oxidizing enzyme.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Garige, Mamatha; Walters, Eric, E-mail: ewalters@howard.edu

The molecular basis for nutraceutical properties of the polyphenol curcumin (Curcuma longa, Turmeric) is complex, affecting multiple factors that regulate cell signaling and homeostasis. Here, we report the effect of curcumin on cellular and developmental mechanisms in the eukaryotic model, Dictyostelium discoideum. Dictyostelium proliferation was inhibited in the presence of curcumin, which also suppressed the prestarvation marker, discoidin I, members of the yakA-mediated developmental signaling pathway, and expression of the extracellular matrix/cell adhesion proteins (DdCAD and csA). This resulted in delayed chemotaxis, adhesion, and development of the organism. In contrast to the inhibitory effects on developmental genes, curcumin induced gstAmore » gene expression, overall GST activity, and generated production of reactive oxygen species. These studies expand our knowledge of developmental and biochemical signaling influenced by curcumin, and lends greater consideration of GST enzyme function in eukaryotic cell signaling, development, and differentiation.« less

RNA sequencing supports distinct reactive oxygen species-mediated pathways of apoptosis by high and low size mass fractions of Bay leaf (Lauris nobilis) in HT-29 cells.

PubMed

Rodd, Annabelle L; Ververis, Katherine; Sayakkarage, Dheeshana; Khan, Abdul W; Rafehi, Haloom; Ziemann, Mark; Loveridge, Shanon J; Lazarus, Ross; Kerr, Caroline; Lockett, Trevor; El-Osta, Assam; Karagiannis, Tom C; Bennett, Louise E

2015-08-01

Anti-proliferative and pro-apoptotic effects of Bay leaf (Laurus nobilis) in mammalian cancer and HT-29 adenocarcinoma cells have been previously attributed to effects of polyphenolic and essential oil chemical species. Recently, we demonstrated differentiated growth-regulating effects of high (HFBL) versus low molecular mass (LFBL) aqueous fractions of bay leaf and now confirm by comparative effects on gene expression, that HFBL and LFBL suppress HT-29 growth by distinct mechanisms. Induction of intra-cellular lesions including DNA strand breakage by extra-cellular HFBL, invoked the hypothesis that iron-mediated reactive oxygen species with capacity to penetrate cell membrane, were responsible for HFBL-mediated effects, supported by equivalent effects of HFBL in combination with γ radiation. Activities of HFBL and LFBL were interpreted to reflect differentiated responses to iron-mediated reactive oxygen species (ROS), occurring either outside or inside cells. In the presence of LFBL, apoptotic death was relatively delayed compared with HFBL. ROS production by LFBL mediated p53-dependent apoptosis and recovery was suppressed by promoting G1/S phase arrest and failure of cellular tight junctions. In comparison, intra-cellular anti-oxidant protection exerted by LFBL was absent for extra-cellular HFBL (likely polysaccharide-rich), which potentiated more rapid apoptosis by producing DNA double strand breaks. Differentiated effects on expression of genes regulating ROS defense and chromatic condensation by LFBL versus HFBL, were observed. The results support ferrous iron in cell culture systems and potentially in vivo, can invoke different extra-cellular versus intra-cellular ROS-mediated chemistries, that may be regulated by exogenous, including dietary species.

Increasing the endogenous NO level causes catalase inactivation and reactivation of intercellular apoptosis signaling specifically in tumor cells.

PubMed

Bauer, Georg

2015-12-01

Tumor cells generate extracellular superoxide anions and are protected against intercellular apoptosis-inducing HOCl- and NO/peroxynitrite signaling through the expression of membrane-associated catalase. This enzyme decomposes H2O2 and thus prevents HOCl synthesis. It efficiently interferes with NO/peroxynitrite signaling through oxidation of NO and decomposition of peroxynitrite. The regulatory potential of catalase at the crosspoint of ROS and RNS chemical biology, as well as its high local concentration on the outside of the cell membrane of tumor cells, establish tight control of intercellular signaling and thus prevent tumor cell apoptosis. Therefore, inhibition of catalase or its inactivation by singlet oxygen reactivate intercellular apoptosis-inducing signaling. Nitric oxide and peroxynitrite are connected with catalase in multiple and meaningful ways, as (i) NO can be oxidated by compound I of catalase, (ii) NO can reversibly inhibit catalase, (iii) peroxynitrite can be decomposed by catalase and (iv) the interaction between peroxynitrite and H2O2 leads to the generation of singlet oxygen that inactivates catalase. Therefore, modulation of the concentration of free NO through addition of arginine, inhibition of arginase, induction of NOS expression or inhibition of NO dioxygenase triggers an autoamplificatory biochemical cascade that is based on initial formation of singlet oxygen, amplification of superoxide anion/H2O2 and NO generation through singlet oxygen dependent stimulation of the FAS receptor and caspase-8. Finally, singlet oxygen is generated at sufficiently high concentration to inactivate protective catalase and to reactivate intercellular apoptosis-inducing ROS signaling. This regulatory network allows to establish several pathways for synergistic interactions, like the combination of modulators of NO metabolism with enhancers of superoxide anion generation, modulators of NO metabolism that act at different targets and between modulators of NO metabolism and direct catalase inhibitors. The latter aspect is explicitely studied for the interaction between catalase inhibiting acetylsalicylic acid and an NO donor. It is also shown that hybrid molecules like NO-aspirin utilize this synergistic potential. Our data open novel approaches for rational tumor therapy based on specific ROS signaling and its control in tumor cells. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Autophagy Primes Neutrophils for Neutrophil Extracellular Trap Formation during Sepsis.

PubMed

Park, So Young; Shrestha, Sanjeeb; Youn, Young-Jin; Kim, Jun-Kyu; Kim, Shin-Yeong; Kim, Hyun Jung; Park, So-Hee; Ahn, Won-Gyun; Kim, Shin; Lee, Myung Goo; Jung, Ki-Suck; Park, Yong Bum; Mo, Eun-Kyung; Ko, Yousang; Lee, Suh-Young; Koh, Younsuck; Park, Myung Jae; Song, Dong-Keun; Hong, Chang-Won

2017-09-01

Neutrophils are key effectors in the host's immune response to sepsis. Excessive stimulation or dysregulated neutrophil functions are believed to be responsible for sepsis pathogenesis. However, the mechanisms regulating functional plasticity of neutrophils during sepsis have not been fully determined. We investigated the role of autophagy in neutrophil functions during sepsis in patients with community-acquired pneumonia. Neutrophils were isolated from patients with sepsis and stimulated with phorbol 12-myristate 13-acetate (PMA). The levels of reactive oxygen species generation, neutrophil extracellular trap (NET) formation, and granule release, and the autophagic status were evaluated. The effect of neutrophil autophagy augmentation was further evaluated in a mouse model of sepsis. Neutrophils isolated from patients who survived sepsis showed an increase in autophagy induction, and were primed for NET formation in response to subsequent PMA stimulation. In contrast, neutrophils isolated from patients who did not survive sepsis showed dysregulated autophagy and a decreased response to PMA stimulation. The induction of autophagy primed healthy neutrophils for NET formation and vice versa. In a mouse model of sepsis, the augmentation of autophagy improved survival via a NET-dependent mechanism. These results indicate that neutrophil autophagy primes neutrophils for increased NET formation, which is important for proper neutrophil effector functions during sepsis. Our study provides important insights into the role of autophagy in neutrophils during sepsis.

Simple radiosensitizing of hypoxic tumor tissues by N2O/Br(-) mixture.

PubMed

Billik, P

2015-07-01

The radiosensitization model of hypoxic tumor tissues based on the N2O/Br(-) mixture is described. The well-documented radiolysis of water in the presence of N2O and Br(-) ions at a low concentration supports this model. An aqueous solution saturated with N2O gas during the radiolysis generates OH radicals in a large extent. In N2O/Br- media at pH<9, Br2 is formed. Br2 hydrolyzes in an aqueous solution to form a very reactive hypobromous (HOBr) acid. Such process is described by the following chemical reaction: H2O + Br(-) + N2O + ionizing radiation (IR) --> HOBr + OH(-). In vivo formed HOBr as a long-lived product with a high biological activity induces the hypoxic tumor cell damage via many unique mechanisms. A local application or inhalation of an N2O-O2 mixture before or during the radiotherapy to enhance the saturation of tissues with N2O is a key prerequisite. Since the extracellular concentration of Br(-) ions is very low (0.02-0.05 mM), an oral or local application of NaBr should be used to shift the extracellular concentration of Br(-) ions to the mM region. Copyright © 2015 Elsevier Ltd. All rights reserved.

Essential oil from leaves of Liquidambar formosana ameliorates inflammatory response in lipopolysaccharide-activated mouse macrophages.

PubMed

Hua, Kuo-Feng; Yang, Tzu-Jung; Chiu, Huan-Wen; Ho, Chen-Lung

2014-06-01

The essential oil from Liquidambar formosana leaves (EOLF) was demonstrated to exhibit anti-inflammatory activity in mouse macrophages. EOLF reduced nitrite oxide generation, secretion levels of tumor necrosis factor-alpha and interleukin-6, and expression levels of prointerleukin-beta, inducible nitric oxide synthase, and cyclooxygenase-2 in lipopolysaccharide (LPS)-activated mouse macrophages. EOLF also reduced NLRP3 inflammasome-derived interleukin-1beta secretion. The underlying mechanisms for the EOLF-mediated anti-inflammatory activity were (1) reduction of LPS-induced reactive oxygen species generation; (2) reduction of LPS-induced activation of c-Jun N-terminal kinase, extracellular signal-regulated kinase, and p38 MAP kinase; (3) reduction of LPS-induced nuclear factor-kappaBeta activation. Furthermore, 25 compounds were identified in the EOLF using GC-FID and GC-MS and the major compounds were terpinen-4-ol (32.0%), beta-pinene (18.0%), gamma-terpinene (13.8%), and alpha-terpinene (9.7%). We found that LPS-induced nitrite oxide generation was inhibited significantly by terpinen-4-ol. Our results indicated that EOLF has anti-inflammatory activity and may provide a molecular rationale for future therapeutic interventions in immune modulation.

Engineering Extracellular Expression Systems in Escherichia coli Based on Transcriptome Analysis and Cell Growth State.

PubMed

Gao, Wen; Yin, Jun; Bao, Lichen; Wang, Qun; Hou, Shan; Yue, Yali; Yao, Wenbing; Gao, Xiangdong

2018-05-18

Escherichia coli extracellular expression systems have a number of advantages over other systems, such as lower pyrogen levels and a simple purification process. Various approaches, such as the generation of leaky mutants via chromosomal engineering, have been explored for this expression system. However, extracellular protein yields in leaky mutants are relatively low compared to that in intracellular expression systems and therefore need to be improved. In this work, we describe the construction, characterization, and mechanism of enhanced extracellular expression in Escherichia coli. On the basis of the localizations, functions, and transcription levels of cell envelope proteins, we systematically elucidated the effects of multiple gene deletions on cell growth and extracellular expression using modified CRISPR/Cas9-based genome editing and a FlAsH labeling assay. High extracellular yields of heterologous proteins of different sizes were obtained by screening multiple gene mutations. The enhancement of extracellular secretion was associated with the derepression of translation and translocation. This work utilized universal methods in the design of extracellular expression systems for genes not directly associated with protein synthesis that were used to generate strains with higher protein expression capability. We anticipate that extracellular expression systems may help to shed light on the poorly understood aspects of these secretion processes as well as to further assist in the construction of engineered prokaryotic cells for efficient extracellular production of heterologous proteins.

Salicin, an extract from white willow bark, inhibits angiogenesis by blocking the ROS-ERK pathways.

PubMed

Kong, Chang-Seok; Kim, Ka-Hyun; Choi, Jae-Sun; Kim, Ja-Eun; Park, Chan; Jeong, Joo-Won

2014-08-01

Salicin has been studied as a potent antiinflammatory agent. Angiogenesis is an essential process for tumor progression, and negative regulation of angiogenesis provides a good strategy for antitumor therapy. However, the potential medicinal value of salicin on antitumorigenic and antiangiogenic effects remain unexplored. In this study, we examined the antitumorigenic and antiangiogenic activity of salicin and its underlying mechanism of action. Salicin suppressed the angiogenic activity of endothelial cells, such as migration, tube formation, and sprouting from an aorta. Moreover, salicin reduced reactive oxygen species production and activation of the extracellular signal-regulated kinase pathway. The expression of vascular endothelial growth factor was also decreased by salicin in endothelial cells. When the salicin was administered to mice, salicin inhibited tumor growth and angiogenesis in a mouse tumor model. Taken together, salicin targets the signaling pathways mediated by reactive oxygen species and extracellular signal-regulated kinase, providing new perspectives into a potent therapeutic agent for hypervascularized tumors. Copyright © 2014 John Wiley & Sons, Ltd.

Decolorization of complex dyes and textile effluent by extracellular enzymes of Cyathus bulleri cultivated on agro-residues/domestic wastes and proposed pathway of degradation of Kiton blue A and reactive orange 16.

PubMed

Vats, Arpita; Mishra, Saroj

2017-04-01

In this study, the white-rot fungus Cyathus bulleri was cultivated on low-cost agro-residues, namely wheat bran (WB), wheat straw (WS), and domestic waste orange peel (OP) for production of ligninolytic enzymes. Of the three substrates, WB and OP served as good materials for the production of laccase with no requirement of additional carbon or nitrogen source. Specific laccase activity of 94.4 U mg -1 extracellular protein and 21.01 U mg -1 protein was obtained on WB and OP, respectively. Maximum decolorization rate of 13.6 μmol h -1  U -1 laccase for reactive black 5 and 22.68 μmol h -1  U -1 laccase for reactive orange 16 (RO) was obtained with the WB culture filtrate, and 11.7 μmol h -1  U -1 laccase for reactive violet 5 was observed with OP culture filtrate. Importantly, Kiton blue A (KB), reported not to be amenable to enzymatic degradation, was degraded by culture filtrate borne activities. Products of degradation of KB and RO were identified by mass spectrometry, and a pathway of degradation proposed. WB-grown culture filtrate decolorized and detoxified real and simulated textile effluents by about 40%. The study highlights the use of inexpensive materials for the production of enzymes effective on dyes and effluents.

Extracellular Alkalinization as a Defense Response in Potato Cells.

PubMed

Moroz, Natalia; Fritch, Karen R; Marcec, Matthew J; Tripathi, Diwaker; Smertenko, Andrei; Tanaka, Kiwamu

2017-01-01

A quantitative and robust bioassay to assess plant defense response is important for studies of disease resistance and also for the early identification of disease during pre- or non-symptomatic phases. An increase in extracellular pH is known to be an early defense response in plants. In this study, we demonstrate extracellular alkalinization as a defense response in potatoes. Using potato suspension cell cultures, we observed an alkalinization response against various pathogen- and plant-derived elicitors in a dose- and time-dependent manner. We also assessed the defense response against a variety of potato pathogens, such as protists ( Phytophthora infestans and Spongospora subterranea ) and fungi ( Verticillium dahliae and Colletotrichum coccodes ). Our results show that extracellular pH increases within 30 min in proportion to the number of pathogen spores added. Consistently with the alkalinization effect, the higher transcription level of several defense-related genes and production of reactive oxygen species was observed. Our results demonstrate that the alkalinization response is an effective marker to study early stages of defense response in potatoes.

Arabidopsis GRI is involved in the regulation of cell death induced by extracellular ROS.

PubMed

Wrzaczek, Michael; Brosché, Mikael; Kollist, Hannes; Kangasjärvi, Jaakko

2009-03-31

Reactive oxygen species (ROS) have important functions in plant stress responses and development. In plants, ozone and pathogen infection induce an extracellular oxidative burst that is involved in the regulation of cell death. However, very little is known about how plants can perceive ROS and regulate the initiation and the containment of cell death. We have identified an Arabidopsis thaliana protein, GRIM REAPER (GRI), that is involved in the regulation of cell death induced by extracellular ROS. Plants with an insertion in GRI display an ozone-sensitive phenotype. GRI is an Arabidopsis ortholog of the tobacco flower-specific Stig1 gene. The GRI protein appears to be processed in leaves with a release of an N-terminal fragment of the protein. Infiltration of the N-terminal fragment of the GRI protein into leaves caused cell death in a superoxide- and salicylic acid-dependent manner. Analysis of the extracellular GRI protein yields information on how plants can initiate ROS-induced cell death during stress response and development.

Arabidopsis GRI is involved in the regulation of cell death induced by extracellular ROS

PubMed Central

Wrzaczek, Michael; Brosché, Mikael; Kollist, Hannes; Kangasjärvi, Jaakko

2009-01-01

Reactive oxygen species (ROS) have important functions in plant stress responses and development. In plants, ozone and pathogen infection induce an extracellular oxidative burst that is involved in the regulation of cell death. However, very little is known about how plants can perceive ROS and regulate the initiation and the containment of cell death. We have identified an Arabidopsis thaliana protein, GRIM REAPER (GRI), that is involved in the regulation of cell death induced by extracellular ROS. Plants with an insertion in GRI display an ozone-sensitive phenotype. GRI is an Arabidopsis ortholog of the tobacco flower-specific Stig1 gene. The GRI protein appears to be processed in leaves with a release of an N-terminal fragment of the protein. Infiltration of the N-terminal fragment of the GRI protein into leaves caused cell death in a superoxide- and salicylic acid-dependent manner. Analysis of the extracellular GRI protein yields information on how plants can initiate ROS-induced cell death during stress response and development. PMID:19279211

Preparation of a functional fluorescent human Fas ligand extracellular domain derivative using a three-dimensional structure guided site-specific fluorochrome conjugation.

PubMed

Muraki, Michiro

2016-01-01

Human Fas ligand extracellular domain has been investigated as an important target protein in the field of medical biotechnology. In a recent study, the author developed an effective method to produce biologically active human Fas ligand extracellular domain derivatives using site-specific chemical modifications. A human Fas ligand extracellular domain derivative containing a reactive cysteine residue within its N-terminal tag sequence, which locates not proximal to the binding interface between the ligand and the receptor in terms of the three-dimensional structure, was modified by Fluorescein-5-Maleimide without impairing the specific binding activity toward human Fas receptor extracellular domain. The purified protein sample free of low molecular-weight contaminants showed a characteristic fluorescence spectrum derived from the attached Fluorescein moieties, and formed a stable binding complex with human Fas receptor extracellular domain-human IgG1 Fc domain fusion protein in solution. The conjugation number of the fluorochrome was estimated to be 2.5 per a single human Fas ligand extracellular domain trimer from the ratio of the absorbance value at 280 nm to that at 495 nm. A functional fluorescent human Fas ligand extracellular domain derivative was prepared via a site-specific conjugation of fluorochrome, which was guided by the three-dimensional structure information on the ligand-receptor complex. Fluorescent derivatives created by this method may contribute to the development of an improved diagnosis system for the diseases related to Fas receptor.

Prediction of Metastasis Using Second Harmonic Generation

DTIC Science & Technology

2016-07-01

extracellular matrix through which metastasizing cells must travel. We and others have demonstrated that tumor collagen structure, as measured with the...algorithm using separate training and validation sets, etc. Keywords: metastasis, overtreatment, extracellular matrix , collagen , second harmonic...optical process called second harmonic generation (SHG), influences tumor metastasis. This suggests that collagen structure may provide prognostic

Subneurotoxic copper(II)-induced NF-κB-dependent microglial activation is associated with mitochondrial ROS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hu, Zhuqin; Yu, Fengxiang; Gong, Ping

2014-04-15

Microglia-mediated neuroinflammation and the associated neuronal damage play critical roles in the pathogenesis of neurodegenerative disorders. Evidence shows an elevated concentration of extracellular copper(II) in the brains of these disorders, which may contribute to neuronal death through direct neurotoxicity. Here we explored whether extracellular copper(II) triggers microglial activation. Primary rat microglia and murine microglial cell line BV-2 cells were cultured and treated with copper(II). The content of tumor necrosis factor-α (TNF-α) and nitric oxide in the medium was determined. Extracellular hydrogen peroxide was quantified by a fluorometric assay with Amplex Red. Mitochondrial superoxide was measured by MitoSOX oxidation. At subneurotoxicmore » concentrations, copper(II) treatment induced a dose- and time-dependent release of TNF-α and nitric oxide from microglial cells, and caused an indirect, microglia-mediated neurotoxicity that was blocked by inhibition of TNF-α and nitric oxide production. Copper(II)-initiated microglial activation was accompanied with reduced IkB-α expression as well as phosphorylation and translocation of nuclear factor-κB (NF-κB) p65 and was blocked by NF-κB inhibitors (BAY11-7082 and SC-514). Moreover, copper(II) treatment evoked a rapid release of hydrogen peroxide from microglial cells, an effect that was not affected by NADPH oxidase inhibitors. N-acetyl-cysteine, a scavenger of reactive oxygen species (ROS), abrogated copper(II)-elicited microglial release of TNF-α and nitric oxide and subsequent neurotoxicity. Importantly, mitochondrial production of superoxide, paralleled to extracellular release of hydrogen peroxide, was induced after copper(II) stimulation. Our findings suggest that extracellular copper(II) at subneurotoxic concentrations could trigger NF-κB-dependent microglial activation and subsequent neurotoxicity. NADPH oxidase-independent, mitochondria-derived ROS may be involved in this activation. - Highlights: • Subneurotoxic copper(II) triggers NF-κB-dependent microglial activation. • This activation leads to hippocampal neuronal death. • This activation may involve mitochondria-derived reactive oxygen species.« less

The Extracellular Matrix of Candida albicans Biofilms Impairs Formation of Neutrophil Extracellular Traps.

PubMed

Johnson, Chad J; Cabezas-Olcoz, Jonathan; Kernien, John F; Wang, Steven X; Beebe, David J; Huttenlocher, Anna; Ansari, Hamayail; Nett, Jeniel E

2016-09-01

Neutrophils release extracellular traps (NETs) in response to planktonic C. albicans. These complexes composed of DNA, histones, and proteins inhibit Candida growth and dissemination. Considering the resilience of Candida biofilms to host defenses, we examined the neutrophil response to C. albicans during biofilm growth. In contrast to planktonic C. albicans, biofilms triggered negligible release of NETs. Time lapse imaging confirmed the impairment in NET release and revealed neutrophils adhering to hyphae and migrating on the biofilm. NET inhibition depended on an intact extracellular biofilm matrix as physical or genetic disruption of this component resulted in NET release. Biofilm inhibition of NETosis could not be overcome by protein kinase C activation via phorbol myristate acetate (PMA) and was associated with suppression of neutrophil reactive oxygen species (ROS) production. The degree of impaired NET release correlated with resistance to neutrophil attack. The clinical relevance of the role for extracellular matrix in diminishing NET production was corroborated in vivo using a rat catheter model. The C. albicans pmr1Δ/Δ, defective in production of matrix mannan, appeared to elicit a greater abundance of NETs by scanning electron microscopy imaging, which correlated with a decreased fungal burden. Together, these findings show that C. albicans biofilms impair neutrophil response through an inhibitory pathway induced by the extracellular matrix.

The Extracellular Matrix of Candida albicans Biofilms Impairs Formation of Neutrophil Extracellular Traps

PubMed Central

Cabezas-Olcoz, Jonathan; Wang, Steven X.; Huttenlocher, Anna; Ansari, Hamayail; Nett, Jeniel E.

2016-01-01

Neutrophils release extracellular traps (NETs) in response to planktonic C. albicans. These complexes composed of DNA, histones, and proteins inhibit Candida growth and dissemination. Considering the resilience of Candida biofilms to host defenses, we examined the neutrophil response to C. albicans during biofilm growth. In contrast to planktonic C. albicans, biofilms triggered negligible release of NETs. Time lapse imaging confirmed the impairment in NET release and revealed neutrophils adhering to hyphae and migrating on the biofilm. NET inhibition depended on an intact extracellular biofilm matrix as physical or genetic disruption of this component resulted in NET release. Biofilm inhibition of NETosis could not be overcome by protein kinase C activation via phorbol myristate acetate (PMA) and was associated with suppression of neutrophil reactive oxygen species (ROS) production. The degree of impaired NET release correlated with resistance to neutrophil attack. The clinical relevance of the role for extracellular matrix in diminishing NET production was corroborated in vivo using a rat catheter model. The C. albicans pmr1Δ/Δ, defective in production of matrix mannan, appeared to elicit a greater abundance of NETs by scanning electron microscopy imaging, which correlated with a decreased fungal burden. Together, these findings show that C. albicans biofilms impair neutrophil response through an inhibitory pathway induced by the extracellular matrix. PMID:27622514

Mitochondrial Superoxide Production Negatively Regulates Neural Progenitor Proliferation and Cerebral Cortical Development

PubMed Central

Hou, Yan; Ouyang, Xin; Wan, Ruiqian; Cheng, Heping; Mattson, Mark P.; Cheng, Aiwu

2012-01-01

Although high amounts of reactive oxygen species (ROS) can damage cells, ROS can also play roles as second messengers, regulating diverse cellular processes. Here we report that embryonic mouse cerebral cortical neural progenitor cells (NPCs) exhibit intermittent spontaneous bursts of mitochondrial superoxide (SO) generation (mitochondrial SO flashes) that require transient opening of membrane permeability transition pores (mPTP). This quantal SO production negatively regulates NPC self-renewal. Mitochondrial SO scavengers and mPTP inhibitors reduce SO flash frequency and enhance NPC proliferation, whereas prolonged mPTP opening and SO generation increase SO flash incidence and decrease NPC proliferation. The inhibition of NPC proliferation by mitochondrial SO involves suppression of extracellular signal-regulated kinases. Moreover, mice lacking SOD2 (SOD2−/− mice) exhibit significantly fewer proliferative NPCs and differentiated neurons in the embryonic cerebral cortex at mid-gestation compared with wild type littermates. Cultured SOD2−/− NPCs exhibit a significant increase in SO flash frequency and reduced NPC proliferation. Taken together, our findings suggest that mitochondrial SO flashes negatively regulate NPC self-renewal in the developing cerebral cortex. PMID:22949407

Molecular mechanism of voltage sensing in voltage-gated proton channels

PubMed Central

Rebolledo, Santiago; Perez, Marta E.

2013-01-01

Voltage-gated proton (Hv) channels play an essential role in phagocytic cells by generating a hyperpolarizing proton current that electrically compensates for the depolarizing current generated by the NADPH oxidase during the respiratory burst, thereby ensuring a sustained production of reactive oxygen species by the NADPH oxidase in phagocytes to neutralize engulfed bacteria. Despite the importance of the voltage-dependent Hv current, it is at present unclear which residues in Hv channels are responsible for the voltage activation. Here we show that individual neutralizations of three charged residues in the fourth transmembrane domain, S4, all reduce the voltage dependence of activation. In addition, we show that the middle S4 charged residue moves from a position accessible from the cytosolic solution to a position accessible from the extracellular solution, suggesting that this residue moves across most of the membrane electric field during voltage activation of Hv channels. Our results show for the first time that the charge movement of these three S4 charges accounts for almost all of the measured gating charge in Hv channels. PMID:23401575

Possible involvement of 12-lipoxygenase activation in glucose-deprivation/reload-treated neurons.

PubMed

Nagasawa, Kazuki; Kakuda, Taichi; Higashi, Youichirou; Fujimoto, Sadaki

2007-12-18

The aim of this study was to clarify whether 12-lipoxygenase (12-LOX) activation was involved in reactive oxygen species (ROS) generation, extensive poly(ADP-ribose) polymerase (PARP) activation and neuronal death induced by glucose-deprivation, followed by glucose-reload (GD/R). The decrease of neuronal viability and accumulation of poly(ADP-ribose) induced by GD/R were prevented 3-aminobenzamide, a representative PARP inhibitor, demonstrating this treatment protocol caused the same oxidative stress with the previously reported one. The PARP activation, ROS generation and decrease of neuron viability induced by GD/R treatment were almost completely abolished by an extracellular zinc chelator, CaEDTA. p47(phox), a cytosolic component of NADPH oxidase was translocated the membrane fraction by GD/R, indicating its activation, but it did not generate detectable ROS. Surprisingly, pharmacological inhibition of NADPH oxidase with apocynin and AEBSF further decreased the decreased neuron viability induced by GD/R. On the other hand, AA861, a 12-LOX inhibitor, prevented ROS generation and decrease of neuron viability caused by GD/R. Interestingly, an antioxidant, N-acetyl-l-cysteine rescued the neurons from GD/R-induced oxidative stress, implying effectiveness of antioxidant administration. These findings suggested that activation of 12-LOX, but not NADPH oxidase, following to zinc release might play an important role in ROS generation and decrease of viability in GD/R-treated neurons.

Friend or foe? Reactive oxygen species production, scavenging and signaling in plant response to environmental stresses.

PubMed

Czarnocka, Weronika; Karpiński, Stanisław

2018-01-10

In the natural environment, plants are exposed to a variety of biotic and abiotic stress conditions that trigger rapid changes in the production and scavenging of reactive oxygen species (ROS). The production and scavenging of ROS is compartmentalized, which means that, depending on stimuli type, they can be generated and eliminated in different cellular compartments such as the apoplast, plasma membrane, chloroplasts, mitochondria, peroxisomes, and endoplasmic reticulum. Although the accumulation of ROS is generally harmful to cells, ROS play an important role in signaling pathways that regulate acclimatory and defense responses in plants, such as systemic acquired acclimation (SAA) and systemic acquired resistance (SAR). However, high accumulations of ROS can also trigger redox homeostasis disturbance which can lead to cell death, and in consequence, to a limitation in biomass and yield production. Different ROS have various half-lifetimes and degrees of reactivity toward molecular components such as lipids, proteins, and nucleic acids. Thus, they play different roles in intra- and extra-cellular signaling. Despite their possible damaging effect, ROS should mainly be considered as signaling molecules that regulate local and systemic acclimatory and defense responses. Over the past two decades it has been proven that ROS together with non-photochemical quenching (NPQ), hormones, Ca 2+ waves, and electrical signals are the main players in SAA and SAR, two physiological processes essential for plant survival and productivity in unfavorable conditions. Copyright © 2018. Published by Elsevier Inc.

Arabidopsis MLO2 is a negative regulator of sensitivity to extracellular reactive oxygen species.

PubMed

Cui, Fuqiang; Wu, Hongpo; Safronov, Omid; Zhang, Panpan; Kumar, Rajeev; Kollist, Hannes; Salojärvi, Jarkko; Panstruga, Ralph; Overmyer, Kirk

2018-04-01

The atmospheric pollutant ozone (O 3 ) is a strong oxidant that causes extracellular reactive oxygen species (ROS) formation, has significant ecological relevance, and is used here as a non-invasive ROS inducer to study plant signalling. Previous genetic screens identified several mutants exhibiting enhanced O 3 sensitivity, but few with enhanced tolerance. We found that loss-of-function mutants in Arabidopsis MLO2, a gene implicated in susceptibility to powdery mildew disease, exhibit enhanced dose-dependent tolerance to O 3 and extracellular ROS, but a normal response to intracellular ROS. This phenotype is increased in a mlo2 mlo6 mlo12 triple mutant, reminiscent of the genetic redundancy of MLO genes in powdery mildew resistance. Stomatal assays revealed that enhanced O 3 tolerance in mlo2 mutants is not caused by altered stomatal conductance. We explored modulation of the mlo2-associated O 3 tolerance, powdery mildew resistance, and early senescence phenotypes by genetic epistasis analysis, involving mutants with known effects on ROS sensitivity or antifungal defence. Mining of publicly accessible microarray data suggests that these MLO proteins regulate accumulation of abiotic stress response transcripts, and transcript accumulation of MLO2 itself is O 3 responsive. In summary, our data reveal MLO2 as a novel negative regulator in plant ROS responses, which links biotic and abiotic stress response pathways. © 2018 John Wiley & Sons Ltd.

Quantitative iTRAQ-based secretome analysis reveals species-specific and temporal shifts in carbon utilization strategies among manganese(II)-oxidizing Ascomycete fungi

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zeiner, Carolyn A.; Purvine, Samuel O.; Zink, Erika M.

Fungi generate a wide range of extracellular hydrolytic and oxidative enzymes and reactive metabolites, collectively known as the secretome, that synergistically drive plant litter decomposition in the environment. While secretome studies of model organisms have greatly expanded our knowledge of these enzymes, few have extended secretome characterization to environmental isolates, particularly filamentous Ascomycetes, or directly compared temporal patterns of enzyme utilization among diverse species. Thus, the mechanisms of carbon (C) degradation by many ubiquitous soil fungi remain poorly understood. Here in this study we use a combination of iTRAQ proteomics and extracellular enzyme activity assays to compare the protein compositionmore » of the secretomes of four manganese(II)-oxidizing Ascomycete fungi over a three-week time course. We demonstrate that the fungi exhibit striking differences in the regulation of extracellular lignocellulose-degrading enzymes among species and over time, revealing species-specific and temporal shifts in C utilization strategies as they degrade the same substrate. Specifically, our findings suggest that Alternaria alternata SRC1lrK2f and Paraconiothyrium sporulosum AP3s5-JAC2a employ sequential enzyme secretion patterns concomitant with decreasing resource availability. Stagonospora sp. SRC1lsM3a preferentially degrades proteinaceous substrate before switching to carbohydrates, and Pyrenochaeta sp. DS3sAY3a utilizes primarily peptidases to aggressively attack carbon sources in a concentrated burst. In conclusion, this work highlights the diversity of operative metabolic strategies among understudied yet ubiquitous cellulose-degrading Ascomycetes, enhancing our understanding of their contribution to C turnover in the environment.« less

Perilla frutescens leaves extract ameliorates ultraviolet radiation-induced extracellular matrix damage in human dermal fibroblasts and hairless mice skin.

PubMed

Bae, Jung-Soo; Han, Mira; Shin, Hee Soon; Kim, Min-Kyoung; Shin, Chang-Yup; Lee, Dong Hun; Chung, Jin Ho

2017-01-04

Perilla frutescens (L.) Britt. (Lamiaceae) is a traditional herb that is consumed in East Asian countries as a traditional medicine. This traditional herb has been documented for centuries to treat various diseases such as depression, allergies, inflammation and asthma. However, the effect of Perilla frutescens on skin has not been characterized well. The present study aimed to investigate the effect of Perilla frutescens leaves extract (PLE) on ultraviolet radiation-induced extracellular matrix damage in human dermal fibroblasts and hairless mice skin. Human dermal fibroblasts and Skh-1 hairless mice were irradiated with UV and treated with PLE. Protein and mRNA levels of various target molecules were analyzed by western blotting and quantitative RT-PCR, respectively. Histological changes of mouse skin were analyzed by H&E staining. To elucidate underlying mechanism of PLE, activator protein-1 (AP-1) DNA binding assay and the measurement of reactive oxygen species (ROS) were performed. PLE significantly inhibited basal and UV-induced MMP-1 and MMP-3 expression dose-dependently, and also decreased UV-induced phosphorylation of extracellular signal-regulated kinases and c-Jun N-terminal kinases. This inhibitory effects of PLE on MMP-1 and MMP-3 were mediated by reduction of ROS generation and AP-1 DNA binding activity induced by UV. Furthermore, PLE promoted type I procollagen production irrespective of UV irradiation. In the UV-irradiated animal model, PLE significantly reduced epidermal skin thickness and MMP-13 expression induced by UV. Our results demonstrate that PLE has the protective effect against UV-induced dermal matrix damage. Therefore, we suggest that PLE can be a potential agent for prevention of skin aging. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Calcite Formation in Soft Coral Sclerites Is Determined by a Single Reactive Extracellular Protein*

PubMed Central

Rahman, M. Azizur; Oomori, Tamotsu; Wörheide, Gert

2011-01-01

Calcium carbonate exists in two main forms, calcite and aragonite, in the skeletons of marine organisms. The primary mineralogy of marine carbonates has changed over the history of the earth depending on the magnesium/calcium ratio in seawater during the periods of the so-called “calcite and aragonite seas.” Organisms that prefer certain mineralogy appear to flourish when their preferred mineralogy is favored by seawater chemistry. However, this rule is not without exceptions. For example, some octocorals produce calcite despite living in an aragonite sea. Here, we address the unresolved question of how organisms such as soft corals are able to form calcitic skeletal elements in an aragonite sea. We show that an extracellular protein called ECMP-67 isolated from soft coral sclerites induces calcite formation in vitro even when the composition of the calcifying solution favors aragonite precipitation. Structural details of both the surface and the interior of single crystals generated upon interaction with ECMP-67 were analyzed with an apertureless-type near-field IR microscope with high spatial resolution. The results show that this protein is the main determining factor for driving the production of calcite instead of aragonite in the biocalcification process and that –OH, secondary structures (e.g. α-helices and amides), and other necessary chemical groups are distributed over the center of the calcite crystals. Using an atomic force microscope, we also explored how this extracellular protein significantly affects the molecular-scale kinetics of crystal formation. We anticipate that a more thorough investigation of the proteinaceous skeleton content of different calcite-producing marine organisms will reveal similar components that determine the mineralogy of the organisms. These findings have significant implications for future models of the crystal structure of calcite in nature. PMID:21768106

Genome, transcriptome, and secretome analysis of wood decay fungus Postia placenta supports unique mechanisms of lignocellulose conversion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Martinez, Diego; Challacombe, Jean; Morgenstern, Ingo

2009-02-04

Brown-rot fungi such as Postia placenta are common inhabitants of forest ecosystems and are also largely responsible for the destructive decay of wooden structures. Rapid depolymerization of cellulose is a distinguishing feature of brown-rot, but the biochemical mechanisms and underlying genetics are poorly understood. Systematic examination of the P. placenta genome, transcriptome, and secretome revealed unique extracellular enzyme systems, including an unusual repertoire of extracellular glycoside hydrolases. Genes encoding exocellobiohydrolases and cellulose-binding domains, typical of cellulolytic microbes, are absent in this efficient cellulose-degrading fungus. When P. placenta was grown in media containing cellulose as sole carbon source, transcripts corresponding tomore » many hemicellulases and to a single putative β-1-4 endoglucanase were expressed at high levels relative to glucose grown cultures. These transcript profiles were confirmed by direct identification of peptides by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Also upregulated under cellulolytic culture conditions were putative iron reductases, quinone reductase, and structurally divergent oxidases potentially involved in extracellular generation of Fe(II) and H2O2. These observations are consistent with a biodegradative role for Fenton chemistry in which Fe(II) and H2O2 react to form hydroxyl radicals, highly reactive oxidants capable of depolymerizing cellulose. The P. placenta genome resources provide unparalleled opportunities for investigating such unusual mechanisms of cellulose conversion. More broadly, the genome offers insight into the diversification of lignocellulose degrading mechanisms in fungi. In particular, comparisons between P. placenta and the closely related white-rot fungus, Phanerochaete chrysosporium support an evolutionary shift from white-rot to brown-rot during which efficient depolymerization of lignin was lost.« less

Genome, transcriptome, and secretome analysis of wood decay fungus postia placenta supports unique mechanisms of lignocellulose conversion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Martinez, Diego; Challacombe, Jean F; Misra, Monica

2008-01-01

Brown-rot fungi such as Postia placenta are common inhabitants of forest ecosystems and are also largely responsible for the destructive decay of wooden structures. Rapid depolymerization of cellulose is a distinguishing feature of brown-rot, but the biochemical mechanisms and underlying genetics are poorly understood. Systematic examination of the P. placenta genome, transcriptome and secretome revealed unique extracellular enzyme systems, including an unusual repertoire of extracellular glycoside hydrolases. Genes encoding exocellobiohydrolases and cellulose-binding domains, typical of cellulolytic microbes, are absent in this efficient cellulose-degrading fungus. When P. placenta was grown in medium containing cellulose as sole carbon source, transcripts corresponding tomore » many hemicellulases and to a single putative {beta}-1-4 endoglucanase were expressed at high levels relative to glucose grown cultures. These transcript profiles were confirmed by direct identification of peptides by liquid chromatography-tandem mass spectrometry (LC{center_dot}MSIMS). Also upregulated during growth on cellulose medium were putative iron reductases, quinone reductase, and structurally divergent oxidases potentially involved in extracellular generation of Fe(II) and H202. These observations are consistent with a biodegradative role for Fenton chemistry in which Fe(II) and H202 react to form hydroxyl radicals, highly reactive oxidants capable of depolymerizing cellulose. The P. placenta genome resources provide unparalleled opportunities for investigating such unusual mechanisms of cellulose conversion. More broadly, the genome offers insight into the diversification of lignocellulose degrading mechanisms in fungi. Comparisons to the closely related white-rot fungus Phanerochaete chrysosporium support an evolutionary shift from white-rot to brown-rot during which the capacity for efficient depolymerization of lignin was lost.« less

Quantitative iTRAQ-based secretome analysis reveals species-specific and temporal shifts in carbon utilization strategies among manganese(II)-oxidizing Ascomycete fungi

DOE PAGES

Zeiner, Carolyn A.; Purvine, Samuel O.; Zink, Erika M.; ...

2017-07-01

Fungi generate a wide range of extracellular hydrolytic and oxidative enzymes and reactive metabolites, collectively known as the secretome, that synergistically drive plant litter decomposition in the environment. While secretome studies of model organisms have greatly expanded our knowledge of these enzymes, few have extended secretome characterization to environmental isolates, particularly filamentous Ascomycetes, or directly compared temporal patterns of enzyme utilization among diverse species. Thus, the mechanisms of carbon (C) degradation by many ubiquitous soil fungi remain poorly understood. Here in this study we use a combination of iTRAQ proteomics and extracellular enzyme activity assays to compare the protein compositionmore » of the secretomes of four manganese(II)-oxidizing Ascomycete fungi over a three-week time course. We demonstrate that the fungi exhibit striking differences in the regulation of extracellular lignocellulose-degrading enzymes among species and over time, revealing species-specific and temporal shifts in C utilization strategies as they degrade the same substrate. Specifically, our findings suggest that Alternaria alternata SRC1lrK2f and Paraconiothyrium sporulosum AP3s5-JAC2a employ sequential enzyme secretion patterns concomitant with decreasing resource availability. Stagonospora sp. SRC1lsM3a preferentially degrades proteinaceous substrate before switching to carbohydrates, and Pyrenochaeta sp. DS3sAY3a utilizes primarily peptidases to aggressively attack carbon sources in a concentrated burst. In conclusion, this work highlights the diversity of operative metabolic strategies among understudied yet ubiquitous cellulose-degrading Ascomycetes, enhancing our understanding of their contribution to C turnover in the environment.« less

Genome, transcriptome, and secretome analysis of wood decay fungus Postia placenta supports unique mechanisms of lignocellulose conversion

PubMed Central

Martinez, Diego; Challacombe, Jean; Morgenstern, Ingo; Hibbett, David; Schmoll, Monika; Kubicek, Christian P.; Ferreira, Patricia; Ruiz-Duenas, Francisco J.; Martinez, Angel T.; Kersten, Phil; Hammel, Kenneth E.; Vanden Wymelenberg, Amber; Gaskell, Jill; Lindquist, Erika; Sabat, Grzegorz; Splinter BonDurant, Sandra; Larrondo, Luis F.; Canessa, Paulo; Vicuna, Rafael; Yadav, Jagjit; Doddapaneni, Harshavardhan; Subramanian, Venkataramanan; Pisabarro, Antonio G.; Lavín, José L.; Oguiza, José A.; Master, Emma; Henrissat, Bernard; Coutinho, Pedro M.; Harris, Paul; Magnuson, Jon Karl; Baker, Scott E.; Bruno, Kenneth; Kenealy, William; Hoegger, Patrik J.; Kües, Ursula; Ramaiya, Preethi; Lucas, Susan; Salamov, Asaf; Shapiro, Harris; Tu, Hank; Chee, Christine L.; Misra, Monica; Xie, Gary; Teter, Sarah; Yaver, Debbie; James, Tim; Mokrejs, Martin; Pospisek, Martin; Grigoriev, Igor V.; Brettin, Thomas; Rokhsar, Dan; Berka, Randy; Cullen, Dan

2009-01-01

Brown-rot fungi such as Postia placenta are common inhabitants of forest ecosystems and are also largely responsible for the destructive decay of wooden structures. Rapid depolymerization of cellulose is a distinguishing feature of brown-rot, but the biochemical mechanisms and underlying genetics are poorly understood. Systematic examination of the P. placenta genome, transcriptome, and secretome revealed unique extracellular enzyme systems, including an unusual repertoire of extracellular glycoside hydrolases. Genes encoding exocellobiohydrolases and cellulose-binding domains, typical of cellulolytic microbes, are absent in this efficient cellulose-degrading fungus. When P. placenta was grown in medium containing cellulose as sole carbon source, transcripts corresponding to many hemicellulases and to a single putative β-1–4 endoglucanase were expressed at high levels relative to glucose-grown cultures. These transcript profiles were confirmed by direct identification of peptides by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Also up-regulated during growth on cellulose medium were putative iron reductases, quinone reductase, and structurally divergent oxidases potentially involved in extracellular generation of Fe(II) and H2O2. These observations are consistent with a biodegradative role for Fenton chemistry in which Fe(II) and H2O2 react to form hydroxyl radicals, highly reactive oxidants capable of depolymerizing cellulose. The P. placenta genome resources provide unparalleled opportunities for investigating such unusual mechanisms of cellulose conversion. More broadly, the genome offers insight into the diversification of lignocellulose degrading mechanisms in fungi. Comparisons with the closely related white-rot fungus Phanerochaete chrysosporium support an evolutionary shift from white-rot to brown-rot during which the capacity for efficient depolymerization of lignin was lost. PMID:19193860

Selective reactivity of monochloramine with extracellular matrix components affects the disinfection of biofilm and detached clusters.

PubMed

Xue, Zheng; Lee, Woo Hyoung; Coburn, Kimberly M; Seo, Youngwoo

2014-04-01

The efficiency of monochloramine disinfection was dependent on the quantity and composition of extracellular polymeric substances (EPS) in biofilms, as monochloramine has a selective reactivity with proteins over polysaccharides. Biofilms with protein-based (Pseudomonas putida) and polysaccharide based EPS (Pseudomonas aeruginosa), as well as biofilms with varied amount of polysaccharide EPS (wild-type and mutant P. aeruginosa), were compared. The different reactivity of EPS components with monochloramine influenced disinfectant penetration, biofilm inactivation, as well as the viability of detached clusters. Monochloramine transport profiling measured by a chloramine-sensitive microelectrode revealed a broader diffusion boundary layer between bulk and biofilm surface in the P. putida biofilm compared to those of P. aeruginosa biofilms. The reaction with proteins in P. putida EPS multiplied both the time and the monochloramine mass required to achieve a full biofilm penetration. Cell viability in biofilms was also spatially influenced by monochloramine diffusion and reaction within biofilms, showing a lower survival in the surface section and a higher persistence in the middle section of the P. putida biofilm compared to the P. aeruginosa biofilms. While polysaccharide EPS promoted biofilm cell viability by obstructing monochloramine reactive sites on bacterial cells, protein EPS hindered monochloramine penetration by reacting with monochloramine and reduced its concentration within biofilms. Furthermore, the persistence of bacterial cells detached from biofilm (over 70% for P. putida and ∼40% for polysaccharide producing P. aeruginosa) suggested that currently recommended monochloramine residual levels may underestimate the risk of water quality deterioration caused by biofilm detachment.

APP Processing Induced by Herpes Simplex Virus Type 1 (HSV-1) Yields Several APP Fragments in Human and Rat Neuronal Cells

PubMed Central

Civitelli, Livia; Argnani, Rafaela; Piacentini, Roberto; Ripoli, Cristian; Manservigi, Roberto; Grassi, Claudio; Garaci, Enrico; Palamara, Anna Teresa

2010-01-01

Lifelong latent infections of the trigeminal ganglion by the neurotropic herpes simplex virus type 1 (HSV-1) are characterized by periodic reactivation. During these episodes, newly produced virions may also reach the central nervous system (CNS), causing productive but generally asymptomatic infections. Epidemiological and experimental findings suggest that HSV-1 might contribute to the pathogenesis of Alzheimer's disease (AD). This multifactorial neurodegenerative disorder is related to an overproduction of amyloid beta (Aβ) and other neurotoxic peptides, which occurs during amyloidogenic endoproteolytic processing of the transmembrane amyloid precursor protein (APP). The aim of our study was to identify the effects of productive HSV-1 infection on APP processing in neuronal cells. We found that infection of SH-SY5Y human neuroblastoma cells and rat cortical neurons is followed by multiple cleavages of APP, which result in the intra- and/or extra-cellular accumulation of various neurotoxic species. These include: i) APP fragments (APP-Fs) of 35 and 45 kDa (APP-F35 and APP-F45) that comprise portions of Aβ; ii) N-terminal APP-Fs that are secreted; iii) intracellular C-terminal APP-Fs; and iv) Aβ1-40 and Aβ1-42. Western blot analysis of infected-cell lysates treated with formic acid suggests that APP-F35 may be an Aβ oligomer. The multiple cleavages of APP that occur in infected cells are produced in part by known components of the amyloidogenic APP processing pathway, i.e., host-cell β-secretase, γ-secretase, and caspase-3-like enzymes. These findings demonstrate that HSV-1 infection of neuronal cells can generate multiple APP fragments with well-documented neurotoxic potentials. It is tempting to speculate that intra- and extracellular accumulation of these species in the CNS resulting from repeated HSV-1 reactivation could, in the presence of other risk factors, play a co-factorial role in the development of AD. PMID:21085580

Endothelial Cell and Platelet Bioenergetics: Effect of Glucose and Nutrient Composition

PubMed Central

Fink, Brian D.; Herlein, Judy A.; O’Malley, Yunxia; Sivitz, William I.

2012-01-01

It has been suggested that cells that are independent of insulin for glucose uptake, when exposed to high glucose or other nutrient concentrations, manifest enhanced mitochondrial substrate oxidation with consequent enhanced potential and generation of reactive oxygen species (ROS); a paradigm that could predispose to vascular complications of diabetes. Here we exposed bovine aortic endothelial (BAE) cells and human platelets to variable glucose and fatty acid concentrations. We then examined oxygen consumption and acidification rates using recently available technology in the form of an extracellular oxygen and proton flux analyzer. Acute or overnight exposure of confluent BAE cells to glucose concentrations from 5.5 to 25 mM did not enhance or change the rate of oxygen consumption (OCR) under basal conditions, during ATP synthesis, or under uncoupled conditions. Glucose also did not alter OCR in sub-confluent cells, in cells exposed to low serum, or in cells treated with added pyruvate. Likewise, overnight exposure to fatty acids of varying saturation had no such effects. Overnight exposure of BAE cells to low glucose concentration decreased maximal uncoupled respiration, but not basal or ATP related oxygen consumption. Labeled glucose oxidation to CO2 increased, but only marginally after high glucose exposure while oleate oxidation to CO2 decreased. Overnight exposure to linolenic acid, but not oleic or linoleic acid increased extracellular acidification consistent with enhanced glycolytic metabolism. We were unable to detect an increase in production of reactive oxygen species (ROS) from BAE cells exposed to high medium glucose. Like BAE cells, exposure of human platelets to glucose did not increase oxygen consumption. As opposed to BAE cells, platelet mitochondria demonstrate less respiratory reserve capacity (beyond that needed for basal metabolism). Our data do not support the concept that exposure to high glucose or fatty acids accelerates mitochondrial oxidative metabolism in endothelial cells or platelets. PMID:22745753

Chemical modification of extracellular matrix by cold atmospheric plasma-generated reactive species affects chondrogenesis and bone formation

PubMed Central

Eisenhauer, Peter; Chernets, Natalie; Song, You; Dobrynin, Danil; Pleshko, Nancy; Steinbeck, Marla J.; Freeman, Theresa A.

2017-01-01

The goal of this study was to investigate whether cold plasma generated by dielectric barrier discharge (DBD) modifies extracellular matrices (ECM) to influence chondrogenesis and endochondral ossification. Replacement of cartilage by bone during endochondral ossification is essential in fetal skeletal development, bone growth and fracture healing. Regulation of this process by the ECM occurs through matrix remodelling, involving a variety of cell attachment molecules and growth factors, which influence cell morphology and protein expression. The commercially available ECM, Matrigel, was treated with microsecond or nanosecond pulsed (µsp or nsp, respectively) DBD frequencies conditions at the equivalent frequencies (1 kHz) or power (~1 W). Recombinant human bone morphogenetic protein-2 was added and the mixture subcutaneously injected into mice to simulate ectopic endochondral ossification. Two weeks later, the masses were extracted and analysed by microcomputed tomography. A significant increase in bone formation was observed in Matrigel treated with µsp DBD compared with control, while a significant decrease in bone formation was observed for both nsp treatments. Histological and immunohistochemical analysis showed Matrigel treated with µsp plasma increased the number of invading cells, the amount of vascular endothelial growth factor and chondrogenesis while the opposite was true for Matrigel treated with nsp plasma. In support of the in vivo Matrigel study, 10 T1/2 cells cultured in vitro on µsp DBD-treated type I collagen showed increased expression of adhesion proteins and activation of survival pathways, which decreased with nsp plasma treatments. These results indicate DBD modification of ECM can influence cellular behaviours to accelerate or inhibit chondrogenesis and endochondral ossification. PMID:27510797

Advanced oxidative protein products induced human keratinocyte apoptosis through the NOX-MAPK pathway.

PubMed

Sun, Baihui; Ding, Ruoting; Yu, Wenlin; Wu, Yanhong; Wang, Bulin; Li, Qin

2016-07-01

Impaired wound healing is a major diabetes-related complication. Keratinocytes play an important role in wound healing. Multiple factors have been proposed that can induce dysfunction in keratinocytes. The focus of present research is at a more specific molecular level. We investigated the role of advanced oxidative protein products (AOPPs) in inducing human immortalized keratinocyte (HaCaT) cell apoptosis and the cellular mechanism underlying the proapoptotic effect of AOPPs. HaCaT cells were treated with increasing concentrations of AOPP-human serum albumin or for increasing time durations. The cell viability was measured using the thiazolyl blue tetrazolium bromide method, and flow cytometry was used to assess the rate of cell apoptosis. A loss of mitochondrial membrane potential (MMP) and an increase in intracellular reactive oxygen species (ROS) were observed through a confocal laser scanning microscope system, and the level of ROS generation was determined using a microplate reader. Nicotinamide adenine dinucleotide phosphate oxidase (NOX)4, extracellular signal-regulated kinase (ERK)1/2, p38 mitogen-activated protein kinase (MAPK), and apoptosis-related downstream protein interactions were investigated using the Western blot analysis. We found that AOPPs triggered HaCaT cell apoptosis and MMP loss. After AOPP treatment, intracellular ROS generation increased in a time- and dose-dependent manner. Proapoptotic proteins, such as Bax, caspase 9/caspase 3, and poly(ADP-ribose) polymerase (PARP)-1 were activated, whereas anti-apoptotic Bcl-2 protein was downregulated. AOPPs also increased NOX4, ERK1/2, and p38 MAPK expression. Taken together, these findings suggest that extracellular AOPP accumulation triggered NOX-dependent ROS production, which activated ERK1/2 and p38 MAPK, and induced HaCaT cell apoptosis by activating caspase 3 and PARP-1.

Alkoxyl- and carbon-centered radicals as primary agents for degrading non-phenolic lignin-substructure model compounds.

PubMed

Ohashi, Yasunori; Uno, Yukiko; Amirta, Rudianto; Watanabe, Takahito; Honda, Yoichi; Watanabe, Takashi

2011-04-07

Lignin degradation by white-rot fungi proceeds via free radical reaction catalyzed by oxidative enzymes and metabolites. Basidiomycetes called selective white-rot fungi degrade both phenolic and non-phenolic lignin substructures without penetration of extracellular enzymes into the cell wall. Extracellular lipid peroxidation has been proposed as a possible ligninolytic mechanism, and radical species degrading the recalcitrant non-phenolic lignin substructures have been discussed. Reactions between the non-phenolic lignin model compounds and radicals produced from azo compounds in air have previously been analysed, and peroxyl radical (PR) is postulated to be responsible for lignin degradation (Kapich et al., FEBS Lett., 1999, 461, 115-119). However, because the thermolysis of azo compounds in air generates both a carbon-centred radical (CR) and a peroxyl radical (PR), we re-examined the reactivity of the three radicals alkoxyl radical (AR), CR and PR towards non-phenolic monomeric and dimeric lignin model compounds. The dimeric lignin model compound is degraded by CR produced by reaction of 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH), which under N(2) atmosphere cleaves the α-β bond in 1-(4-ethoxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)-1,3-propanediol to yield 4-ethoxy-3-methoxybenzaldehyde. However, it is not degraded by the PR produced by reaction of Ce(4+)/tert-BuOOH. In addition, it is degraded by AR produced by reaction of Ti(3+)/tert-BuOOH. PR and AR are generated in the presence and absence of veratryl alcohol, respectively. Rapid-flow ESR analysis of the radical species demonstrates that AR but not PR reacts with the lignin model compound. Thus, AR and CR are primary agents for the degradation of non-phenolic lignin substructures.

Does abnormal glycogen structure contribute to increased susceptibility to seizures in epilepsy?

PubMed Central

DiNuzzo, Mauro; Mangia, Silvia; Maraviglia, Bruno; Giove, Federico

2014-01-01

Epilepsy is a family of brain disorders with a largely unknown etiology and high percentage of pharmacoresistance. The clinical manifestations of epilepsy are seizures, which originate from aberrant neuronal synchronization and hyperexcitability. Reactive astrocytosis, a hallmark of the epileptic tissue, develops into loss-of-function of glutamine synthetase, impairment of glutamate-glutamine cycle and increase in extracellular and astrocytic glutamate concentration. Here, we argue that chronically elevated intracellular glutamate level in astrocytes is instrumental to alterations in the metabolism of glycogen and leads to the synthesis of polyglucosans. Unaccessibility of glycogen-degrading enzymes to these insoluble molecules compromises the glycogenolysis-dependent reuptake of extracellular K+ by astrocytes, thereby leading to increased extracellular K+ and associated membrane depolarization. Based on current knowledge, we propose that the deterioration in structural homogeneity of glycogen particles is relevant to disruption of brain K+ homeostasis and increased susceptibility to seizures in epilepsy. PMID:24643875

Does abnormal glycogen structure contribute to increased susceptibility to seizures in epilepsy?

PubMed

DiNuzzo, Mauro; Mangia, Silvia; Maraviglia, Bruno; Giove, Federico

2015-02-01

Epilepsy is a family of brain disorders with a largely unknown etiology and high percentage of pharmacoresistance. The clinical manifestations of epilepsy are seizures, which originate from aberrant neuronal synchronization and hyperexcitability. Reactive astrocytosis, a hallmark of the epileptic tissue, develops into loss-of-function of glutamine synthetase, impairment of glutamate-glutamine cycle and increase in extracellular and astrocytic glutamate concentration. Here, we argue that chronically elevated intracellular glutamate level in astrocytes is instrumental to alterations in the metabolism of glycogen and leads to the synthesis of polyglucosans. Unaccessibility of glycogen-degrading enzymes to these insoluble molecules compromises the glycogenolysis-dependent reuptake of extracellular K(+) by astrocytes, thereby leading to increased extracellular K(+) and associated membrane depolarization. Based on current knowledge, we propose that the deterioration in structural homogeneity of glycogen particles is relevant to disruption of brain K(+) homeostasis and increased susceptibility to seizures in epilepsy.

Natural extracellular nanovesicles and photodynamic molecules: is there a future for drug delivery?

PubMed

Kusuzaki, Katsuyuki; Matsubara, Takao; Murata, Hiroaki; Logozzi, Mariantonia; Iessi, Elisabetta; Di Raimo, Rossella; Carta, Fabrizio; Supuran, Claudiu T; Fais, Stefano

2017-12-01

Photodynamic molecules represent an alternative approach for cancer therapy for their property (i) to be photo-reactive; (ii) to be not-toxic for target cells in absence of light; (iii) to accumulate specifically into tumour tissues; (iv) to be activable by a light beam only at the tumour site and (v) to exert cytotoxic activity against tumour cells. However, to date their clinical use is limited by the side effects elicited by systemic administration. Extracellular vesicles are endogenous nanosized-carriers that have been recently introduced as a natural delivery system for therapeutic molecules. We have recently shown the ability of human exosomes to deliver photodynamic molecules. Therefore, this review focussed on extracellular vesicles as a novel strategy for the delivery of photodynamic molecules at cancer sites. This completely new approach may enhance the delivery and decrease the toxicity of photodynamic molecules, therefore, represent the future for photodynamic therapy for cancer treatment.

Reactive oxygen species formation and bystander effects in gradient irradiation on human breast cancer cells.

PubMed

Zhang, Dongqing; Zhou, Tingyang; He, Feng; Rong, Yi; Lee, Shin Hee; Wu, Shiyong; Zuo, Li

2016-07-05

Ionizing radiation (IR) in cancer radiotherapy can induce damage to neighboring cells via non-targeted effects by irradiated cells. These so-called bystander effects remain an area of interest as it may provide enhanced efficacy in killing carcinomas with minimal radiation. It is well known that reactive oxygen species (ROS) are ubiquitous among most biological activities. However, the role of ROS in bystander effects has not been thoroughly elucidated. We hypothesized that gradient irradiation (GI) has enhanced therapeutic effects via the ROS-mediated bystander pathways as compared to uniform irradiation (UI). We evaluated ROS generation, viability, and apoptosis in breast cancer cells (MCF-7) exposed to UI (5 Gy) or GI (8-2 Gy) in radiation fields at 2, 24 and 48 h after IR. We found that extracellular ROS release induced by GI was higher than that by UI at both 24 h (p < 0.001) and 48 h (p < 0.001). More apoptosis and less viability were observed in GI when compared to UI at either 24 h or 48 h after irradiation. The mean effective doses (ED) of GI were ~130% (24 h) and ~48% (48 h) higher than that of UI, respectively. Our results suggest that GI is superior to UI regarding redox mechanisms, ED, and toxic dosage to surrounding tissues.

Probucol inhibited Nox2 expression and attenuated podocyte injury in type 2 diabetic nephropathy of db/db mice.

PubMed

Zhou, Guangyu; Wang, Yanqiu; He, Ping; Li, Detian

2013-01-01

The present study was conducted to investigate the effects of probucol on the progression of diabetic nephropathy and the underlying mechanism in type 2 diabetic db/db mice. Eight weeks db/db mice were treated with regular diet or probucol-containing diet (1%) for 12 weeks. Non-diabetic db/m mice were used as controls. We examined body weight, blood glucose, and urinary albumin. At 20 weeks, experimental mice were sacrificed and their blood and kidneys were extracted for the analysis of blood chemistry, kidney histology, oxidative stress marker, and podocyte marker. As a result, 24 h urinary albumin excretions were reduced after probucol treatment. There were improvements of extracellular matrix accumulation and fibronectin and collagen IV deposition in glomeruli in the probucol-treated db/db mice. The reduction of nephrin and the loss of podocytes were effectively prevented by probucol in db/db mice. Furthermore, probucol significantly decreased the production of thiobarbituric acid-reactive substances (TBARS), an index of reactive oxygen species (ROS) generation and down-regulated the expression of Nox2. Taken together, our findings support that probucol may have the potential to protect against type 2 diabetic nephropathy via amelioration of podocyte injury and reduction of oxidative stress.

Point Mutations in c-Myc Uncouple Neoplastic Transformation from Multiple Other Phenotypes in Rat Fibroblasts

PubMed Central

Graves, J. Anthony; Rothermund, Kristi; Wang, Tao; Qian, Wei; Van Houten, Bennett; Prochownik, Edward V.

2010-01-01

Deregulation of c-Myc (Myc) occurs in many cancers. In addition to transforming various cell types, Myc also influences additional transformation-associated cellular phenotypes including proliferation, survival, genomic instability, reactive oxygen species production, and metabolism. Although Myc is wild type in most cancers (wtMyc), it occasionally acquires point mutations in certain lymphomas. Some of these mutations confer a survival advantage despite partially attenuating proliferation and transformation. Here, we have evaluated four naturally-occurring or synthetic point mutations of Myc for their ability to affect these phenotypes, as well as to promote genomic instability, to generate reactive oxygen species and to up-regulate aerobic glycolysis and oxidative phosphorylation. Our findings indicate that many of these phenotypes are genetically and functionally independent of one another and are not necessary for transformation. Specifically, the higher rate of glucose metabolism known to be associated with wtMyc deregulation was found to be independent of transformation. One mutation (Q131R) was greatly impaired for nearly all of the studied Myc phenotypes, yet was able to retain some ability to transform. These findings indicate that, while the Myc phenotypes examined here make additive contributions to transformation, none, with the possible exception of increased reliance on extracellular glutamine for survival, are necessary for achieving this state. PMID:21060841

Diabetes-Induced Reactive Oxygen Species: Mechanism of Their Generation and Role in Renal Injury

PubMed Central

Fakhruddin, Selim; Alanazi, Wael

2017-01-01

Diabetes induces the onset and progression of renal injury through causing hemodynamic dysregulation along with abnormal morphological and functional nephron changes. The most important event that precedes renal injury is an increase in permeability of plasma proteins such as albumin through a damaged glomerular filtration barrier resulting in excessive urinary albumin excretion (UAE). Moreover, once enhanced UAE begins, it may advance renal injury from progression of abnormal renal hemodynamics, increased glomerular basement membrane (GBM) thickness, mesangial expansion, extracellular matrix accumulation, and glomerulosclerosis to eventual end-stage renal damage. Interestingly, all these pathological changes are predominantly driven by diabetes-induced reactive oxygen species (ROS) and abnormal downstream signaling molecules. In diabetic kidney, NADPH oxidase (enzymatic) and mitochondrial electron transport chain (nonenzymatic) are the prominent sources of ROS, which are believed to cause the onset of albuminuria followed by progression to renal damage through podocyte depletion. Chronic hyperglycemia and consequent ROS production can trigger abnormal signaling pathways involving diverse signaling mediators such as transcription factors, inflammatory cytokines, chemokines, and vasoactive substances. Persistently, increased expression and activation of these signaling molecules contribute to the irreversible functional and structural changes in the kidney resulting in critically decreased glomerular filtration rate leading to eventual renal failure. PMID:28164134

Contribution of cytochrome P450 1B1 to hypertension and associated pathophysiology: a novel target for antihypertensive agents.

PubMed

Malik, Kafait U; Jennings, Brett L; Yaghini, Fariborz A; Sahan-Firat, Seyhan; Song, Chi Young; Estes, Anne M; Fang, Xiao R

2012-08-01

The aim of this review is to discuss the contribution of cytochrome P450 (CYP) 1B1 in vascular smooth muscle cell growth, hypertension, and associated pathophysiology. CYP1B1 is expressed in cardiovascular and renal tissues, and mediates angiotensin II (Ang II)-induced activation of NADPH oxidase and generation of reactive oxygen species (ROS), and vascular smooth muscle cell migration, proliferation, and hypertrophy. Moreover, CYP1B1 contributes to the development and/or maintenance of hypertension produced by Ang II-, deoxycorticosterone (DOCA)-salt-, and N(ω)-nitro-L-arginine methyl ester-induced hypertension and in spontaneously hypertensive rats. The pathophysiological changes, including cardiovascular hypertrophy, increased vascular reactivity, endothelial and renal dysfunction, injury and inflammation associated with Ang II- and/or DOCA-salt induced hypertension in rats, and Ang II-induced hypertension in mice are minimized by inhibition of CYP1B1 activity with 2,4,3',5'-tetramethoxystilbene or by Cyp1b1 gene disruption in mice. These pathophysiological changes appear to be mediated by increased production of ROS via CYP1B1-dependent NADPH oxidase activity and extracellular signal-regulated kinase 1/2, p38 mitogen-activated protein kinase, and c-Src. Copyright © 2011 Elsevier Inc. All rights reserved.

Contribution of Cytochrome P450 1B1 to Hypertension and Associated Pathophysiology: A Novel Target for Antihypertensive Agents

PubMed Central

Malik, Kafait U.; Jennings, Brett L.; Yaghini, Fariborz A.; Sahan-Firat, Seyhan; Song, Chi Young; Estes, Anne M.; Fang, Xiao R.

2012-01-01

The aim of this review is to discuss the contribution of cytochrome P450 (CYP) 1B1 in vascular smooth muscle cell growth, hypertension, and associated pathophysiology. CYP1B1 is expressed in cardiovascular and renal tissues, and mediates angiotensin II (Ang II)-induced activation of NADPH oxidase and generation of reactive oxygen species (ROS), and vascular smooth muscle cell migration, proliferation, and hypertrophy. Moreover, CYP1B1 contributes to the development and/or maintenance of hypertension produced by Ang II-, deoxycorticosterone Nω-nitro-(DOCA)-salt-, and L-arginine methyl ester-induced hypertension and in spontaneously hypertensive rats. The pathophysiological changes, including cardiovascular hypertrophy, increased vascular reactivity, endothelial and renal dysfunction, injury and inflammation associated with Ang II- and/or DOCA-salt induced hypertension in rats, and Ang II-induced hypertension in mice are minimized by inhibition of CYP1B1 activity with 2,4,3′,5′-tetramethoxystilbene or by Cyp1b1 gene disruption in mice. These pathophysiological changes appear to be mediated by increased production of ROS via CYP1B1-dependent NADPH oxidase activity and extracellular signal-regulated kinase 1/2, p38 mitogen-activated protein kinase, and c-Src. PMID:22210049

Immunization with a novel chimeric peptide representing B and T cell epitopes from HER2 extracellular domain (HER2 ECD) for breast cancer.

PubMed

Mahdavi, Manijeh; Keyhanfar, Mehrnaz; Jafarian, Abbas; Mohabatkar, Hassan; Rabbani, Mohammad

2014-12-01

Because of direct stimulating immune system against disease, vaccination or active immunotherapy is preferable compared to passive immunotherapy. For this purpose, a newly designed chimeric peptide containing epitopes for both B and T cells from HER2 ECD subdomain III was proposed. To evaluate the effects of the active immunization, a discontinuous B cell epitope peptide was selected based on average antigenicity by bioinformatics analysis. The selected peptide was collinearly synthesized as a chimera with a T helper epitope from the protein sequence of measles virus fusion (208-302) using the GPSL linker. Three mice were immunized with the chimeric peptide. Reactive antibodies with HER2 protein in ELISA and immunofluorescence assays with no cross-reactivity were generated. The 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay indicated that the anti-peptide sera had inhibitory effects on proliferation of SK-BR-3 cells. Hence, the newly designed, discontinuous chimeric peptide representing B and T cell epitopes from subdomain III of HER2-ECD can form the basis for future vaccines design, where these data can be applied for monoclonal antibody production targeting the distinct epitope of HER2 receptor compared to the two broadly used anti-HER2 monoclonal antibodies, Herceptin and pertuzumab.

Nontypeable Haemophilus influenzae Induces Sustained Lung Oxidative Stress and Protease Expression

PubMed Central

King, Paul T.; Sharma, Roleen; O’Sullivan, Kim; Selemidis, Stavros; Lim, Steven; Radhakrishna, Naghmeh; Lo, Camden; Prasad, Jyotika; Callaghan, Judy; McLaughlin, Peter; Farmer, Michael; Steinfort, Daniel; Jennings, Barton; Ngui, James; Broughton, Bradley R. S.; Thomas, Belinda; Essilfie, Ama-Tawiah; Hickey, Michael; Holmes, Peter W.; Hansbro, Philip; Bardin, Philip G.; Holdsworth, Stephen R.

2015-01-01

Nontypeable Haemophilus influenzae (NTHi) is a prevalent bacterium found in a variety of chronic respiratory diseases. The role of this bacterium in the pathogenesis of lung inflammation is not well defined. In this study we examined the effect of NTHi on two important lung inflammatory processes 1), oxidative stress and 2), protease expression. Bronchoalveolar macrophages were obtained from 121 human subjects, blood neutrophils from 15 subjects, and human-lung fibroblast and epithelial cell lines from 16 subjects. Cells were stimulated with NTHi to measure the effect on reactive oxygen species (ROS) production and extracellular trap formation. We also measured the production of the oxidant, 3-nitrotyrosine (3-NT) in the lungs of mice infected with this bacterium. NTHi induced widespread production of 3-NT in mouse lungs. This bacterium induced significantly increased ROS production in human fibroblasts, epithelial cells, macrophages and neutrophils; with the highest levels in the phagocytic cells. In human macrophages NTHi caused a sustained, extracellular production of ROS that increased over time. The production of ROS was associated with the formation of macrophage extracellular trap-like structures which co-expressed the protease metalloproteinase-12. The formation of the macrophage extracellular trap-like structures was markedly inhibited by the addition of DNase. In this study we have demonstrated that NTHi induces lung oxidative stress with macrophage extracellular trap formation and associated protease expression. DNase inhibited the formation of extracellular traps. PMID:25793977

Mitochondrion-Derived Reactive Oxygen Species Lead to Enhanced Amyloid Beta Formation

PubMed Central

Schütt, Tanja; Kurz, Christopher; Eckert, Schamim H.; Schiller, Carola; Occhipinti, Angelo; Mai, Sören; Jendrach, Marina; Eckert, Gunter P.; Kruse, Shane E.; Palmiter, Richard D.; Brandt, Ulrich; Dröse, Stephan; Wittig, Ilka; Willem, Michael; Haass, Christian; Reichert, Andreas S.; Müller, Walter E.

2012-01-01

Abstract Aims: Intracellular amyloid beta (Aβ) oligomers and extracellular Aβ plaques are key players in the progression of sporadic Alzheimer's disease (AD). Still, the molecular signals triggering Aβ production are largely unclear. We asked whether mitochondrion-derived reactive oxygen species (ROS) are sufficient to increase Aβ generation and thereby initiate a vicious cycle further impairing mitochondrial function. Results: Complex I and III dysfunction was induced in a cell model using the respiratory inhibitors rotenone and antimycin, resulting in mitochondrial dysfunction and enhanced ROS levels. Both treatments lead to elevated levels of Aβ. Presence of an antioxidant rescued mitochondrial function and reduced formation of Aβ, demonstrating that the observed effects depended on ROS. Conversely, cells overproducing Aβ showed impairment of mitochondrial function such as comprised mitochondrial respiration, strongly altered morphology, and reduced intracellular mobility of mitochondria. Again, the capability of these cells to generate Aβ was partly reduced by an antioxidant, indicating that Aβ formation was also ROS dependent. Moreover, mice with a genetic defect in complex I, or AD mice treated with a complex I inhibitor, showed enhanced Aβ levels in vivo. Innovation: We show for the first time that mitochondrion-derived ROS are sufficient to trigger Aβ production in vitro and in vivo. Conclusion: Several lines of evidence show that mitochondrion-derived ROS result in enhanced amyloidogenic amyloid precursor protein processing, and that Aβ itself leads to mitochondrial dysfunction and increased ROS levels. We propose that starting from mitochondrial dysfunction a vicious cycle is triggered that contributes to the pathogenesis of sporadic AD. Antioxid. Redox Signal. 16, 1421–1433. PMID:22229260

The importance of extracellular speciation and corrosion of copper nanoparticles on lung cell membrane integrity.

PubMed

Hedberg, Jonas; Karlsson, Hanna L; Hedberg, Yolanda; Blomberg, Eva; Odnevall Wallinder, Inger

2016-05-01

Copper nanoparticles (Cu NPs) are increasingly used in various biologically relevant applications and products, e.g., due to their antimicrobial and catalytic properties. This inevitably demands for an improved understanding on their interactions and potential toxic effects on humans. The aim of this study was to investigate the corrosion of copper nanoparticles in various biological media and to elucidate the speciation of released copper in solution. Furthermore, reactive oxygen species (ROS) generation and lung cell (A549 type II) membrane damage induced by Cu NPs in the various media were studied. The used biological media of different complexity are of relevance for nanotoxicological studies: Dulbecco's modified eagle medium (DMEM), DMEM(+) (includes fetal bovine serum), phosphate buffered saline (PBS), and PBS+histidine. The results show that both copper release and corrosion are enhanced in DMEM(+), DMEM, and PBS+histidine compared with PBS alone. Speciation results show that essentially no free copper ions are present in the released fraction of Cu NPs in neither DMEM(+), DMEM nor histidine, while labile Cu complexes form in PBS. The Cu NPs were substantially more membrane reactive in PBS compared to the other media and the NPs caused larger effects compared to the same mass of Cu ions. Similarly, the Cu NPs caused much more ROS generation compared to the released fraction only. Taken together, the results suggest that membrane damage and ROS formation are stronger induced by Cu NPs and by free or labile Cu ions/complexes compared with Cu bound to biomolecules. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Parasitization by Scleroderma guani influences expression of superoxide dismutase genes in Tenebrio molitor

USDA-ARS?s Scientific Manuscript database

Superoxide dismutase (SOD) is an antioxidant enzyme involved in detoxifying reactive oxygen species. In this study, we identified genes encoding the extracellular and intracellular copper-zinc SODs (ecCuZnSOD and icCuZnSOD) and a manganese SOD (MnSOD) in the yellow mealworm beetle, Tenebrio molitor....

PPARδ inhibits UVB-induced secretion of MMP-1 through MKP-7-mediated suppression of JNK signaling.

PubMed

Ham, Sun A; Kang, Eun S; Lee, Hanna; Hwang, Jung S; Yoo, Taesik; Paek, Kyung S; Park, Chankyu; Kim, Jin-Hoi; Lim, Dae-Seog; Seo, Han G

2013-11-01

In the present study, we investigated the role of peroxisome proliferator-activated receptor (PPAR) δ in modulating matrix-degrading metalloproteinases and other mechanisms underlying photoaging processes in the skin. In human dermal fibroblasts (HDFs), activation of PPARδ by its specific ligand GW501516 markedly attenuated UVB-induced secretion of matrix metalloproteinase (MMP)-1, concomitant with decreased generation of reactive oxygen species. These effects were significantly reduced in the presence of PPARδ small interfering RNA and GSK0660. Furthermore, c-Jun N-terminal kinase (JNK), but not p38 or extracellular signal-regulated kinase, mediated PPARδ-dependent inhibition of MMP-1 secretion in HDFs exposed to UVB. PPARδ-mediated messenger RNA stabilization of mitogen-activated protein kinase phosphatase (MKP)-7 was responsible for the GW501516-mediated inhibition of JNK signaling. Inhibition of UVB-induced secretion of MMP-1 by PPARδ was associated with the restoration of types I and III collagen to levels approaching those in cells not exposed to UVB. Finally, in HR-1 hairless mice exposed to UVB, administration of GW501516 significantly reduced wrinkle formation and skin thickness, downregulated MMP-1 and JNK phosphorylation, and restored the levels of MKP-7, types I and III collagen. These results suggest that PPARδ-mediated inhibition of MMP-1 secretion prevents some effects of photoaging and maintains the integrity of skin by inhibiting the degradation of the collagenous extracellular matrix.

Reactive Oxygen Species-Mediated Cellular Stress Response and Lipid Accumulation in Oleaginous Microorganisms: The State of the Art and Future Perspectives

PubMed Central

Shi, Kun; Gao, Zhen; Shi, Tian-Qiong; Song, Ping; Ren, Lu-Jing; Huang, He; Ji, Xiao-Jun

2017-01-01

Microbial oils, which are mainly extracted from yeasts, molds, and algae, have been of considerable interest as food additives and biofuel resources due to their high lipid content. While these oleaginous microorganisms generally produce only small amounts of lipids under optimal growth conditions, their lipid accumulation machinery can be induced by environmental stresses, such as nutrient limitation and an inhospitable physical environmental. As common second messengers of many stress factors, reactive oxygen species (ROS) may act as a regulator of cellular responses to extracellular environmental signaling. Furthermore, increasing evidence indicates that ROS may act as a mediator of lipid accumulation, which is associated with dramatic changes in the transcriptome, proteome, and metabolome. However, the specific mechanisms of ROS involvement in the crosstalk between extracellular stress signaling and intracellular lipid synthesis require further investigation. Here, we summarize current knowledge on stress-induced lipid biosynthesis and the putative role of ROS in the control of lipid accumulation in oleaginous microorganisms. Understanding such links may provide guidance for the development of stress-based strategies to enhance microbial lipid production. PMID:28507542

Extracellular ultrathin fibers sensitive to intracellular reactive oxygen species: Formation of intercellular membrane bridges

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jung, Se-Hui; Park, Jin-Young; Joo, Jung-Hoon

2011-07-15

Membrane bridges are key cellular structures involved in intercellular communication; however, dynamics for their formation are not well understood. We demonstrated the formation and regulation of novel extracellular ultrathin fibers in NIH3T3 cells using confocal and atomic force microscopy. At adjacent regions of neighboring cells, phorbol 12-myristate 13-acetate (PMA) and glucose oxidase induced ultrathin fiber formation, which was prevented by Trolox, a reactive oxygen species (ROS) scavenger. The height of ROS-sensitive ultrathin fibers ranged from 2 to 4 nm. PMA-induced formation of ultrathin fibers was inhibited by cytochalasin D, but not by Taxol or colchicine, indicating that ultrathin fibers mainlymore » comprise microfilaments. PMA-induced ultrathin fibers underwent dynamic structural changes, resulting in formation of intercellular membrane bridges. Thus, these fibers are formed by a mechanism(s) involving ROS and involved in formation of intercellular membrane bridges. Furthermore, ultrastructural imaging of ultrathin fibers may contribute to understanding the diverse mechanisms of cell-to-cell communication and the intercellular transfer of biomolecules, including proteins and cell organelles.« less

Lebein, a Snake Venom Disintegrin, Induces Apoptosis in Human Melanoma Cells

PubMed Central

Hammouda, Manel B.; Montenegro, María F.; Sánchez-del-Campo, Luis; Zakraoui, Ons; Aloui, Zohra; Riahi-Chebbi, Ichrak; Karoui, Habib; Rodríguez-López, José Neptuno; Essafi-Benkhadir, Khadija

2016-01-01

Melanoma, the most threatening form of skin cancer, has a very poor prognosis and is characterized by its very invasive and chemoresistant properties. Despite the recent promising news from the field of immunotherapy, there is an urgent need for new therapeutic approaches that are free of resistance mechanisms and side effects. Anti-neoplasic properties have been highlighted for different disintegrins from snake venom including Lebein; however, the exact effect of Lebein on melanoma has not yet been defined. In this study, we showed that Lebein blocks melanoma cell proliferation and induces a more differentiated phenotype with inhibition of extracellular signal-regulated kinase (ERK) phosphorylation and microphthalmia-associated transcription factor (MITF) overexpression. Melanoma cells became detached but were less invasive with upregulation of E-cadherin after Lebein exposure. Lebein induced a caspase-independent apoptotic program with apoptosis inducing factor (AIF), BCL-2-associated X protein (BAX) and Bim overexpression together with downregulation of B-cell lymphoma-2 (BCL-2). It generated a distinct response in reactive oxygen species (ROS) generation and p53 levels depending on the p53 cell line status (wild type or mutant). Therefore, we propose Lebein as a new candidate for development of potential therapies for melanoma. PMID:27399772

Saturated fatty acid palmitate induces extracellular release of histone H3: A possible mechanistic basis for high-fat diet-induced inflammation and thrombosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shrestha, Chandan; Department of Laboratory and Vascular Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima; Ito, Takashi

Highlights: •High-fat diet feeding and palmitate induces the release of nuclear protein histone H3. •ROS production and JNK signaling mediates the release of histone H3. •Extracellular histones induces proinflammatory and procoagulant response. -- Abstract: Chronic low-grade inflammation is a key contributor to high-fat diet (HFD)-related diseases, such as type 2 diabetes, non-alcoholic steatohepatitis, and atherosclerosis. The inflammation is characterized by infiltration of inflammatory cells, particularly macrophages, into obese adipose tissue. However, the molecular mechanisms by which a HFD induces low-grade inflammation are poorly understood. Here, we show that histone H3, a major protein component of chromatin, is released into themore » extracellular space when mice are fed a HFD or macrophages are stimulated with the saturated fatty acid palmitate. In a murine macrophage cell line, RAW 264.7, palmitate activated reactive oxygen species (ROS) production and JNK signaling. Inhibitors of these pathways dampened palmitate-induced histone H3 release, suggesting that the extracellular release of histone H3 was mediated, in part, through ROS and JNK signaling. Extracellular histone activated endothelial cells toexpress the adhesion molecules ICAM-1 and VCAM-1 and the procoagulant molecule tissue factor, which are known to contribute to inflammatory cell recruitment and thrombosis. These results suggest the possible contribution of extracellular histone to the pathogenesis of HFD-induced inflammation and thrombosis.« less

FRAS1-related extracellular matrix 3 (FREM3) single-nucleotide polymorphism effects on gene expression, amygdala reactivity and perceptual processing speed: An accelerated aging pathway of depression risk

PubMed Central

Nikolova, Yuliya S.; Iruku, Swetha P.; Lin, Chien-Wei; Conley, Emily Drabant; Puralewski, Rachel; French, Beverly; Hariri, Ahmad R.; Sibille, Etienne

2015-01-01

The A allele of the FRAS1-related extracellular matrix protein 3 (FREM3) rs7676614 single nucleotide polymorphism (SNP) was linked to major depressive disorder (MDD) in an early genome-wide association study (GWAS), and to symptoms of psychomotor retardation in a follow-up investigation. In line with significant overlap between age- and depression-related molecular pathways, parallel work has shown that FREM3 expression in postmortem human brain decreases with age. Here, we probe the effect of rs7676614 on amygdala reactivity and perceptual processing speed, both of which are altered in depression and aging. Amygdala reactivity was assessed using a face-matching BOLD fMRI paradigm in 365 Caucasian participants in the Duke Neurogenetics Study (DNS) (192 women, mean age 19.7 ± 1.2). Perceptual processing speed was indexed by reaction times in the same task and the Trail Making Test (TMT). The effect of rs7676614 on FREM3 mRNA brain expression levels was probed in a postmortem cohort of 169 Caucasian individuals (44 women, mean age 50.8 ± 14.9). The A allele of rs7676614 was associated with blunted amygdala reactivity to faces, slower reaction times in the face-matching condition (p < 0.04), as well as marginally slower performance on TMT Part B (p = 0.056). In the postmortem cohort, the T allele of rs6537170 (proxy for the rs7676614 A allele), was associated with trend-level reductions in gene expression in Brodmann areas 11 and 47 (p = 0.066), reminiscent of patterns characteristic of older age. The low-expressing allele of another FREM3 SNP (rs1391187) was similarly associated with reduced amygdala reactivity and slower TMT Part B speed, in addition to reduced BA47 activity and extraversion (p < 0.05). Together, these results suggest common genetic variation associated with reduced FREM3 expression may confer risk for a subtype of depression characterized by reduced reactivity to environmental stimuli and slower perceptual processing speed, possibly suggestive of accelerated aging. PMID:26441752

Platelet lysate gel and endothelial progenitors stimulate microvascular network formation in vitro: tissue engineering implications.

PubMed

Fortunato, Tiago M; Beltrami, Cristina; Emanueli, Costanza; De Bank, Paul A; Pula, Giordano

2016-05-04

Revascularisation is a key step for tissue regeneration and complete organ engineering. We describe the generation of human platelet lysate gel (hPLG), an extracellular matrix preparation from human platelets able to support the proliferation of endothelial colony forming cells (ECFCs) in 2D cultures and the formation of a complete microvascular network in vitro in 3D cultures. Existing extracellular matrix preparations require addition of high concentrations of recombinant growth factors and allow only limited formation of capillary-like structures. Additional advantages of our approach over existing extracellular matrices are the absence of any animal product in the composition hPLG and the possibility of obtaining hPLG from patients to generate homologous scaffolds for re-implantation. This discovery has the potential to accelerate the development of regenerative medicine applications based on implantation of microvascular networks expanded ex vivo or the generation of fully vascularised organs.

Platelet lysate gel and endothelial progenitors stimulate microvascular network formation in vitro: tissue engineering implications

PubMed Central

Fortunato, Tiago M.; Beltrami, Cristina; Emanueli, Costanza; De Bank, Paul A.; Pula, Giordano

2016-01-01

Revascularisation is a key step for tissue regeneration and complete organ engineering. We describe the generation of human platelet lysate gel (hPLG), an extracellular matrix preparation from human platelets able to support the proliferation of endothelial colony forming cells (ECFCs) in 2D cultures and the formation of a complete microvascular network in vitro in 3D cultures. Existing extracellular matrix preparations require addition of high concentrations of recombinant growth factors and allow only limited formation of capillary-like structures. Additional advantages of our approach over existing extracellular matrices are the absence of any animal product in the composition hPLG and the possibility of obtaining hPLG from patients to generate homologous scaffolds for re-implantation. This discovery has the potential to accelerate the development of regenerative medicine applications based on implantation of microvascular networks expanded ex vivo or the generation of fully vascularised organs. PMID:27141997

Skeletal muscle expresses the extracellular cyclic AMP–adenosine pathway

PubMed Central

Chiavegatti, T; Costa, V L; Araújo, M S; Godinho, R O

2007-01-01

Background and purpose: cAMP is a key intracellular signalling molecule that regulates multiple processes of the vertebrate skeletal muscle. We have shown that cAMP can be actively pumped out from the skeletal muscle cell. Since in other tissues, cAMP efflux had been associated with extracellular generation of adenosine, in the present study we have assessed the fate of interstitial cAMP and the existence of an extracellular cAMP-adenosine signalling pathway in skeletal muscle. Experimental approach: cAMP efflux and/or its extracellular degradation were analysed by incubating rat cultured skeletal muscle with exogenous cAMP, forskolin or isoprenaline. cAMP and its metabolites were quantified by radioassay or HPLC, respectively. Key results: Incubation of cells with exogenous cAMP was followed by interstitial accumulation of 5′-AMP and adenosine, a phenomenon inhibited by selective inhibitors of ecto-phosphodiesterase (DPSPX) and ecto-nucleotidase (AMPCP). Activation of adenylyl cyclase (AC) in cultured cells with forskolin or isoprenaline increased cAMP efflux and extracellular generation of 5′-AMP and adenosine. Extracellular cAMP-adenosine pathway was also observed after direct and receptor-dependent stimulation of AC in rat extensor muscle ex vivo. These events were attenuated by probenecid, an inhibitor of ATP binding cassette family transporters. Conclusions and implications: Our results show the existence of an extracellular biochemical cascade that converts cAMP into adenosine. The functional relevance of this extracellular signalling system may involve a feedback modulation of cellular response initiated by several G protein-coupled receptor ligands, amplifying cAMP influence to a paracrine mode, through its metabolite, adenosine. PMID:18157164

ABCG1-mediated generation of extracellular cholesterol microdomains[S

PubMed Central

Freeman, Sebastian R.; Jin, Xueting; Anzinger, Joshua J.; Xu, Qing; Purushothaman, Sonya; Fessler, Michael B.; Addadi, Lia; Kruth, Howard S.

2014-01-01

Previous studies have demonstrated that the ATP-binding cassette transporters (ABC)A1 and ABCG1 function in many aspects of cholesterol efflux from macrophages. In this current study, we continued our investigation of extracellular cholesterol microdomains that form during enrichment of macrophages with cholesterol. Human monocyte-derived macrophages and mouse bone marrow-derived macrophages, differentiated with macrophage colony-stimulating factor (M-CSF) or granulocyte macrophage colony-stimulation factor (GM-CSF), were incubated with acetylated LDL (AcLDL) to allow for cholesterol enrichment and processing. We utilized an anti-cholesterol microdomain monoclonal antibody to reveal pools of unesterified cholesterol, which were found both in the extracellular matrix and associated with the cell surface, that we show function in reverse cholesterol transport. Coincubation of AcLDL with 50 μg/ml apoA-I eliminated all extracellular and cell surface-associated cholesterol microdomains, while coincubation with the same concentration of HDL only removed extracellular matrix-associated cholesterol microdomains. Only at an HDL concentration of 200 µg/ml did HDL eliminate the cholesterol microdomains that were cell-surface associated. The deposition of cholesterol microdomains was inhibited by probucol, but it was increased by the liver X receptor (LXR) agonist TO901317, which upregulates ABCA1 and ABCG1. Extracellular cholesterol microdomains did not develop when ABCG1-deficient mouse bone marrow-derived macrophages were enriched with cholesterol. Our findings show that generation of extracellular cholesterol microdomains is mediated by ABCG1 and that reverse cholesterol transport occurs not only at the cell surface but also within the extracellular space. PMID:24212237

Extracellular determinants of anion discrimination of the Cl-/H+ antiporter protein CLC-5.

PubMed

De Stefano, Silvia; Pusch, Michael; Zifarelli, Giovanni

2011-12-23

Mammalian CLC proteins comprise both Cl- channels and Cl-/H+ antiporters that carry out fundamental physiological tasks by transporting Cl- across plasma membrane and intracellular compartments. The NO3- over Cl- preference of a plant CLC transporter has been pinpointed to a conserved serine residue located at Scen and it is generally assumed that the other two binding sites of CLCs, Sext and Sin, do not substantially contribute to anion selectivity. Here we show for the Cl-/H+ antiporter CLC-5 that the conserved and extracellularly exposed Lys210 residue is critical to determine the anion specificity for transport activity. In particular, mutations that neutralize or invert the charge at this position reverse the NO3- over Cl- preference of WT CLC-5 at a concentration of 100 mm, but do not modify the coupling stoichiometry with H+. The importance of the electrical charge is shown by chemical modification of K210C with positively charged cysteine-reactive compounds that reintroduce the WT preference for Cl-. At saturating extracellular anion concentrations, neutralization of Lys210 is of little impact on the anion preference, suggesting an important role of Lys210 on the association rate of extracellular anions to Sext.

GABA and Glutamate Uptake and Metabolism in Retinal Glial (Müller) Cells

PubMed Central

Bringmann, Andreas; Grosche, Antje; Pannicke, Thomas; Reichenbach, Andreas

2013-01-01

Müller cells, the principal glial cells of the retina, support the synaptic activity by the uptake and metabolization of extracellular neurotransmitters. Müller cells express uptake and exchange systems for various neurotransmitters including glutamate and γ-aminobutyric acid (GABA). Müller cells remove the bulk of extracellular glutamate in the inner retina and contribute to the glutamate clearance around photoreceptor terminals. By the uptake of glutamate, Müller cells are involved in the shaping and termination of the synaptic activity, particularly in the inner retina. Reactive Müller cells are neuroprotective, e.g., by the clearance of excess extracellular glutamate, but may also contribute to neuronal degeneration by a malfunctioning or even reversal of glial glutamate transporters, or by a downregulation of the key enzyme, glutamine synthetase. This review summarizes the present knowledge about the role of Müller cells in the clearance and metabolization of extracellular glutamate and GABA. Some major pathways of GABA and glutamate metabolism in Müller cells are described; these pathways are involved in the glutamate-glutamine cycle of the retina, in the defense against oxidative stress via the production of glutathione, and in the production of substrates for the neuronal energy metabolism. PMID:23616782

Pathways for Intracellular Generation of Oxidants and Tyrosine Nitration by a Macrophage Cell Line†

PubMed Central

Palazzolo-Ballance, Amy M.; Suquet, Christine; Hurst, James K.

2008-01-01

Two transformed murine macrophage cell lines (RAW 264.7 ATCC TIB-71 and CRL-2278) were examined for oxidant production at various times following activation by using a set of fluorescence and ESR-active probes. Stimulation with a soluble agonist or activation with bacterial lipopolysaccharide plus γ-interferon caused only very small initial increases in O2 consumption above basal rates; however, at 2-4 h post-activation, respiration increased to 2-3 fold and remained at these elevated levels over the subsequent lifetime of the cell (20-30 h). Oxidation reactions were confined primarily within the cell, as was demonstrated by using phagocytosable dichlorodihydrofluorescein-conjugated latex beads and cyclic hydroxylamines with differing membrane permeabilities. From the intrinsic reactivities of these probes and the time course of their oxidations, one infers induction of apparent peroxidase activity beginning at ∼2 h post-activation, coinciding with the increase in overall respiratory rate; this acquired capability was accompanied by accumulation of a stable horseradish peroxidase-reactive oxidant, presumably H2O2, in the extracellular medium,. Nitrite ion rapidly accumulated in the extracellular medium over a period of 5-8 h post-activation in both cell lines, indicating the presence of active nitric oxide synthase (iNOS) during that period. Prostaglandin endoperoxide H synthase (COX-2) activity was detected at 15-20 h post-activation by use of sensitive peroxide assay in conjunction with a COX-2 specific inhibitor (DuP-697). Superoxide formation was detected by reaction with hydroethidine within the first hour following activation, but not thereafter. Consistent with the absence of significant respiratory stimulation, the amount of O2·- formed was very small; comparative reactions of cyclic hydroxylamine probes indicated that virtually none of the O2·- was discharged into the external medium. Myeloperoxidase (MPO) activity was probed at various times post-activation by using fluorescein-conjugated polyacrylamide beads, which efficiently trap MPO-generated HOCl in neutrophils to give stable chlorofluorescein products. However, chlorination of the dye was not detected under any conditions in RAW cells, virtually precluding MPO involvement in their intracellular reactions. This same probe was used to determine changes in intraphagosomal pH, which increased slowly from ∼6.5 to ∼8.2 over a 20 h post-phagocytosis period. The cumulative data suggest activation is followed by sequential induction of an endogenous peroxidase, iNOS, and COX-2, with NADPH oxidase-derived O2·- playing a minimal role in direct generation of intracellular oxidants. To account for reported observations of intracellular tyrosine nitration late in the life cycles of macrophages, we propose a novel mechanism wherein iNOS-generated NO2- is used by COX-2 to produce NO2· as a terminal microbicidal oxidant and nitrating agent. PMID:17530864

Oxidative Stressors Modify the Response of Streptococcus mutans to Its Competence Signal Peptides

PubMed Central

De Furio, Matthew; Ahn, Sang Joon

2017-01-01

ABSTRACT The dental caries pathogen Streptococcus mutans is continually exposed to several types of stress in the oral biofilm environment. Oxidative stress generated by reactive oxygen species has a major impact on the establishment, persistence, and virulence of S. mutans. Here, we combined fluorescent reporter-promoter fusions with single-cell imaging to study the effects of reactive oxygen species on activation of genetic competence in S. mutans. Exposure to paraquat, which generates superoxide anion, produced a qualitatively different effect on activation of expression of the gene for the master competence regulator, ComX, than did treatment with hydrogen peroxide (H2O2), which can yield hydroxyl radical. Paraquat suppressed peptide-mediated induction of comX in a progressive and cumulative fashion, whereas the response to H2O2 displayed a strong threshold behavior. Low concentrations of H2O2 had little effect on induction of comX or the bacteriocin gene cipB, but expression of these genes declined sharply if extracellular H2O2 exceeded a threshold concentration. These effects were not due to decreased reporter gene fluorescence. Two different threshold concentrations were observed in the response to H2O2, depending on the gene promoter that was analyzed and the pathway by which the competence regulon was stimulated. The results show that paraquat and H2O2 affect the S. mutans competence signaling pathway differently, and that some portions of the competence signaling pathway are more sensitive to oxidative stress than others. IMPORTANCE Streptococcus mutans inhabits the oral biofilm, where it plays an important role in the development of dental caries. Environmental stresses such as oxidative stress influence the growth of S. mutans and its important virulence-associated behaviors, such as genetic competence. S. mutans competence development is a complex behavior that involves two different signaling peptides and can exhibit cell-to-cell heterogeneity. Although oxidative stress is known to influence S. mutans competence, it is not understood how oxidative stress interacts with the peptide signaling or affects heterogeneity. In this study, we used fluorescent reporters to probe the effect of reactive oxygen species on competence signaling at the single-cell level. Our data show that different reactive oxygen species have different effects on S. mutans competence, and that some portions of the signaling pathway are more acutely sensitive to oxidative stress than others. PMID:28887419

Reaction kinetics and targeting to cellular glutathione S-transferase of the glutathione peroxidase mimetic PhSeZnCl and its D,L-polylactide microparticle formulation.

PubMed

Bartolini, D; Piroddi, M; Tidei, C; Giovagnoli, S; Pietrella, D; Manevich, Y; Tew, K D; Giustarini, D; Rossi, R; Townsend, D M; Santi, C; Galli, F

2015-01-01

Catalytic properties and cellular effects of the glutathione peroxidase (GPx)-mimetic compound PhSeZnCl or its d,l-lactide polymer microencapsulation form (M-PhSeZnCl) were investigated and compared with the prototypical Se-organic compounds ebselen and diselenide (PhSe)2. PhSeZnCl was confirmed to catalyze the ping-pong reaction of GPx with higher Vmax than ebselen and (PhSe)2, but the catalytic efficiency calculated for the cosubstrates glutathione (GSH) and H2O2, and particularly the high reactivity against thiols (lowest KM for GSH in the series of test molecules), suggested poor biological applicability of PhSeZnCl as a GPx mimetic. Cytotoxicity of PhSeZnCl was demonstrated in various cancer cell lines via increased reactive oxygen species (ROS) generation, depletion of intracellular thiols, and induction of apoptosis. Experiments carried out in GSH S-transferase P (GSTP)-overexpressing K562 human erythroleukemia cells and in GSTP1-1-knockout murine embryonic fibroblasts (MEFs) demonstrated that this cytosolic enzyme represents a preferential target of the redox disturbances produced by this Se-compound with a key role in controlling H2O2 generation and the perturbation of stress/survival kinase signaling. Microencapsulation was adopted as a strategy to control the thiol reactivity and oxidative stress effects of PhSeZnCl, then assessing applications alternative to anticancer. The uptake of this "depowered" GPx-mimetic formulation, which occurred through an endocytosis-like mechanism, resulted in a marked reduction of cytotoxicity. In MCF-7 cells transfected with different allelic variants of GSTP, M-PhSeZnCl lowered the burst of cellular ROS induced by the exposure to extracellular H2O2, and the extent of this effect changed between the GSTP variants. Microencapsulation is a straightforward strategy to mitigate the toxicity of thiol-reactive Se-organic drugs that enhanced the antioxidant and cellular protective effects of PhSeZnCl. A mechanistic linkage of these effects with the expression pattern and signaling properties of GSTP . This has overcome the GPx-mimetic paradigm proposed for Se-organic drugs with a more pragmatic concept of GSTP signaling modulators. Copyright © 2014 Elsevier Inc. All rights reserved.

Reaction kinetics and targeting to cellular glutathione S-transferase of the glutathione peroxidase mimetic PhSeZnCl and its d,l-polylactide microparticle formulation

PubMed Central

Bartolini, D.; Piroddi, M.; Tidei, C.; Giovagnoli, S.; Pietrella, D.; Manevich, Y.; Tew, K.D.; Giustarini, D.; Rossi, R.; Townsend, D.M.; Santi, C.; Galli, F.

2015-01-01

Catalytic properties and cellular effects of the glutathione peroxidase (GPx)-mimetic compound PhSeZnCl or its d,l-lactide polymer microencapsulation form (M-PhSeZnCl) were investigated and compared with the prototypical Se-organic compounds ebselen and diselenide (PhSe)2. PhSeZnCl was confirmed to catalyze the ping-pong reaction of GPx with higher Vmax than ebselen and (PhSe)2, but the catalytic efficiency calculated for the cosubstrates glutathione (GSH) and H2O2, and particularly the high reactivity against thiols (lowest KM for GSH in the series of test molecules), suggested poor biological applicability of PhSeZnCl as a GPx mimetic. Cytotoxicity of PhSeZnCl was demonstrated in various cancer cell lines via increased reactive oxygen species (ROS) generation, depletion of intracellular thiols, and induction of apoptosis. Experiments carried out in GSH S-transferase P (GSTP)-overexpressing K562 human erythroleukemia cells and in GSTP1-1-knockout murine embryonic fibroblasts (MEFs) demonstrated that this cytosolic enzyme represents a preferential target of the redox disturbances produced by this Se-compound with a key role in controlling H2O2 generation and the perturbation of stress/survival kinase signaling. Microencapsulation was adopted as a strategy to control the thiol reactivity and oxidative stress effects of PhSeZnCl, then assessing applications alternative to anticancer. The uptake of this “depowered” GPx-mimetic formulation, which occurred through an endocytosis-like mechanism, resulted in a marked reduction of cytotoxicity. In MCF-7 cells transfected with different allelic variants of GSTP, M-PhSeZnCl lowered the burst of cellular ROS induced by the exposure to extracellular H2O2, and the extent of this effect changed between the GSTP variants. Microencapsulation is a straightforward strategy to mitigate the toxicity of thiol-reactive Se-organic drugs that enhanced the antioxidant and cellular protective effects of PhSeZnCl. A mechanistic linkage of these effects with the expression pattern and signaling properties of GSTP. This has overcome the GPx-mimetic paradigm proposed for Se-organic drugs with a more pragmatic concept of GSTP signaling modulators. PMID:25452145

Natural Product Anacardic Acid from Cashew Nut Shells Stimulates Neutrophil Extracellular Trap Production and Bactericidal Activity.

PubMed

Hollands, Andrew; Corriden, Ross; Gysler, Gabriela; Dahesh, Samira; Olson, Joshua; Raza Ali, Syed; Kunkel, Maya T; Lin, Ann E; Forli, Stefano; Newton, Alexandra C; Kumar, Geetha B; Nair, Bipin G; Perry, J Jefferson P; Nizet, Victor

2016-07-01

Emerging antibiotic resistance among pathogenic bacteria is an issue of great clinical importance, and new approaches to therapy are urgently needed. Anacardic acid, the primary active component of cashew nut shell extract, is a natural product used in the treatment of a variety of medical conditions, including infectious abscesses. Here, we investigate the effects of this natural product on the function of human neutrophils. We find that anacardic acid stimulates the production of reactive oxygen species and neutrophil extracellular traps, two mechanisms utilized by neutrophils to kill invading bacteria. Molecular modeling and pharmacological inhibitor studies suggest anacardic acid stimulation of neutrophils occurs in a PI3K-dependent manner through activation of surface-expressed G protein-coupled sphingosine-1-phosphate receptors. Neutrophil extracellular traps produced in response to anacardic acid are bactericidal and complement select direct antimicrobial activities of the compound. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Memory Trace Reactivation and Behavioral Response during Retrieval Are Differentially Modulated by Amygdalar Glutamate Receptors Activity: Interaction between Amygdala and Insular Cortex

ERIC Educational Resources Information Center

Osorio-Gómez, Daniel; Guzmán-Ramos, Kioko; Bermúdez-Rattoni, Federico

2017-01-01

The insular cortex (IC) is required for conditioned taste aversion (CTA) retrieval. However, it remains unknown which cortical neurotransmitters levels are modified upon CTA retrieval. Using in vivo microdialysis, we observed that there were clear elevations in extracellular glutamate, norepinephrine, and dopamine in and around the center of the…

Extracellular cell stress (heat shock) proteins-immune responses and disease: an overview.

PubMed

Pockley, A Graham; Henderson, Brian

2018-01-19

Extracellular cell stress proteins are highly conserved phylogenetically and have been shown to act as powerful signalling agonists and receptors for selected ligands in several different settings. They also act as immunostimulatory 'danger signals' for the innate and adaptive immune systems. Other studies have shown that cell stress proteins and the induction of immune reactivity to self-cell stress proteins can attenuate disease processes. Some proteins (e.g. Hsp60, Hsp70, gp96) exhibit both inflammatory and anti-inflammatory properties, depending on the context in which they encounter responding immune cells. The burgeoning literature reporting the presence of stress proteins in a range of biological fluids in healthy individuals/non-diseased settings, the association of extracellular stress protein levels with a plethora of clinical and pathological conditions and the selective expression of a membrane form of Hsp70 on cancer cells now supports the concept that extracellular cell stress proteins are involved in maintaining/regulating organismal homeostasis and in disease processes and phenotype. Cell stress proteins, therefore, form a biologically complex extracellular cell stress protein network having diverse biological, homeostatic and immunomodulatory properties, the understanding of which offers exciting opportunities for delivering novel approaches to predict, identify, diagnose, manage and treat disease.This article is part of the theme issue 'Heat shock proteins as modulators and therapeutic targets of chronic disease: an integrated perspective'. © 2017 The Author(s).

Extracellular matrix controls neuronal features that mediate the persistence of fear.

PubMed

Pignataro, Annabella; Pagano, Roberto; Guarneri, Giorgia; Middei, Silvia; Ammassari-Teule, Martine

2017-12-01

Degradation of the chondroitin sulfate proteoglycans of the extracellular matrix (ECM) by injections of the bacterial enzyme chondroitinase ABC (ChABC) in the basolateral amygdala (BLA) does not impair fear memory formation but accelerates its extinction and disrupts its reactivation. These observations suggest that the treatment might selectively interfere with the post-extinction features of neurons that mediate the reinstatement of fear. Here, we report that ChABC mice show regular fear memory and memory-driven c-fos activation and dendritic spine formation in the BLA. These mice then rapidly extinguish their fear response and exhibit a post-extinction concurrent reduction in c-fos activation and large dendritic spines that extends to the anterior cingulate cortex 7 days later. At this remote time point, fear renewal and fear retrieval are impaired. These findings show that a non-cellular component of the brain tissue controls post-extinction levels of neuronal activity and spine enlargement in the regions sequentially remodelled during the formation of recent and remote fear memory. By preventing BLA and aCC neurons to retain neuronal features that serve to reactivate an extinguished fear memory, ECM digestion might offer a therapeutic strategy for durable attenuation of traumatic memories.

Data on how several physiological parameters of stored red blood cells are similar in glucose 6-phosphate dehydrogenase deficient and sufficient donors.

PubMed

Tzounakas, Vassilis L; Kriebardis, Anastasios G; Georgatzakou, Hara T; Foudoulaki-Paparizos, Leontini E; Dzieciatkowska, Monika; Wither, Matthew J; Nemkov, Travis; Hansen, Kirk C; Papassideri, Issidora S; D'Alessandro, Angelo; Antonelou, Marianna H

2016-09-01

This article contains data on the variation in several physiological parameters of red blood cells (RBCs) donated by eligible glucose-6-phosphate dehydrogenase (G6PD) deficient donors during storage in standard blood bank conditions compared to control, G6PD sufficient (G6PD(+)) cells. Intracellular reactive oxygen species (ROS) generation, cell fragility and membrane exovesiculation were measured in RBCs throughout the storage period, with or without stimulation by oxidants, supplementation of N-acetylcysteine and energy depletion, following incubation of stored cells for 24 h at 37 °C. Apart from cell characteristics, the total or uric acid-dependent antioxidant capacity of the supernatant in addition to extracellular potassium concentration was determined in RBC units. Finally, procoagulant activity and protein carbonylation levels were measured in the microparticles population. Further information can be found in "Glucose 6-phosphate dehydrogenase deficient subjects may be better "storers" than donors of red blood cells" [1].

Oxidants produced by methylglyoxal-modified collagen trigger ER stress and apoptosis in skin fibroblasts.

PubMed

Nowotny, Kerstin; Castro, José Pedro; Hugo, Martín; Braune, Sabine; Weber, Daniela; Pignitter, Marc; Somoza, Veronika; Bornhorst, Julia; Schwerdtle, Tanja; Grune, Tilman

2018-05-20

Methylglyoxal (MG), a highly reactive dicarbonyl, interacts with proteins to form advanced glycation end products (AGEs). AGEs include a variety of compounds which were shown to have damaging potential and to accumulate in the course of different conditions such as diabetes mellitus and aging. After confirming collagen as a main target for MG modifications in vivo within the extracellular matrix, we show here that MG-collagen disrupts fibroblast redox homeostasis and induces endoplasmic reticulum (ER) stress and apoptosis. In particular, MG-collagen-induced apoptosis is associated with the activation of the PERK-eIF2α pathway and caspase-12. MG-collagen contributes to altered redox homeostasis by directly generating hydrogen peroxide and oxygen-derived free radicals. The induction of ER stress in human fibroblasts was confirmed using collagen extracts isolated from old mice in which MG-derived AGEs were enriched. In conclusion, MG-derived AGEs represent one factor contributing to diminished fibroblast function during aging. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Calcium and ROS: A mutual interplay

PubMed Central

Görlach, Agnes; Bertram, Katharina; Hudecova, Sona; Krizanova, Olga

2015-01-01

Calcium is an important second messenger involved in intra- and extracellular signaling cascades and plays an essential role in cell life and death decisions. The Ca2+ signaling network works in many different ways to regulate cellular processes that function over a wide dynamic range due to the action of buffers, pumps and exchangers on the plasma membrane as well as in internal stores. Calcium signaling pathways interact with other cellular signaling systems such as reactive oxygen species (ROS). Although initially considered to be potentially detrimental byproducts of aerobic metabolism, it is now clear that ROS generated in sub-toxic levels by different intracellular systems act as signaling molecules involved in various cellular processes including growth and cell death. Increasing evidence suggests a mutual interplay between calcium and ROS signaling systems which seems to have important implications for fine tuning cellular signaling networks. However, dysfunction in either of the systems might affect the other system thus potentiating harmful effects which might contribute to the pathogenesis of various disorders. PMID:26296072

Chromatin remodeling and histone modification in the conversion of oligodendrocyte precursors to neural stem cells

PubMed Central

Kondo, Toru; Raff, Martin

2004-01-01

We showed previously that purified rat oligodendrocyte precursor cells (OPCs) can be induced by extracellular signals to convert to multipotent neural stem-like cells (NSLCs), which can then generate both neurons and glial cells. Because the conversion of precursor cells to stem-like cells is of both intellectual and practical interest, it is important to understand its molecular basis. We show here that the conversion of OPCs to NSLCs depends on the reactivation of the sox2 gene, which in turn depends on the recruitment of the tumor suppressor protein Brca1 and the chromatin-remodeling protein Brahma (Brm) to an enhancer in the sox2 promoter. Moreover, we show that the conversion is associated with the modification of Lys 4 and Lys 9 of histone H3 at the same enhancer. Our findings suggest that the conversion of OPCs to NSLCs depends on progressive chromatin remodeling, mediated in part by Brca1 and Brm. PMID:15574597

Chitosan multiple addition enhances laccase production from Trametes versicolor.

PubMed

Adekunle, Abiodun Emmanuel; Wang, Feng; Hu, Jianhua; Ma, Anzhou; Guo, Chen; Zhuang, Guoqiang; Liu, Chun-Zhao

2015-10-01

Chitosan multiple addition strategy was developed to improve laccase production from Trametes versicolor cultures. The optimized multiple addition strategy was carried out by two-time addition of 0.1 g L(-1) chitosan to a 2-day-old culture media, with 24-h interval between the treatments. Under these conditions, laccase activity of 644.9 U l(-1) was achieved on the seventh day and laccase production was improved by 93.5 % higher than the control. Chitosan treatment increased reactive oxygen species generation and extracellular protein concentration in the treated mycelia. In contrast, the inducer inhibited the mycelia growth. The result of the quantitative reverse transcription polymerase chain reaction showed that the copy number of the laccase gene transcript increased by 16.7-fold in the treated mycelia relative to the control. This study provides insight into some of the intrinsic metabolic processes involved in the upregulation of laccase production in the presence of chitosan inducer in fungal culture.

Peroxisome proliferator-activated receptor δ inhibits Porphyromonas gingivalis lipopolysaccharide-induced activation of matrix metalloproteinase-2 by downregulating NADPH oxidase 4 in human gingival fibroblasts.

PubMed

Yoo, T; Ham, S A; Hwang, J S; Lee, W J; Paek, K S; Oh, J W; Kim, J H; Do, J T; Han, C W; Kim, J H; Seo, H G

2016-10-01

We investigated the roles of peroxisome proliferator-activated receptor δ (PPARδ) in Porphyromonas gingivalis-derived lipopolysaccharide (Pg-LPS)-induced activation of matrix metalloproteinase 2 (MMP-2). In human gingival fibroblasts (HGFs), activation of PPARδ by GW501516, a specific ligand of PPARδ, inhibited Pg-LPS-induced activation of MMP-2 and generation of reactive oxygen species (ROS), which was associated with reduced expression of NADPH oxidase 4 (Nox4). These effects were significantly smaller in the presence of small interfering RNA targeting PPARδ or the specific PPARδ inhibitor GSK0660, indicating that PPARδ is involved in these events. In addition, modulation of Nox4 expression by small interfering RNA influenced the effect of PPARδ on MMP-2 activity, suggesting a mechanism in which Nox4-derived ROS modulates MMP-2 activity. Furthermore, c-Jun N-terminal kinase and p38, but not extracellular signal-regulated kinase, mediated PPARδ-dependent inhibition of MMP-2 activity in HGFs treated with Pg-LPS. Concomitantly, PPARδ-mediated inhibition of MMP-2 activity was associated with the restoration of types I and III collagen to levels approaching those in HGFs not treated with Pg-LPS. These results indicate that PPARδ-mediated downregulation of Nox4 modulates cellular redox status, which in turn plays a critical role in extracellular matrix homeostasis through ROS-dependent regulation of MMP-2 activity. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Reprogramming to a pluripotent state modifies mesenchymal stem cell resistance to oxidative stress

PubMed Central

Asensi, Karina D; Fortunato, Rodrigo S; dos Santos, Danúbia S; Pacheco, Thaísa S; de Rezende, Danielle F; Rodrigues, Deivid C; Mesquita, Fernanda C P; Kasai-Brunswick, Tais H; de Carvalho, Antonio C Campos; Carvalho, Denise P; Carvalho, Adriana B; Goldenberg, Regina C dos S

2014-01-01

Properties of induced pluripotent stem cells (iPSC) have been extensively studied since their first derivation in 2006. However, the modification in reactive oxygen species (ROS) production and detoxification caused by reprogramming still needs to be further elucidated. The objective of this study was to compare the response of iPSC generated from menstrual blood–derived mesenchymal stem cells (mb-iPSC), embryonic stem cells (H9) and adult menstrual blood–derived mesenchymal stem cells (mbMSC) to ROS exposure and investigate the effects of reprogramming on cellular oxidative stress (OS). mbMSC were extremely resistant to ROS exposure, however, mb-iPSC were 10-fold less resistant to H2O2, which was very similar to embryonic stem cell sensitivity. Extracellular production of ROS was also similar in mb-iPSC and H9 and almost threefold lower than in mbMSC. Furthermore, intracellular amounts of ROS were higher in mb-iPSC and H9 when compared with mbMSC. As the ability to metabolize ROS is related to antioxidant enzymes, we analysed enzyme activities in these cell types. Catalase and superoxide dismutase activities were reduced in mb-iPSC and H9 when compared with mbMSC. Finally, cell adhesion under OS conditions was impaired in mb-iPSC when compared with mbMSC, albeit similar to H9. Thus, reprogramming leads to profound modifications in extracellular ROS production accompanied by loss of the ability to handle OS. PMID:24528612

Suppression of T24 human bladder cancer cells by ROS from locally delivered hematoporphyrin-containing polyurethane films.

PubMed

Kim, Dohyun; Lee, Mi Hee; Koo, Min-Ah; Kwon, Byeong-Ju; Kim, Min Sung; Seon, Gyeung Mi; Hong, Seung Hee; Park, Jong-Chul

2018-06-13

Systemic injection of a photosensitizer is a general method in photodynamic therapy, but it has complications due to the unintended systemic distribution and remnants of photosensitizers. This study focused on the possibility of suppressing luminal proliferative cells by excessive reactive oxygen species from locally delivered photosensitizer with biocompatible polyurethane, instead of the systemic injection method. We used human bladder cancer cells, hematoporphyrin as the photosensitizer, and polyurethane film as the photosensitizer-delivering container. The light source was a self-made LED (510 nm, 5 mW cm-2) system. The cancer cells were cultured on different doses of hematoporphyrin-containing polyurethane film and irradiated with LED for 15 minutes and 30 minutes each. After irradiating with LED and incubating for 24 hours, cell viability analysis, cell cycle analysis, apoptosis assay, intracellular and extracellular ROS generation study and western blot were performed. The cancer cell suppression effects of different concentrations of the locally delivered hematoporphyrin with PDT were compared. Apoptosis dominant cancer cell suppressions were shown to be hematoporphyrin dose-dependent. However, after irradiation, intracellular ROS amounts were similar in all the groups having different doses of hematoporphyrin, but these values were definitely higher than those in the control group. Excessive extracellular ROS from the intended, locally delivered photosensitizer for photodynamic treatment application had an inhibitory effect on luminal proliferative cancer cells. This method can be another possibility for PDT application on contactable or attachable lesions.

Imaging articular cartilage using second harmonic generation microscopy

NASA Astrophysics Data System (ADS)

Mansfield, Jessica C.; Winlove, C. Peter; Knapp, Karen; Matcher, Stephen J.

2006-02-01

Sub cellular resolution images of equine articular cartilage have been obtained using both second harmonic generation microscopy (SHGM) and two-photon fluorescence microscopy (TPFM). The SHGM images clearly map the distribution of the collagen II fibers within the extracellular matrix while the TPFM images show the distribution of endogenous two-photon fluorophores in both the cells and the extracellular matrix, highlighting especially the pericellular matrix and bright 2-3μm diameter features within the cells. To investigate the source of TPF in the extracellular matrix experiments have been carried out to see if it may originate from the proteoglycans. Pure solutions of the following proteoglycans hyaluronan, chondroitin sulfate and aggrecan have been imaged, only the aggrecan produced any TPF and here the intensity was not great enough to account for the TPF in the extracellular matrix. Also cartilage samples were subjected to a process to remove proteoglycans and cellular components. After this process the TPF from the samples had decreased by a factor of two, with respect to the SHG intensity.

Proteolytic processing of lysyl oxidase-like-2 in the extracellular matrix is required for crosslinking of basement membrane collagen IV.

PubMed

López-Jiménez, Alberto J; Basak, Trayambak; Vanacore, Roberto M

2017-10-13

Lysyl oxidase-like-2 (LOXL2) is an enzyme secreted into the extracellular matrix that crosslinks collagens by mediating oxidative deamination of lysine residues. Our previous work demonstrated that this enzyme crosslinks the 7S domain, a structural domain that stabilizes collagen IV scaffolds in the basement membrane. Despite its relevant role in extracellular matrix biosynthesis, little is known about the structural requirements of LOXL2 that enable collagen IV crosslinking. In this study, we demonstrate that LOXL2 is processed extracellularly by serine proteases, generating a 65-kDa form lacking the first two scavenger receptor cysteine-rich domains. Site-specific mutagenesis to prevent proteolytic processing generated a full-length enzyme that is active in vitro toward a soluble substrate, but fails to crosslink insoluble collagen IV within the extracellular matrix. In contrast, the processed form of LOXL2 binds to collagen IV and crosslinks the 7S domain. Together, our data demonstrate that proteolytic processing is an important event that allows LOXL2-mediated crosslinking of basement membrane collagen IV. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Generation and functional analysis of monoclonal antibodies against the second extracellular loop of human M3 muscarinic acetylcholine receptor.

PubMed

Tsuboi, Hiroto; Nakamura, Yumi; Iizuka, Mana; Matsuo, Naomi; Matsumoto, Isao; Sumida, Takayuki

2012-04-01

The M3 muscarinic acetylcholine receptor (M3R) plays a crucial role in the activation of salivary and lachrymal glands. The M3R contains four extracellular domains (the N-terminal, and the first, second, and third extracellular loops), and we recently detected antibodies against each of these four domains in a subgroup of patients with Sjögren's syndrome (SS). Functional analysis indicated that the influence of such anti-M3R antibodies on salivary secretion might differ based on the epitopes to which they bind. To clarify the relationship between B-cell epitopes on the M3R and its function, we generated two hybridomas producing anti-M3R monoclonal antibodies against the second extracellular loop of M3R (anti-M3R(2nd) mAbs) and analyzed their function by Ca(2+)-influx assays, using a human salivary gland (HSG) cell line. These two anti-M3R(2nd) mAbs suppressed Ca(2+)-influx in the HSG cells induced by cevimeline stimulation, suggesting that autoantibodies against the second extracellular loop of M3R could be involved in salivary dysfunction in patients with SS.

Ca2+ influx-mediated dilation of the endoplasmic reticulum and c-FLIPL downregulation trigger CDDO-Me–induced apoptosis in breast cancer cells

PubMed Central

Lee, A Reum; Yoon, Mi Jin; Cho, Hyeseong; Lee, Jong-Soo; Choi, Kyeong Sook

2015-01-01

The synthetic triterpenoid 2-cyano-3, 12-dioxooleana-1, 9(11)-dien-C28-methyl ester (CDDO-Me) is considered a promising anti-tumorigenic compound. In this study, we show that treatment with CDDO-Me induces progressive endoplasmic reticulum (ER)-derived vacuolation in various breast cancer cells and ultimately kills these cells by inducing apoptosis. We found that CDDO-Me–induced increases in intracellular Ca2+ levels, reflecting influx from the extracellular milieu, make a critical contribution to ER-derived vacuolation and subsequent cell death. In parallel with increasing Ca2+ levels, CDDO-Me markedly increased the generation of reactive oxygen species (ROS). Interestingly, there exists a reciprocal positive-regulatory loop between Ca2+ influx and ROS generation that triggers ER stress and ER dilation in response to CDDO-Me. In addition, CDDO-Me rapidly reduced the protein levels of c-FLIPL (cellular FLICE-inhibitory protein) and overexpression of c-FLIPL blocked CDDO-Me–induced cell death, but not vacuolation. These results suggest that c-FLIPL downregulation is a key contributor to CDDO-Me–induced apoptotic cell death, independent of ER-derived vacuolation. Taken together, our results show that ER-derived vacuolation via Ca2+ influx and ROS generation as well as caspase activation via c-FLIPL downregulation are responsible for the potent anticancer effects of CDDO-Me on breast cancer cells. PMID:26053096

Ca2+ influx-mediated dilation of the endoplasmic reticulum and c-FLIPL downregulation trigger CDDO-Me-induced apoptosis in breast cancer cells.

PubMed

Jeong, Soo Ah; Kim, In Young; Lee, A Reum; Yoon, Mi Jin; Cho, Hyeseong; Lee, Jong-Soo; Choi, Kyeong Sook

2015-08-28

The synthetic triterpenoid 2-cyano-3, 12-dioxooleana-1, 9(11)-dien-C28-methyl ester (CDDO-Me) is considered a promising anti-tumorigenic compound. In this study, we show that treatment with CDDO-Me induces progressive endoplasmic reticulum (ER)-derived vacuolation in various breast cancer cells and ultimately kills these cells by inducing apoptosis. We found that CDDO-Me-induced increases in intracellular Ca2+ levels, reflecting influx from the extracellular milieu, make a critical contribution to ER-derived vacuolation and subsequent cell death. In parallel with increasing Ca2+ levels, CDDO-Me markedly increased the generation of reactive oxygen species (ROS). Interestingly, there exists a reciprocal positive-regulatory loop between Ca2+ influx and ROS generation that triggers ER stress and ER dilation in response to CDDO-Me. In addition, CDDO-Me rapidly reduced the protein levels of c-FLIPL (cellular FLICE-inhibitory protein) and overexpression of c-FLIPL blocked CDDO-Me-induced cell death, but not vacuolation. These results suggest that c-FLIPL downregulation is a key contributor to CDDO-Me-induced apoptotic cell death, independent of ER-derived vacuolation. Taken together, our results show that ER-derived vacuolation via Ca2+ influx and ROS generation as well as caspase activation via c-FLIPL downregulation are responsible for the potent anticancer effects of CDDO-Me on breast cancer cells.

Enhancement of hippocampal mossy fiber activity in zinc deficiency and its influence on behavior.

PubMed

Takeda, Atsushi; Itoh, Hiromasa; Yamada, Kohei; Tamano, Haruna; Oku, Naoto

2008-10-01

The extracellular concentration of glutamate in the hippocampus is increased by hippocampal perfusion with CaEDTA, a membrane-impermeable zinc chelator, suggesting that the activity of glutamatergic neurons in the hippocampus are influenced by the extracellular concentrations of zinc. In the present study, the relationship between the extracellular concentrations of zinc and mossy fiber activity in the hippocampus was examined in mice and rats fed a zinc-deficient diet for 4 weeks. Timm's stain, by which histochemically reactive zinc in the presynaptic vesicles is detected, was attenuated in the hippocampus in zinc deficiency. The extracellular signal of ZnAF-2, a membrane-impermeable zinc indicator, was also lower in the hippocampal CA3, suggesting that the basal extracellular concentrations of zinc are lower maintained in zinc deficiency. To check mossy fiber activity after 4-week zinc deprivation, the decrease in the signal of FM4-64, an indicator of presynaptic activity (exocytosis), at mossy fiber synapses was measured under the condition of spontaneous depolarization. The decrease was significantly facilitated by zinc deficiency, suggesting that the basal exocytosis at mossy fiber synapses is enhanced by zinc deficiency. On the other hand, the increase in anxiety-like behavior was observed in the open-field test after 4-week zinc deprivation. The present study demonstrates that the decrease in the basal extracellular concentrations of zinc may be linked to the enhancement of the basal mossy fiber activity in zinc deficiency. This decrease seems to be also involved in neuropsychological behavior in zinc deficiency.

Reactive Oxygen Species on the Early Earth and Survival of Bacteria

NASA Technical Reports Server (NTRS)

Balk, Melikea; Mason, Paul; Stams, Alfons J. M.; Smidt, Hauke; Freund, Friedemann; Rothschild, Lynn

2011-01-01

An oxygen-rich atmosphere appears to have been a prerequisite for complex, multicellular life to evolve on Earth and possibly elsewhere in the Universe. However it remains unclear how free oxygen first became available on the early Earth. A potentially important, and as yet poorly constrained pathway, is the production of oxygen through the weathering of rocks and release into the near-surface environment. Reactive Oxygen Species (ROS), as precursors to molecular oxygen, are a key step in this process, and may have had a decisive impact on the evolution of life, present and past. ROS are generated from minerals in igneous rocks during hydrolysis of peroxy defects, which consist of pairs of oxygen anions oxidized to the valence state -1 and during (bio) transformations of iron sulphide minerals. ROS are produced and consumed by intracellular and extracellular reactions of Fe, Mn, C, N, and S species. We propose that, despite an overall reducing or neutral oxidation state of the macroenvironment and the absence of free O2 in the atmosphere, organisms on the early Earth had to cope with ROS in their microenvironments. They were thus under evolutionary pressure to develop enzymatic and other defences against the potentially dangerous, even lethal effects of oxygen and its derived ROS. Conversely it appears that microorganisms learned to take advantage of the enormous reactive potential and energy gain provided by nascent oxygen. We investigate how oxygen might be released through weathering. We test microorganisms in contact with rock surfaces and iron sulphides. We model bacteria such as Deionococcus radiodurans and Desulfotomaculum, Moorella and Bacillus species for their ability to grow or survive in the presence of ROS. We examine how early Life might have adapted to oxygen.

β-Phenylethylamine requires the dopamine transporter to increase extracellular dopamine in Caenorhabditis elegans dopaminergic neurons.

PubMed

Hossain, Murad; Wickramasekara, Rochelle N; Carvelli, Lucia

2014-07-01

β-Phenylethylamine (βPEA) is an endogenous amine that has been shown to increase the synaptic levels of dopamine (DA). A number of in vitro and behavioral studies suggest the dopamine transporter (DAT) plays a role in the effects generated by βPEA, however the mechanism through which βPEA affects DAT has not yet been elucidated. Here, we used Caenorhabditis (C.) elegans DAT (DAT-1) expressing LLC-pk1 cells and neuronal cultures to investigate whether the βPEA-induced increase of extracellular DA required DAT-1. Our data show that βPEA increases extracellular dopamine both in DAT-1 transfected cells and cultures of differentiated neurons. RTI-55, a cocaine homologue and DAT inhibitor, completely blocked the βPEA-induced effect in transfected cells. However in neuronal cultures, RTI-55 only partly inhibited the increase of extracellular DA generated by βPEA. These results suggest that βPEA requires DAT-1 and other, not yet identified proteins, to increase extracellular DA when tested in a native system. Furthermore, our results suggest that βPEA-induced increase of extracellular DA does not require functional monoamine vesicles as genetic ablation of the C. elegans homologue vesicular monoamine transporter, cat-1, did not compromise the ability of βPEA to increase extracellular DA. Finally, our electrophysiology data show that βPEA caused fast-rising and self-inactivating amperometric currents in a subset of wild-type DA neurons but not in neurons isolated from dat-1 knockout animals. Taken together, these data demonstrate that in both DA neurons and heterogeneous cultures of differentiated C. elegans neurons, βPEA releases cytoplasmic DA through DAT-1 to ultimately increase the extracellular concentration of DA. Copyright © 2013 Elsevier Ltd. All rights reserved.

β-phenylethylamine Requires the Dopamine Transporter to Increase Extracellular Dopamine in C. elegans Dopaminergic Neurons

PubMed Central

Hossain, Murad; Wickramasekara, Rochelle N.; Carvelli, Lucia

2013-01-01

β-phenylethylamine (βPEA) is an endogenous amine that has been shown to increase the synaptic levels of dopamine (DA). A number of in vitro and behavioral studies suggest the dopamine transporter (DAT) plays a role in the effects generated by βPEA, however the mechanism through which βPEA affects DAT has not yet been elucidated. Here, we used Caenorhabditis (C.) elegans DAT (DAT-1) expressing LLC-pk1 cells and neuronal cultures to investigate whether the βPEA-induced increase of extracellular DA required DAT-1. Our data show that βPEA increases extracellular dopamine both in DAT-1 transfected cells and cultures of differentiated neurons. RTI-55, a cocaine homologue and DAT inhibitor, completely blocked the βPEA-induced effect in transfected cells. However in neuronal cultures, RTI-55 only partly inhibited the increase of extracellular DA generated by βPEA. These results suggest that βPEA requires DAT-1 and other, not yet identified proteins, to increase extracellular DA when tested in a native system. Furthermore, our results suggest that βPEA-induced increase of extracellular DA does not require functional monoamine vesicles as genetic ablation of the C. elegans homologue vesicular monoamine transporter, cat-1, did not compromise the ability of βPEA to increase extracellular DA. Finally, our electrophysiology data show that βPEA caused fast-rising and self-inactivating amperometric currents in a subset of wild-type DA neurons but not in neurons isolated from dat-1 knockout animals. Taken together, these data demonstrate that in both DA neurons and heterogeneous cultures of differentiated C. elegans neurons, βPEA releases cytoplasmic DA through DAT-1 to ultimately increase the extracellular concentration of DA. PMID:24161617

Characterizing Myeloid Cell Activation in NF1 Vasculopathy

DTIC Science & Technology

2017-07-01

stimulation of its receptor (CCR2) and the generation of reactive oxygen species, which are generated in excessive quantities by neurofibromin-deficient...macrophages via monocyte chemotactic peptide-1 (MCP-1) stimulation of its receptor (CCR2) and the generation of reactive oxygen species, which are...neurofibromatosis; stenosis; aneurysm; MCP-1; CCR2; reactive oxygen species; superoxide; macrophages; monocytes; arteries; cardiovascular disease Major

Impact of plasma jet vacuum ultraviolet radiation on reactive oxygen species generation in bio-relevant liquids

NASA Astrophysics Data System (ADS)

Jablonowski, H.; Bussiahn, R.; Hammer, M. U.; Weltmann, K.-D.; von Woedtke, Th.; Reuter, S.

2015-12-01

Plasma medicine utilizes the combined interaction of plasma produced reactive components. These are reactive atoms, molecules, ions, metastable species, and radiation. Here, ultraviolet (UV, 100-400 nm) and, in particular, vacuum ultraviolet (VUV, 10-200 nm) radiation generated by an atmospheric pressure argon plasma jet were investigated regarding plasma emission, absorption in a humidified atmosphere and in solutions relevant for plasma medicine. The energy absorption was obtained for simple solutions like distilled water (dH2O) or ultrapure water and sodium chloride (NaCl) solution as well as for more complex ones, for example, Rosewell Park Memorial Institute (RPMI 1640) cell culture media. As moderate stable reactive oxygen species, hydrogen peroxide (H2O2) was studied. Highly reactive oxygen radicals, namely, superoxide anion (O2•-) and hydroxyl radicals (•OH), were investigated by the use of electron paramagnetic resonance spectroscopy. All species amounts were detected for three different treatment cases: Plasma jet generated VUV and UV radiation, plasma jet generated UV radiation without VUV part, and complete plasma jet including all reactive components additionally to VUV and UV radiation. It was found that a considerable amount of radicals are generated by the plasma generated photoemission. From the experiments, estimation on the low hazard potential of plasma generated VUV radiation is discussed.

Osteopontin expression and clinicopathologic correlation of oral hyperplastic reactive lesions: An institutional 6-year retrospective study.

PubMed

Narwal, Anjali; Bala, Shashi

2017-01-01

Reactive proliferations of oral cavity comprise pyogenic granuloma (PG), fibrous hyperplasia (FH), peripheral ossifying fibroma (POF), and peripheral giant-cell granuloma (PGCG). They often pose diagnostic challenges due to their overlapping clinical and histopathological features. This study was conducted to determine the frequency and clinicopathological correlation of reactive hyperplastic lesions in the oral cavity reported in our institute and compared it with other previous studies. Further evaluation of osteopontin (OPN) expression in normal gingival tissue and different types of focal reactive lesions was also done. Data of all reactive hyperplasias were retrieved, reviewed, and analyzed for age, gender, clinical presentation, and site of location. Presence and distribution of OPN were assessed using immunohistochemistry in these reactive lesions. Two hundred and forty-eight reactive lesions were comprised of FH (38%), PG (23%), POF (13%), and PGCG (7%). FH was more common in males (55%) whereas other reactive lesions were more in females (68%-73%). The most frequently involved site was gingiva (59%), and most common clinical presentation was sessile growth on gingiva. OPN expression was minimal in normal gingiva. Few cases of FH, PG, and all cases of POF showed positivity for OPN in inflammatory cells, stromal cells, extracellular matrix, and in calcifications. Reactive hyperplastic lesions of oral cavity are mucosal responses to chronic low-grade irritation caused by plaque, calculus, and any other irritant. It is helpful to know their frequency and presentation as their early identification enables accurate patient evaluation and management.

A novel approach to teach the generation of bioelectrical potentials from a descriptive and quantitative perspective.

PubMed

Rodriguez-Falces, Javier

2013-12-01

In electrophysiology studies, it is becoming increasingly common to explain experimental observations using both descriptive methods and quantitative approaches. However, some electrophysiological phenomena, such as the generation of extracellular potentials that results from the propagation of the excitation source along the muscle fiber, are difficult to describe and conceptualize. In addition, most traditional approaches aimed at describing extracellular potentials consist of complex mathematical machinery that gives no chance for physical interpretation. The aim of the present study is to present a new method to teach the formation of extracellular potentials around a muscle fiber from both a descriptive and quantitative perspective. The implementation of this method was tested through a written exam and a satisfaction survey. The new method enhanced the ability of students to visualize the generation of bioelectrical potentials. In addition, the new approach improved students' understanding of how changes in the fiber-to-electrode distance and in the shape of the excitation source are translated into changes in the extracellular potential. The survey results show that combining general principles of electrical fields with accurate graphic imagery gives students an intuitive, yet quantitative, feel for electrophysiological signals and enhances their motivation to continue their studies in the biomedical engineering field.

Effects of peptides on generation of reactive oxygen species in subcellular fractions of Drosophila melanogaster.

PubMed

Khavinson, V K; Myl'nikov, S V; Oparina, T I; Arutyunyan, A V

2001-07-01

We studied the effects of Epithalon (Ala-Glu-Asp-Gly) and Vilon (Lys-Glu) on free radical processes in highly inbred HA(+)line of Drosophila melanogaster. Vilon inhibited generation of reactive oxygen species in mitochondria, but stimulated this process in the cytosol. We found sex- and age-related differences in the generation of reactive oxygen species and cytosol antioxidant activity.

Low Reactive Level Laser Therapy for Mesenchymal Stromal Cells Therapies

PubMed Central

Kushibiki, Toshihiro; Hirasawa, Takeshi; Okawa, Shinpei; Ishihara, Miya

2015-01-01

Low reactive level laser therapy (LLLT) is mainly focused on the activation of intracellular or extracellular chromophore and the initiation of cellular signaling by using low power lasers. Over the past forty years, it was realized that the laser therapy had the potential to improve wound healing and reduce pain and inflammation. In recent years, the term LLLT has become widely recognized in the field of regenerative medicine. In this review, we will describe the mechanisms of action of LLLT at a cellular level and introduce the application to mesenchymal stem cells and mesenchymal stromal cells (MSCs) therapies. Finally, our recent research results that LLLT enhanced the MSCs differentiation to osteoblast will also be described. PMID:26273309

Imaging extracellular ATP with a genetically-encoded, ratiometric fluorescent sensor

PubMed Central

Conley, Jason M.

2017-01-01

Extracellular adenosine triphosphate (ATP) is a key purinergic signal that mediates cell-to-cell communication both within and between organ systems. We address the need for a robust and minimally invasive approach to measuring extracellular ATP by re-engineering the ATeam ATP sensor to be expressed on the cell surface. Using this approach, we image real-time changes in extracellular ATP levels with a sensor that is fully genetically-encoded and does not require an exogenous substrate. In addition, the sensor is ratiometric to allow for reliable quantitation of extracellular ATP fluxes. Using live-cell microscopy, we characterize sensor performance when expressed on cultured Neuro2A cells, and we measure both stimulated release of ATP and its clearance by ectonucleotidases. Thus, this proof-of-principle demonstrates a first-generation sensor to report extracellular ATP dynamics that may be useful for studying purinergic signaling in living specimens. PMID:29121644

A novel extracellular multicopper oxidase from Phanerochaete chrysosporium with ferroxidase activity

Treesearch

Luis F. Larrondo; Loreto Salas; Francisco Melo; Rafael Vicuna; Daniel Cullen

2003-01-01

Lignin degradation by the white rot basidiomycete Phanerochaete chrysosporium involves various extracellular oxidative enzymes, including lignin peroxidase, manganese peroxidase, and a peroxide-generating enzyme, glyoxal oxidase. Recent studies have suggested that laccases also may be produced by this fungus, but these conclusions have been controversial. We identified...

Acidosis induces reprogramming of cellular metabolism to mitigate oxidative stress

PubMed Central

2013-01-01

Background A variety of oncogenic and environmental factors alter tumor metabolism to serve the distinct cellular biosynthetic and bioenergetic needs present during oncogenesis. Extracellular acidosis is a common microenvironmental stress in solid tumors, but little is known about its metabolic influence, particularly when present in the absence of hypoxia. In order to characterize the extent of tumor cell metabolic adaptations to acidosis, we employed stable isotope tracers to examine how acidosis impacts glucose, glutamine, and palmitate metabolism in breast cancer cells exposed to extracellular acidosis. Results Acidosis increased both glutaminolysis and fatty acid β-oxidation, which contribute metabolic intermediates to drive the tricarboxylic acid cycle (TCA cycle) and ATP generation. Acidosis also led to a decoupling of glutaminolysis and novel glutathione (GSH) synthesis by repressing GCLC/GCLM expression. We further found that acidosis redirects glucose away from lactate production and towards the oxidative branch of the pentose phosphate pathway (PPP). These changes all serve to increase nicotinamide adenine dinucleotide phosphate (NADPH) production and counter the increase in reactive oxygen species (ROS) present under acidosis. The reduced novel GSH synthesis under acidosis may explain the increased demand for NADPH to recycle existing pools of GSH. Interestingly, acidosis also disconnected novel ribose synthesis from the oxidative PPP, seemingly to reroute PPP metabolites to the TCA cycle. Finally, we found that acidosis activates p53, which contributes to both the enhanced PPP and increased glutaminolysis, at least in part, through the induction of G6PD and GLS2 genes. Conclusions Acidosis alters the cellular metabolism of several major metabolites, which induces a significant degree of metabolic inflexibility. Cells exposed to acidosis largely rely upon mitochondrial metabolism for energy generation to the extent that metabolic intermediates are redirected away from several other critical metabolic processes, including ribose and glutathione synthesis. These alterations lead to both a decrease in cellular proliferation and increased sensitivity to ROS. Collectively, these data reveal a role for p53 in cellular metabolic reprogramming under acidosis, in order to permit increased bioenergetic capacity and ROS neutralization. Understanding the metabolic adaptations that cancer cells make under acidosis may present opportunities to generate anti-tumor therapeutic agents that are more tumor-specific. PMID:24359630

Rapamycin Protects Skin Fibroblasts from Ultraviolet B-Induced Photoaging by Suppressing the Production of Reactive Oxygen Species.

PubMed

Qin, Dengke; Ren, Runjian; Jia, Chuanlong; Lu, Yongzhou; Yang, Qingjian; Chen, Liang; Wu, Xinyuan; Zhu, Jingjing; Guo, Yu; Yang, Ping; Zhou, Yiqun; Zhu, Ningwen; Bi, Bo; Liu, Tianyi

2018-01-01

Ultraviolet B (UVB) irradiation alters multiple molecular pathways in the skin, thereby inducing skin photoaging. Murine dermal fibroblasts (MDFs) were subjected to a series of 4 sub-cytotoxic UVB doses (120 mJ/cm2), resulting in changes in cell shape, DNA damage, cell cycle arrest, extracellular matrix variations, reactive oxygen species (ROS) generation, and alterations in major intracellular antioxidant and cellular autophagy levels. Rapamycin (RAPA) is a new macrolide immunosuppressive agent that is primarily used in oncology, cardiology, and transplantation medicine and has been found to extend the lifespan of genetically heterogeneous mice. Several studies have shown that RAPA may have anti-aging effects in cells and organisms. Thus, in this study, we explored the effects and mechanisms of RAPA against the photoaging process using a well-established cellular photoaging model. We developed a stress-induced premature senescence (SIPS) model through repeated exposure of MDFs to ultraviolet B (UVB) irradiation. The cells were cultured in the absence or presence of RAPA for 48 h. Senescent phenotypes were assessed by examining cell viability, cell morphology, senescence-associated β-galactosidase (SA-β-gal) expression, cell cycle progression, intracellular ROS production, matrix metalloproteinase (MMP) synthesis and degradation, extracellular matrix (ECM) component protein expression, alterations in major intracellular antioxidant levels, and the cellular autophagy level. Compared with the UVB group, pretreatment with RAPA (5 µM) significantly decreased the staining intensity and percentage of SA-β-gal-positive cells and preserved the elongated cell shape. Moreover, cells pretreated with RAPA showed inhibition of the reduction in the type I collagen content by blocking the UVB-induced upregulation of MMP expression. RAPA also decreased photoaging cell cycle arrest and downregulated p53 and p21 expression. RAPA application significantly attenuated irradiation-induced ROS release by modulating intracellular antioxidants and increasing the autophagy level. Our study demonstrated that RAPA elicited oxidative damage in vitro by reducing ROS accumulation in photoaged fibroblasts. The anti-aging effect can be attributed to the maintenance of normal antioxidant and cellular autophagy levels. However, determination of the definitive mechanism requires further study. © 2018 The Author(s). Published by S. Karger AG, Basel.

Secure provision of reactive power ancillary services in competitive electricity markets

NASA Astrophysics Data System (ADS)

El-Samahy, Ismael

The research work presented in this thesis discusses various complex issues associated with reactive power management and pricing in the context of new operating paradigms in deregulated power systems, proposing appropriate policy solutions. An integrated two-level framework for reactive power management is set forth, which is both suitable for a competitive market and ensures a secure and reliable operation of the associated power system. The framework is generic in nature and can be adopted for any electricity market structure. The proposed hierarchical reactive power market structure comprises two stages: procurement of reactive power resources on a seasonal basis, and real-time reactive power dispatch. The main objective of the proposed framework is to provide appropriate reactive power support from service providers at least cost, while ensuring a secure operation of the power system. The proposed procurement procedure is based on a two-step optimization model. First, the marginal benefits of reactive power supply from each provider, with respect to system security, are obtained by solving a loadability-maximization problem subject to transmission security constraints imposed by voltage and thermal limits. Second, the selected set of generators is determined by solving an optimal power flow (OPF)-based auction. This auction maximizes a societal advantage function comprising generators' offers and their corresponding marginal benefits with respect to system security, and considering all transmission system constraints. The proposed procedure yields the selected set of generators and zonal price components, which would form the basis for seasonal contracts between the system operator and the selected reactive power service providers. The main objective of the proposed reactive power dispatch model is to minimize the total payment burden on the Independent System Operator (ISO), which is associated with reactive power dispatch. The real power generation is decoupled and assumed to be fixed during the reactive power dispatch procedures; however, the effect of reactive power on real power is considered in the model by calculating the required reduction in real power output of a generator due to an increase in its reactive power supply. In this case, real power generation is allowed to be rescheduled, within given limits, from the already dispatched levels obtained from the energy market clearing process. The proposed dispatch model achieves the main objective of an ISO in a competitive electricity market, which is to provide the required reactive power support from generators at least cost while ensuring a secure operation of the power system. The proposed reactive power procurement and dispatch models capture both the technical and economic aspects of power system operation in competitive electricity markets; however, from an optimization point of view, these models represent non-convex mixed integer non-linear programming (MINLP) problems due to the presence of binary variables associated with the different regions of reactive power operation in a synchronous generator. Such MINLP optimization problems are difficult to solve, especially for an actual power system. A novel Generator Reactive Power Classification (GRPC) algorithm is proposed in this thesis to address this issue, with the advantage of iteratively solving the optimization models as a series of non-linear programming (NLP) sub-problems. The proposed reactive power procurement and dispatch models are implemented and tested on the CIGRE 32-bus system, with several case studies that represent different practical operating scenarios. The developed models are also compared with other approaches for reactive power provision, and the results demonstrate the robustness and effectiveness of the proposed model. The results clearly reveal the main features of the proposed models for optimal provision of reactive power ancillary service, in order to suit the requirements of an ISO under today's stressed system conditions in a competitive market environment.

Extracellular Determinants of Anion Discrimination of the Cl−/H+ Antiporter Protein CLC-5*

PubMed Central

De Stefano, Silvia; Pusch, Michael; Zifarelli, Giovanni

2011-01-01

Mammalian CLC proteins comprise both Cl− channels and Cl−/H+ antiporters that carry out fundamental physiological tasks by transporting Cl− across plasma membrane and intracellular compartments. The NO3− over Cl− preference of a plant CLC transporter has been pinpointed to a conserved serine residue located at Scen and it is generally assumed that the other two binding sites of CLCs, Sext and Sin, do not substantially contribute to anion selectivity. Here we show for the Cl−/H+ antiporter CLC-5 that the conserved and extracellularly exposed Lys210 residue is critical to determine the anion specificity for transport activity. In particular, mutations that neutralize or invert the charge at this position reverse the NO3− over Cl− preference of WT CLC-5 at a concentration of 100 mm, but do not modify the coupling stoichiometry with H+. The importance of the electrical charge is shown by chemical modification of K210C with positively charged cysteine-reactive compounds that reintroduce the WT preference for Cl−. At saturating extracellular anion concentrations, neutralization of Lys210 is of little impact on the anion preference, suggesting an important role of Lys210 on the association rate of extracellular anions to Sext. PMID:21921031

Monitoring intra- and extracellular redox capacity of intact barley aleurone layers responding to phytohormones.

PubMed

Mark, Christina; Zór, Kinga; Heiskanen, Arto; Dufva, Martin; Emnéus, Jenny; Finnie, Christine

2016-12-15

Redox regulation is important for numerous processes in plant cells including abiotic stress, pathogen defence, tissue development, seed germination and programmed cell death. However, there are few methods allowing redox homeostasis to be addressed in whole plant cells, providing insight into the intact in vivo environment. An electrochemical redox assay that applies the menadione-ferricyanide double mediator is used to assess changes in the intracellular and extracellular redox environment in living aleurone layers of barley (Hordeum vulgare cv. Himalaya) grains, which respond to the phytohormones gibberellic acid and abscisic acid. Gibberellic acid is shown to elicit a mobilisation of electrons as detected by an increase in the reducing capacity of the aleurone layers. By taking advantage of the membrane-permeable menadione/menadiol redox pair to probe the membrane-impermeable ferricyanide/ferrocyanide redox pair, the mobilisation of electrons was dissected into an intracellular and an extracellular, plasma membrane-associated component. The intracellular and extracellular increases in reducing capacity were both suppressed when the aleurone layers were incubated with abscisic acid. By probing redox levels in intact plant tissue, the method provides a complementary approach to assays of reactive oxygen species and redox-related enzyme activities in tissue extracts. Copyright © 2016 Elsevier Inc. All rights reserved.

Targeting the extracellular matrix of ovarian cancer using functionalized, drug loaded lyophilisomes.

PubMed

van der Steen, Sophieke C H A; Raavé, René; Langerak, Sjoerd; van Houdt, Laurens; van Duijnhoven, Sander M J; van Lith, Sanne A M; Massuger, Leon F A G; Daamen, Willeke F; Leenders, William P; van Kuppevelt, Toin H

2017-04-01

Epithelial ovarian cancer is characterized by a high mortality rate and is in need for novel therapeutic avenues to improve patient outcome. The tumor's extracellular matrix ("stroma") offers new possibilities for targeted drug-delivery. Recently we identified highly sulfated chondroitin sulfate (CS-E) as a component abundantly present in the ovarian cancer extracellular matrix, and as a novel target for anti-cancer therapy. Here, we report on the functionalization of drug-loaded lyophilisomes (albumin-based biocapsules) to specifically target the stroma of ovarian carcinomas with the potential to eliminate cancer cells. To achieve specific targeting, we conjugated single chain antibodies reactive with CS-E to lyophilisomes using a two-step approach comprising sortase-mediated ligation and bioorthogonal click chemistry. Antibody-functionalized lyophilisomes specifically targeted the ovarian cancer stroma through CS-E. In a CS-E rich micro-environment in vitro lyophilisomes induced cell death by extracellular release of doxorubicin which localized to the nucleus. Immunohistochemistry identified CS-E rich stroma in a variety of solid tumors other than ovarian cancer, including breast, lung and colon cancer indicating the potential versatility of matrix therapy and the use of highly sulfated chondroitin sulfates in cancer stroma as a micro-environmental hook for targeted drug delivery. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Application of a clot-based assay to measure the procoagulant activity of stored allogeneic red blood cell concentrates

PubMed Central

Wannez, Adeline; Bailly, Nicolas; Alpan, Lutfiye; Gheldof, Damien; Douxfils, Jonathan; Deneys, Véronique; Bihin, Benoît; Chatelain, Bernard; Dogné, Jean-Michel; Chatelain, Christian; Mullier, François

2018-01-01

Background Thrombotic effects are possible complications of red blood cell transfusion. The generation and accumulation of procoagulant red blood cell extracellular vesicles during storage may play an important role in these thrombotic effects. The objective of this study was to assess the value of a simple phospholipid-dependent clot-based assay (STA®-Procoag-PPL) to estimate the procoagulant activity of stored red blood cells and changes in this activity during storage of the blood component. Materials and methods Extracellular vesicles from 12 red blood cell concentrates were isolated at 13 storage time-points and characterised by quantitative and functional methods: the degree of haemolysis (direct spectrophotometry), the quantification and determination of cellular origin (flow cytometry) and the procoagulant activity (thrombin generation and STA®-Procoag-PPL assays) were assessed. Results The mean clotting time of extracellular vesicles isolated from red blood cell concentrates decreased from 117.2±3.6 sec on the day of collection to 33.8±1.3 sec at the end of the storage period. This illustrates the phospholipid-dependent procoagulant activity of these extracellular vesicles, as confirmed by thrombin generation. Results of the peak of thrombin and the STA®-Procoag-PPL were well correlated (partial r=−0.41. p<0.001). In parallel, an exponential increase of the number of red blood cell-derived extracellular vesicles from 1,779/μL to 218,451/μL was observed. Discussion The STA®-Procoag-PPL is a potentially useful technique for assessing the procoagulant activity of a red blood cell concentrate. PMID:28287378

Impact of plasma jet vacuum ultraviolet radiation on reactive oxygen species generation in bio-relevant liquids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jablonowski, H.; Hammer, M. U.; Reuter, S.

Plasma medicine utilizes the combined interaction of plasma produced reactive components. These are reactive atoms, molecules, ions, metastable species, and radiation. Here, ultraviolet (UV, 100–400 nm) and, in particular, vacuum ultraviolet (VUV, 10–200 nm) radiation generated by an atmospheric pressure argon plasma jet were investigated regarding plasma emission, absorption in a humidified atmosphere and in solutions relevant for plasma medicine. The energy absorption was obtained for simple solutions like distilled water (dH{sub 2}O) or ultrapure water and sodium chloride (NaCl) solution as well as for more complex ones, for example, Rosewell Park Memorial Institute (RPMI 1640) cell culture media. As moderate stablemore » reactive oxygen species, hydrogen peroxide (H{sub 2}O{sub 2}) was studied. Highly reactive oxygen radicals, namely, superoxide anion (O{sub 2}{sup •−}) and hydroxyl radicals ({sup •}OH), were investigated by the use of electron paramagnetic resonance spectroscopy. All species amounts were detected for three different treatment cases: Plasma jet generated VUV and UV radiation, plasma jet generated UV radiation without VUV part, and complete plasma jet including all reactive components additionally to VUV and UV radiation. It was found that a considerable amount of radicals are generated by the plasma generated photoemission. From the experiments, estimation on the low hazard potential of plasma generated VUV radiation is discussed.« less

Evidence for the in vivo polymerization of ependymin: a brain extracellular glycoprotein.

PubMed

Shashoua, V E; Hesse, G W; Milinazzo, B

1990-07-09

Ependymin, a glycoprotein of the brain extracellular fluid, has been implicated in synaptic changes associated with the consolidation process of long-term memory formation and the activity-dependent sharpening of connections of regenerating optic nerve. In vitro experiments have demonstrated that ependymin has the capacity to form fibrous insoluble polymers (FIP) when the solvent Ca2+ concentration is reduced by the addition of EGTA. Such products, once formed, do not dissolve in 2% sodium dodecyl sulfate (SDS) in 5 M urea. This property was used to develop a method for isolating brain FIP. A reproducible quantity of FIP was found in goldfish and mouse brain. This was highly concentrated in the synaptosomal fraction and had identical immunoreactivity properties to FIP obtained by the polymerization of pure ependymin in vitro as well as a cross-reactivity to other protein components of the extracellular matrix such as fibronectin and laminin. Labeling studies with [35S]methionine showed that labeled FIP aggregates are synthesized in vivo and become associated with the synaptosomal fraction. A comparison of the amino acid sequence of ependymin with those for proteins of the extracellular matrix indicated that common sequences 5-6 amino acids long exist in the molecules. These homologies may explain why antibodies to fibronectin, laminin and tubulin can recognize the FIP prepared from pure ependymin. These results suggest that ependymin can polymerize in vivo to form FIP aggregates which have similar immunoreactivity properties to major components of the brain extracellular matrix.

Public antibodies to malaria antigens generated by two LAIR1 insertion modalities.

PubMed

Pieper, Kathrin; Tan, Joshua; Piccoli, Luca; Foglierini, Mathilde; Barbieri, Sonia; Chen, Yiwei; Silacci-Fregni, Chiara; Wolf, Tobias; Jarrossay, David; Anderle, Marica; Abdi, Abdirahman; Ndungu, Francis M; Doumbo, Ogobara K; Traore, Boubacar; Tran, Tuan M; Jongo, Said; Zenklusen, Isabelle; Crompton, Peter D; Daubenberger, Claudia; Bull, Peter C; Sallusto, Federica; Lanzavecchia, Antonio

2017-08-31

In two previously described donors, the extracellular domain of LAIR1, a collagen-binding inhibitory receptor encoded on chromosome 19 (ref. 1), was inserted between the V and DJ segments of an antibody. This insertion generated, through somatic mutations, broadly reactive antibodies against RIFINs, a type of variant antigen expressed on the surface of Plasmodium falciparum-infected erythrocytes. To investigate how frequently such antibodies are produced in response to malaria infection, we screened plasma from two large cohorts of individuals living in malaria-endemic regions. Here we report that 5-10% of malaria-exposed individuals, but none of the European blood donors tested, have high levels of LAIR1-containing antibodies that dominate the response to infected erythrocytes without conferring enhanced protection against febrile malaria. By analysing the antibody-producing B cell clones at the protein, cDNA and gDNA levels, we characterized additional LAIR1 insertions between the V and DJ segments and discovered a second insertion modality whereby the LAIR1 exon encoding the extracellular domain and flanking intronic sequences are inserted into the switch region. By exon shuffling, this mechanism leads to the production of bispecific antibodies in which the LAIR1 domain is precisely positioned at the elbow between the VH and CH1 domains. Additionally, in one donor the genomic DNA encoding the VH and CH1 domains was deleted, leading to the production of a camel-like LAIR1-containing antibody. Sequencing of the switch regions of memory B cells from European blood donors revealed frequent templated inserts originating from transcribed genes that, in rare cases, comprised exons with orientations and frames compatible with expression. These results reveal different modalities of LAIR1 insertion that lead to public and dominant antibodies against infected erythrocytes and suggest that insertion of templated DNA represents an additional mechanism of antibody diversification that can be selected in the immune response against pathogens and exploited for B cell engineering.

Perspectives for Developing New Tuberculosis Vaccines Derived from the Pathogenesis of Tuberculosis: I. Basic Principles, II. Preclinical Testing, and III. Clinical Testing

PubMed Central

Dannenberg, Arthur M.; Dey, Bappaditya

2013-01-01

Part I. Basic Principles. TB vaccines cannot prevent establishment of the infection. They can only prevent an early pulmonary tubercle from developing into clinical disease. A more effective new vaccine should optimize both cell-mediated immunity (CMI) and delayed-type hypersensitivity (DTH) better than any existing vaccine. The rabbit is the only laboratory animal in which all aspects of the human disease can be reproduced: namely, the prevention of most primary tubercles, the arrestment of most primary tubercles, the formation of the tubercle’s solid caseous center, the liquefaction of this center, the formation of cavities and the bronchial spread of the disease. In liquefied caseum, virulent tubercle bacilli can multiply extracellularly, especially in the liquefied caseum next to the inner wall of a cavity where oxygen is plentiful. The bacilli in liquefied caseum cannot be reached by the increased number of activated macrophages produced by TB vaccines. Therefore, new TB vaccines will have little or no effect on the extracellular bacillary growth within liquefied caseum. TB vaccines can only increase the host’s ability to stop the development of new TB lesions that arise from the bronchial spread of tubercle bacilli from the cavity to other parts of the lung. Therefore, effective TB vaccines do not prevent the reactivation of latent TB. Such vaccines only control (or reduce) the number of metastatic lesions that result after the primary TB lesion was reactivated by the liquefaction process. (Note: the large number of tubercle bacilli growing extracellularly in liquefied caseum gives rise to mutations that enable antimicrobial resistance—which is a major reason why TB still exists today). Part II. Preclinical Testing. The counting of grossly visible tubercles in the lungs of rabbits after the inhalation of virulent human-type tubercle bacilli is the most pertinent preclinical method to assess the efficacy of new TB vaccines (because an effective vaccine will stop the growth of developing tubercles before while they are still microscopic in size). Unfortunately, rabbits are rarely used in preclinical vaccine trials, despite their relative ease of handling and human-like response to this infection. Mice do not generate an effective DTH response, and guinea pigs do not generate an effective CMI response. Only the rabbits and most humans can establish the proper amount of DTH and CMI that is necessary to contain this infection. Therefore, rabbits should be included in all pre-clinical testing of new TB vaccines. New drugs (and/or immunological procedures) to reduce liquefaction and cavity formation are urgently needed. A simple intradermal way to select such drugs or procedures is described herein. Part III. Clinical Testing. Vaccine trials would be much more precise if the variations in human populations (listed herein) were taken into consideration. BCG and successful new TB vaccines should always increase host resistance to TB in naive subjects. This is a basic immunological principle. The efficacies of new and old TB vaccines are often not recognized, because these variations were not identified in the populations evaluated. PMID:26343850

Calcium Signaling Is Involved in Cadmium-Induced Neuronal Apoptosis via Induction of Reactive Oxygen Species and Activation of MAPK/mTOR Network

PubMed Central

Luo, Yan; Chen, Zi; Liu, Lei; Zhou, Hongyu; Chen, Wenxing; Shen, Tao; Han, Xiuzhen; Chen, Long; Huang, Shile

2011-01-01

Cadmium (Cd), a toxic environmental contaminant, induces oxidative stress, leading to neurodegenerative disorders. Recently we have demonstrated that Cd induces neuronal apoptosis in part by activation of the mitogen-activated protein kineses (MAPK) and mammalian target of rapamycin (mTOR) pathways. However, the underlying mechanism remains elusive. Here we show that Cd elevated intracellular calcium ion ([Ca2+]i) level in PC12, SH-SY5Y cells and primary murine neurons. BAPTA/AM, an intracellular Ca2+ chelator, abolished Cd-induced [Ca2+]i elevation, and blocked Cd activation of MAKPs including extracellular signal-regulated kinase 1/2 (Erk1/2), c-Jun N-terminal kinase (JNK) and p38, and mTOR-mediated signaling pathways, as well as cell death. Pretreatment with the extracellular Ca2+ chelator EGTA also prevented Cd-induced [Ca2+]i elevation, MAPK/mTOR activation, as well as cell death, suggesting that Cd-induced extracellular Ca2+ influx plays a critical role in contributing to neuronal apoptosis. In addition, calmodulin (CaM) antagonist trifluoperazine (TFP) or silencing CaM attenuated the effects of Cd on MAPK/mTOR activation and cell death. Furthermore, Cd-induced [Ca2+]i elevation or CaM activation resulted in induction of reactive oxygen species (ROS). Pretreatment with BAPTA/AM, EGTA or TFP attenuated Cd-induced ROS and cleavage of caspase-3 in the neuronal cells. Our findings indicate that Cd elevates [Ca2+]i, which induces ROS and activates MAPK and mTOR pathways, leading to neuronal apoptosis. The results suggest that regulation of Cd-disrupted [Ca2+]i homeostasis may be a new strategy for prevention of Cd-induced neurodegenerative diseases. PMID:21544200

Behavioral Response Generation and Selection of Rejected-Reactive Aggressive, Rejected-Nonaggressive, and Average Status Children.

ERIC Educational Resources Information Center

Wood, C. Nannette; Gross, Alan M.

2002-01-01

Examines response decision processes of rejected-reactive aggressive, rejected-nonaggressive and average children in terms of the presence or absence of behavioral response alternatives. Congruent with previous research, rejected-reactive aggressive children made significantly more hostile attributions and generated a higher number of aggressive…

Controlling Material Reactivity Using Architecture

DOE PAGES

Sullivan, Kyle T.; Zhu, Cheng; Duoss, Eric B.; ...

2015-12-16

3D-printing methods are used to generate reactive material architectures. We observed several geometric parameters in order to influence the resultant flame propagation velocity, indicating that the architecture can be utilized to control reactivity. Two different architectures, channels and hurdles, are generated, and thin films of thermite are deposited onto the surface. Additionally, the architecture offers a route to control, at will, the energy release rate in reactive composite materials.

Ischemic postconditioning: from receptor to end-effector.

PubMed

Cohen, Michael V; Downey, James M

2011-03-01

Ischemic preconditioning, a robust cardioprotective intervention, has limited clinical efficacy because it must be initiated before myocardial ischemia. Conversely, ischemic postconditioning, repeated brief reocclusions of a coronary artery after release of prolonged coronary occlusion, provides cardioprotection in clinically feasible settings, that is, coronary angioplasty. Ischemic postconditioning's signaling is being investigated to identify pharmacological triggers that could be used without angioplasty. In initial minutes of reperfusion H(+) washes out of previously ischemic cells. pH rises enabling mitochondrial permeability transition pores (MPTPs) to form leading to cessation of ATP production and cell necrosis. Coronary reocclusions maintain sufficient acidosis to keep MPTP closed while signaling is initiated that can generate endogenous antagonists of MPTP formation even after cellular pH normalizes. Reintroduction of oxygen generates reactive oxygen species that activate protein kinase C to increase sensitivity of adenosine A(2b) receptors allowing adenosine released from ischemic cells to bind leading to activation of phosphatidylinositol 3-kinase and extracellular signal-regulated kinase 1/2. Phosphatidylinositol 3-kinase activation results in phosphorylation of Akt promoting activation of nitric oxide synthase and nitric oxide production, which inhibits glycogen synthase kinase-3β, perhaps the final cytosolic signaling step before inhibition of MPTP formation. Interference with MPTP may be the final step that determines cell salvage.

Mechanism of short-term ERK activation by electromagnetic fields at mobile phone frequencies.

PubMed

Friedman, Joseph; Kraus, Sarah; Hauptman, Yirmi; Schiff, Yoni; Seger, Rony

2007-08-01

The exposure to non-thermal microwave electromagnetic fields generated by mobile phones affects the expression of many proteins. This effect on transcription and protein stability can be mediated by the MAPK (mitogen-activated protein kinase) cascades, which serve as central signalling pathways and govern essentially all stimulated cellular processes. Indeed, long-term exposure of cells to mobile phone irradiation results in the activation of p38 as well as the ERK (extracellular-signal-regulated kinase) MAPKs. In the present study, we have studied the immediate effect of irradiation on the MAPK cascades, and found that ERKs, but not stress-related MAPKs, are rapidly activated in response to various frequencies and intensities. Using signalling inhibitors, we delineated the mechanism that is involved in this activation. We found that the first step is mediated in the plasma membrane by NADH oxidase, which rapidly generates ROS (reactive oxygen species). These ROS then directly stimulate MMPs (matrix metalloproteinases) and allow them to cleave and release Hb-EGF [heparin-binding EGF (epidermal growth factor)]. This secreted factor activates the EGF receptor, which in turn further activates the ERK cascade. Thus this study demonstrates for the first time a detailed molecular mechanism by which electromagnetic irradiation from mobile phones induces the activation of the ERK cascade and thereby induces transcription and other cellular processes.

Silver Nanoparticles: Two-Faced Neuronal Differentiation-Inducing Material in Neuroblastoma (SH-SY5Y) Cells.

PubMed

Abdal Dayem, Ahmed; Lee, Soo Bin; Choi, Hye Yeon; Cho, Ssang-Goo

2018-05-15

We have previously demonstrated the potential of biologically synthesized silver nanoparticles (AgNP) in the induction of neuronal differentiation of human neuroblastoma, SH-SY5Y cells; we aimed herein to unveil its molecular mechanism in comparison to the well-known neuronal differentiation-inducing agent, all-trans-retinoic acid (RA). AgNP-treated SH-SY5Y cells showed significantly higher reactive oxygen species (ROS) generation, stronger mitochondrial membrane depolarization, lower dual-specificity phosphatase expression, higher extracellular-signal-regulated kinase (ERK) phosphorylation, lower AKT phosphorylation, and lower expression of the genes encoding the antioxidant enzymes than RA-treated cells. Notably, pretreatment with N -acetyl-l-cysteine significantly abolished AgNP-induced neuronal differentiation, but not in that induced by RA. ERK inhibition, but not AKT inhibition, suppresses neurite growth that is induced by AgNP. Taken together, our results uncover the pivotal contribution of ROS in the AgNP-induced neuronal differentiation mechanism, which is different from that of RA. However, the negative consequence of AgNP-induced neurite growth may be high ROS generation and the downregulation of the expression of the genes encoding the antioxidant enzymes, which prompts the future consideration and an in-depth study of the application of AgNP-differentiated cells in neurodegenerative disease therapy.

Novel mutants of Erwinia carotovora subsp. carotovora defective in the production of plant cell wall degrading enzymes generated by Mu transpososome-mediated insertion mutagenesis.

PubMed

Laasik, Eve; Ojarand, Merli; Pajunen, Maria; Savilahti, Harri; Mäe, Andres

2005-02-01

As in Erwinia carotovora subsp. carotovora the regulation details of the main virulence factors, encoding extracellular enzymes that degrade the plant cell wall, is only rudimentally understood, we performed a genetic screen to identify novel candidate genes involved in the process. Initially, we used Mu transpososome-mediated mutagenesis approach to generate a comprehensive transposon insertion mutant library of ca. 10000 clones and screened the clones for the loss of extracellular enzyme production. Extracellular enzymes production was abolished by mutations in the chromosomal helEcc, trkAEcc yheLEcc, glsEcc, igaAEcc and cysQEcc genes. The findings reported here demonstrate that we have isolated six new representatives that belong to the pool of genes modulating the production of virulence factors in E. carotovora.

Chemically reactive species in liquids generated by atmospheric-pressure plasmas and their roles in plasma medicine

NASA Astrophysics Data System (ADS)

Hamaguchi, Satoshi

2013-07-01

Plasmas whose gas temperatures are close to room temperature may be generated in ambient air or a gas at atmospheric pressure with the use of low-frequency high voltage or low-power radio-frequency (RF) or microwave power applied to electrodes. Such plasmas can serve as a powerful source of free radicals and/or chemically reactive species that arise from atoms and molecules of the ambient gas. Recently use of such plasmas for medical purposes has attracted much attention as they can be implemented in possible medical devices that can cause blood coagulation, heal wounds, facilitate angiogenesis, sterilize surgical devices as well as living tissues without harming healthy cells, and selectively inactivate cancer cells. Especially of interest among reactive species generated by atmospheric-pressure plasmas (APP) are reactive oxygen species (ROS) and reactive nitrogen species (RNS) that are generated in liquid phase. Since most living tissues and cells are immersed in liquids (such as blood or culture media), reactive species generated by APPs in the gas phase are transported to the liquid phase and possibly converted to different types of reactive species therein before causing some influence on the tissues or cells. In this study, the rate equations are solved to evaluate concentrations of various reactive species in pure water that are originated by plasma reactions in atmosphere and possible effects of such species (including ROS/RNS) on living tissues and cells are discussed.

Copper radical oxidases and related extracellular oxidoreductases of wood-decay Agaricomycetes

Treesearch

Phil Kersten; Dan Cullen

2014-01-01

Extracellular peroxide generation, a key component of oxidative lignocellulose degradation, has been attributed to various enzymes including the copper radical oxidases. Encoded by a family of structurally related sequences, the genes are widely distributed among wood decay fungi including three recently completed polypore genomes. In all cases, core catalytic residues...

Increases in extracellular zinc in the amygdala in acquisition and recall of fear experience and their roles in response to fear.

PubMed

Takeda, A; Tamano, H; Imano, S; Oku, N

2010-07-14

The amygdala is enriched with histochemically reactive zinc, which is dynamically coupled with neuronal activity and co-released with glutamate. The dynamics of the zinc in the amygdala was analyzed in rats, which were subjected to inescapable stress, to understand the role of the zinc in emotional behavior. In the communication box, two rats were subjected to foot shock stress and anxiety stress experiencing emotional responses of foot-shocked rat under amygdalar perfusion. Extracellular zinc was increased by foot shock stress, while decreased by anxiety stress, suggesting that the differential changes in extracellular zinc are associated with emotional behavior. In rats conditioned with foot shock, furthermore, extracellular zinc was increased again in the recall of fear (foot shock) in the same box without foot shock. When this recall was performed under perfusion with CaEDTA, a membrane-impermeable zinc chelator, to examine the role of the increase in extracellular zinc, the time of freezing behavior was more increased, suggesting that zinc released in the lateral amygdala during the recall of fear participates in freezing behavior. To examine the role of the increase in extracellular zinc during fear conditioning, fear conditioning was also performed under perfusion with CaEDTA. The time of freezing behavior was more increased in the contextual recall, suggesting that zinc released in the lateral nucleus during fear conditioning also participates in freezing behavior in the recall. In brain slice experiment, CaEDTA enhanced presynaptic activity (exocytosis) in the lateral nucleus after activation of the entorhinal cortex. The present paper demonstrates that zinc released in the lateral amygdala may participate in emotional behavior in response to fear. Copyright 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

A comprehensive approach to reactive power scheduling in restructured power systems

NASA Astrophysics Data System (ADS)

Shukla, Meera

Financial constraints, regulatory pressure, and need for more economical power transfers have increased the loading of interconnected transmission systems. As a consequence, power systems have been operated close to their maximum power transfer capability limits, making the system more vulnerable to voltage instability events. The problem of voltage collapse characterized by a severe local voltage depression is generally believed to be associated with inadequate VAr support at key buses. The goal of reactive power planning is to maintain a high level of voltage security, through installation of properly sized and located reactive sources and their optimal scheduling. In case of vertically-operated power systems, the reactive requirement of the system is normally satisfied by using all of its reactive sources. But in case of different scenarios of restructured power systems, one may consider a fixed amount of exchange of reactive power through tie lines. Reviewed literature suggests a need for optimal scheduling of reactive power generation for fixed inter area reactive power exchange. The present work proposed a novel approach for reactive power source placement and a novel approach for its scheduling. The VAr source placement technique was based on the property of system connectivity. This is followed by development of optimal reactive power dispatch formulation which facilitated fixed inter area tie line reactive power exchange. This formulation used a Line Flow-Based (LFB) model of power flow analysis. The formulation determined the generation schedule for fixed inter area tie line reactive power exchange. Different operating scenarios were studied to analyze the impact of VAr management approach for vertically operated and restructured power systems. The system loadability, losses, generation and the cost of generation were the performance measures to study the impact of VAr management strategy. The novel approach was demonstrated on IEEE 30 bus system.

Murine Macrophages Use Oxygen- and Nitric Oxide-Dependent Mechanisms To Synthesize S-Nitroso-Albumin and To Kill Extracellular Trypanosomes

PubMed Central

Gobert, Alain P.; Semballa, Silla; Daulouede, Sylvie; Lesthelle, Sophie; Taxile, Murielle; Veyret, Bernard; Vincendeau, Philippe

1998-01-01

Reactive nitrogen intermediates were synthesized spontaneously in cultures of macrophages from Trypanosoma brucei brucei-infected mice by an inducible nitric oxide (NO) synthase. This was inhibited by the addition of nitro-l-arginine. In this paper, we report the kinetics of the fixation of macrophage-derived NO on bovine serum albumin by using an enzyme-linked immunosorbent assay. S nitrosylation was confirmed by the Saville reaction, using mercuric chloride. It is known that reactive oxygen intermediates (ROI) are also synthesized by stimulated macrophages. The fact that NO is able to bind cysteine only under aerobic conditions led us to investigate the role of macrophage-derived ROI in the formation of S-nitrosylated proteins by activated macrophages. The immunoenzymatic signal decreased by 66 and 30% when superoxide dismutase and catalase, respectively, were added to the culture medium of macrophages from infected mice. In addition, the decrease in S-nitrosylated albumin formation correlated with the protection of extracellular trypanosomes from the cytostatic and cytotoxic activity of NO. Melatonin, a hydroxyl radical scavenger resulting from the decomposition of peroxynitrous acid, had no effect. All these data support the concept that an interaction between NO and ROI promoted the production of S-nitroso-albumin by activated macrophages from infected mice. PMID:9712749

Osteopontin expression and clinicopathologic correlation of oral hyperplastic reactive lesions: An institutional 6-year retrospective study

PubMed Central

Narwal, Anjali; Bala, Shashi

2017-01-01

Background and Objective: Reactive proliferations of oral cavity comprise pyogenic granuloma (PG), fibrous hyperplasia (FH), peripheral ossifying fibroma (POF), and peripheral giant-cell granuloma (PGCG). They often pose diagnostic challenges due to their overlapping clinical and histopathological features. This study was conducted to determine the frequency and clinicopathological correlation of reactive hyperplastic lesions in the oral cavity reported in our institute and compared it with other previous studies. Further evaluation of osteopontin (OPN) expression in normal gingival tissue and different types of focal reactive lesions was also done. Materials and Methods: Data of all reactive hyperplasias were retrieved, reviewed, and analyzed for age, gender, clinical presentation, and site of location. Presence and distribution of OPN were assessed using immunohistochemistry in these reactive lesions. Results: Two hundred and forty-eight reactive lesions were comprised of FH (38%), PG (23%), POF (13%), and PGCG (7%). FH was more common in males (55%) whereas other reactive lesions were more in females (68%–73%). The most frequently involved site was gingiva (59%), and most common clinical presentation was sessile growth on gingiva. OPN expression was minimal in normal gingiva. Few cases of FH, PG, and all cases of POF showed positivity for OPN in inflammatory cells, stromal cells, extracellular matrix, and in calcifications. Conclusion: Reactive hyperplastic lesions of oral cavity are mucosal responses to chronic low-grade irritation caused by plaque, calculus, and any other irritant. It is helpful to know their frequency and presentation as their early identification enables accurate patient evaluation and management. PMID:29391712

Mouse embryonic stem cell culture for generation of three-dimensional retinal and cortical tissues.

PubMed

Eiraku, Mototsugu; Sasai, Yoshiki

2011-12-15

Generation of compound tissues with complex structures is a major challenge in cell biology. In this article, we describe a protocol for mouse embryonic stem cell (ESC) culture for in vitro generation of three-dimensional retinal tissue, comparing it with the culture protocol for cortical tissue generation. Dissociated ESCs are reaggregated in a 96-well plate with reduced cell-plate adhesion and cultured as floating aggregates. Retinal epithelium is efficiently generated when ESC aggregates are cultured in serum-free medium containing extracellular matrix proteins, spontaneously forming hemispherical vesicles and then progressively transforming into a shape reminiscent of the embryonic optic cup in 9-10 d. In long-term culture, the ESC-derived optic cup generates a fully stratified retinal tissue consisting of all major neural retinal components. In contrast, the cortical differentiation culture can be started without exogenous extracellular matrix proteins, and it generates stratified cortical epithelia consisting of four distinct layers in 13 d.

Characterization and isolation of an extracellular serine protease from the tomato pathogen Colletotrichum coccodes, and it's role in pathogenicity

USGS Publications Warehouse

Redman, Regina S.; Rodriguez, Rusty J.

2002-01-01

Extracellular enzymes play an important role in the pathogenicity and virulence of phytopathogenic fungi. Several isolates of Colletotrichum coccodes causal agent of anthracnose on tomato, were screened to determine the relationship between protease activity and virulence. A direct relationship was observed between extracellular protease activity and the induction of disease symptoms of fruit and mortality in plants. Isolate Cc155 exhibited the highest protease activity after five days of growth in protease induction medium and produced an extracellular serine protease (sp78) that was 78 kDa, auto-degradative, glucose repressible, and non-glycosylated. To determine the role of sp78 in pathogenicity, a UV-induced extracellular protease deficient mutant (np155) was generated from the wildtype isolate Cc155. Np155 maintained growth rates comparable to Cc155 and produced wildtype levels of extracellular cellulase but did not produce extracellular protease. Unlike Cc155, np155 caused no disease symptoms on tomato fruit and 0% mortality on tomato seedlings. These results suggest that extracellular protease activity is required for pathogenicity and virulence of C. coccodes and that the elimination of protease activity transforms a virulent pathogen to a non-pathogenic endophyte.

Characterization and isolation of an extracellular serine protease from the tomato pathogen Colletotrichum coccodes, and it's role in pathogenicity

USGS Publications Warehouse

Redman, R.S.; Rodriguez, R.J.

2002-01-01

Extracellular enzymes play an important role in the pathogenicity and virulence of phytopathogenic fungi. Several isolates of Colletotrichum coccodes, causal agent of anthracnose on tomato, were screened to determine the relationship between protease activity and virulence. A direct relationship was observed between extracellular protease activity and the induction of disease symptoms of fruit and mortality in plants. Isolate Cc155 exhibited the highest protease activity after five days of growth in protease induction medium and produced an extracellular serine protease (sp78) that was 78 kDa, auto-degradative, glucose repressible, and non-glycosylated. To determine the role of sp78 in pathogenicity, a uv-induced extracellular protease deficient mutant (np155) was generated from the wildtype isolate Cc155. Np155 maintained growth rates comparable to Cc155 and produced wildtype levels of extracellular cellulase but did not produce extracellular protease. Unlike Cc155, np155 caused no disease symptoms on tomato fruit and 0% mortality on tomato seedlings. These results suggest that extracellular protease activity is required for pathogenicity and virulence of C. coccodes, and that the elimination of protease activity transforms a virulent pathogen to a non-pathogenic endophyte.

Formation of hydrogen peroxide in cell culture media by apple polyphenols and its effect on antioxidant biomarkers in the colon cell line HT-29.

PubMed

Bellion, Phillip; Olk, Melanie; Will, Frank; Dietrich, Helmut; Baum, Matthias; Eisenbrand, Gerhard; Janzowski, Christine

2009-10-01

Beneficial health effects of diets containing fruits have partly been attributed to polyphenols which display a spectrum of bioactive effects, including antioxidant activity. However, polyphenols can also exert prooxidative effects in vitro. In this study, polyphenol-mediated hydrogen peroxide (H(2)O(2)) formation was determined after incubation of apple juice extracts (AEs) and polyphenols in cell culture media. Effects of extracellular H(2)O(2 )on total glutathione (tGSH; =GSH + GSSG) and cellular reactive oxygen species (ROS) level of HT-29 cells were studied by coincubation +/- catalase (CAT). AEs ( > or =30 microg/mL) significantly generated H(2)O(2) in DMEM, depending on their composition. Similarly, H(2)O(2) was measured for individual apple polyphenols/degradation products (phenolic acids > epicatechin, flavonols > dihydrochalcones). Highest concentrations were generated by compounds bearing the o-catechol moiety. H(2)O(2) formation was found to be pH dependent; addition of CAT caused a complete decomposition of H(2)O(2) whereas superoxide dismutase was less/not effective. At incubation of HT-29 cells with quercetin (1-100 microM), generated H(2)O(2) slightly contributed to antioxidant cell protection by modulation of tGSH- and ROS-level. In conclusion, H(2)O(2) generation in vitro by polyphenols has to be taken into consideration when interpreting results of such cell culture experiments. Unphysiologically high polyphenol concentrations, favoring substantial H(2)O(2 )formation, are not expected to be met in vivo, even under conditions of high end nutritional uptake.

Extracellular ATP Acts on Jasmonate Signaling to Reinforce Plant Defense.

PubMed

Tripathi, Diwaker; Zhang, Tong; Koo, Abraham J; Stacey, Gary; Tanaka, Kiwamu

2018-01-01

Damaged cells send various signals to stimulate defense responses. Recent identification and genetic studies of the plant purinoceptor, P2K1 (also known as DORN1), have demonstrated that extracellular ATP is a signal involved in plant stress responses, including wounding, perhaps to evoke plant defense. However, it remains largely unknown how extracellular ATP induces plant defense responses. Here, we demonstrate that extracellular ATP induces plant defense mediated through activation of the intracellular signaling of jasmonate (JA), a well-characterized defense hormone. In Arabidopsis ( Arabidopsis thaliana ) leaves, ATP pretreatment induced resistance against the necrotrophic fungus, Botrytis cinerea The induced resistance was enhanced in the P2K1 receptor overexpression line, but reduced in the receptor mutant, dorn1 - 3 Mining the transcriptome data revealed that ATP induces a set of JA-induced genes. In addition, the P2K1-associated coexpression network contains defense-related genes, including those encoding jasmonate ZIM-domain (JAZ) proteins, which play key roles as repressors of JA signaling. We examined whether extracellular ATP impacts the stability of JAZ1 in Arabidopsis. The results showed that the JAZ1 stability decreased in response to ATP addition in a proteasome-dependent manner. This reduction required intracellular signaling via second messengers-cytosolic calcium, reactive oxygen species, and nitric oxide. Interestingly, the ATP-induced JAZ1 degradation was attenuated in the JA receptor mutant, coi1 , but not in the JA biosynthesis mutant, aos , or upon addition of JA biosynthesis inhibitors. Immunoprecipitation analysis demonstrated that ATP increases the interaction between COI1 and JAZ1, suggesting direct cross talk between extracellular ATP and JA in intracellular signaling events. Taken together, these results suggest that extracellular ATP signaling directly impacts the JA signaling pathway to maximize plant defense responses. © 2018 American Society of Plant Biologists. All Rights Reserved.

Quantitative iTRAQ-based secretome analysis reveals species-specific and temporal shifts in carbon utilization strategies among manganese(II)-oxidizing Ascomycete fungi

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zeiner, Carolyn A.; Purvine, Samuel O.; Zink, Erika M.

Fungi generate a wide range of extracellular hydrolytic and oxidative enzymes and reactive metabolites, collectively known as the secretome, that synergistically drive plant litter decomposition in the environment. While secretome studies of model organisms have greatly expanded our knowledge of these enzymes, few have extended secretome characterization to environmental isolates or directly compared temporal patterns of enzyme utilization among diverse species. Thus, the mechanisms of carbon (C) degradation by many ubiquitous soil fungi remain poorly understood. Here we use a combination of iTRAQ proteomics and custom bioinformatic analyses to compare the protein composition of the secretomes of four manganese(II)-oxidizing Ascomycetemore » fungi over a three-week time course. We demonstrate that although the fungi produce a similar suite of extracellular enzymes, they exhibit striking differences in the regulation of these enzymes among species and over time, revealing species-specific and temporal shifts in C utilization strategies as they degrade the same substrate. Specifically, our findings suggest that Paraconiothyrium sporulosum AP3s5-JAC2a and Alternaria alternata SRC1lrK2f employ sequential enzyme secretion patterns concomitant with decreasing resource availability, Stagonospora sp. SRC1lsM3a preferentially degrades proteinaceous substrate before switching to carbohydrates, and Pyrenochaeta sp. DS3sAY3a utilizes primarily peptidases to aggressively attack carbon sources in a concentrated burst. This work highlights the diversity of operative metabolic strategies among cellulose-degrading Ascomycetes and enhances our understanding of their role in C turnover in the environment.« less

Controlling Material Reactivity Using Architecture.

PubMed

Sullivan, Kyle T; Zhu, Cheng; Duoss, Eric B; Gash, Alexander E; Kolesky, David B; Kuntz, Joshua D; Lewis, Jennifer A; Spadaccini, Christopher M

2016-03-09

3D-printing methods are used to generate reactive material architectures. Several geometric parameters are observed to influence the resultant flame propagation velocity, indicating that the architecture can be utilized to control reactivity. Two different architectures, channels and hurdles, are generated, and thin films of thermite are deposited onto the surface. The architecture offers an additional route to control, at will, the energy release rate in reactive composite materials. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Oxidative Stressors Modify the Response of Streptococcus mutans to Its Competence Signal Peptides.

PubMed

De Furio, Matthew; Ahn, Sang Joon; Burne, Robert A; Hagen, Stephen J

2017-11-15

The dental caries pathogen Streptococcus mutans is continually exposed to several types of stress in the oral biofilm environment. Oxidative stress generated by reactive oxygen species has a major impact on the establishment, persistence, and virulence of S. mutans Here, we combined fluorescent reporter-promoter fusions with single-cell imaging to study the effects of reactive oxygen species on activation of genetic competence in S. mutans Exposure to paraquat, which generates superoxide anion, produced a qualitatively different effect on activation of expression of the gene for the master competence regulator, ComX, than did treatment with hydrogen peroxide (H 2 O 2 ), which can yield hydroxyl radical. Paraquat suppressed peptide-mediated induction of comX in a progressive and cumulative fashion, whereas the response to H 2 O 2 displayed a strong threshold behavior. Low concentrations of H 2 O 2 had little effect on induction of comX or the bacteriocin gene cipB , but expression of these genes declined sharply if extracellular H 2 O 2 exceeded a threshold concentration. These effects were not due to decreased reporter gene fluorescence. Two different threshold concentrations were observed in the response to H 2 O 2 , depending on the gene promoter that was analyzed and the pathway by which the competence regulon was stimulated. The results show that paraquat and H 2 O 2 affect the S. mutans competence signaling pathway differently, and that some portions of the competence signaling pathway are more sensitive to oxidative stress than others. IMPORTANCE Streptococcus mutans inhabits the oral biofilm, where it plays an important role in the development of dental caries. Environmental stresses such as oxidative stress influence the growth of S. mutans and its important virulence-associated behaviors, such as genetic competence. S. mutans competence development is a complex behavior that involves two different signaling peptides and can exhibit cell-to-cell heterogeneity. Although oxidative stress is known to influence S. mutans competence, it is not understood how oxidative stress interacts with the peptide signaling or affects heterogeneity. In this study, we used fluorescent reporters to probe the effect of reactive oxygen species on competence signaling at the single-cell level. Our data show that different reactive oxygen species have different effects on S. mutans competence, and that some portions of the signaling pathway are more acutely sensitive to oxidative stress than others. Copyright © 2017 American Society for Microbiology.

Cold atmospheric plasma (CAP), a novel physicochemical source, induces neural differentiation through cross-talk between the specific RONS cascade and Trk/Ras/ERK signaling pathway.

PubMed

Jang, Ja-Young; Hong, Young June; Lim, Junsup; Choi, Jin Sung; Choi, Eun Ha; Kang, Seongman; Rhim, Hyangshuk

2018-02-01

Plasma, formed by ionization of gas molecules or atoms, is the most abundant form of matter and consists of highly reactive physicochemical species. In the physics and chemistry fields, plasma has been extensively studied; however, the exact action mechanisms of plasma on biological systems, including cells and humans, are not well known. Recent evidence suggests that cold atmospheric plasma (CAP), which refers to plasma used in the biomedical field, may regulate diverse cellular processes, including neural differentiation. However, the mechanism by which these physicochemical signals, elicited by reactive oxygen and nitrogen species (RONS), are transmitted to biological system remains elusive. In this study, we elucidated the physicochemical and biological (PCB) connection between the CAP cascade and Trk/Ras/ERK signaling pathway, which resulted in neural differentiation. Excited atomic oxygen in the plasma phase led to the formation of RONS in the PCB network, which then interacted with reactive atoms in the extracellular liquid phase to form nitric oxide (NO). Production of large amounts of superoxide radical (O 2 - ) in the mitochondria of cells exposed to CAP demonstrated that extracellular NO induced the reversible inhibition of mitochondrial complex IV. We also demonstrated that cytosolic hydrogen peroxide, formed by O 2 - dismutation, act as an intracellular messenger to specifically activate the Trk/Ras/ERK signaling pathway. This study is the first to elucidate the mechanism linking physicochemical signals from the CAP cascade to the intracellular neural differentiation signaling pathway, providing physical, chemical and biological insights into the development of therapeutic techniques to treat neurological diseases. Copyright © 2017 Elsevier Ltd. All rights reserved.

Well-known surface and extracellular antigens of pathogenic microorganisms among the immunodominant proteins of the infectious microalgae Prototheca zopfii

PubMed Central

Irrgang, Alexandra; Murugaiyan, Jayaseelan; Weise, Christoph; Azab, Walid; Roesler, Uwe

2015-01-01

Microalgae of the genus Prototheca (P.) are associated with rare but severe infections (protothecosis) and represent a potential zoonotic risk. Genotype (GT) 2 of P. zopfii has been established as pathogenic agent for humans, dogs, and cattle, whereas GT1 is considered to be non-pathogenic. Since pathogenesis is poorly understood, the aim of this study was to determine immunogenic proteins and potential virulence factors of P. zopfii GT2. Therefore, 2D western blot analyses with sera and isolates of two dogs naturally infected with P. zopfii GT2 have been performed. Cross-reactivity was determined by including the type strains of P. zopfii GT2, P. zopfii GT1, and P. blaschkeae, a close relative of P. zopfii, which is known to cause subclinical forms of bovine mastitis. The sera showed a high strain-, genotype-, and species-cross-reactivity. A total of 198 immunogenic proteins have been analyzed via MALDI—TOF MS. The majority of the 86 identified proteins are intracellularly located (e.g., malate dehydrogenase, oxidoreductase, 3-dehydroquinate synthase) but some antigens and potential virulence factors, known from other pathogens, have been found (e.g., phosphomannomutase, triosephosphate isomerase). One genotype-specific antigen could be identified as heat shock protein 70 (Hsp70), a well-known antigen of eukaryotic pathogens with immunological importance when located extracellularly. Both sera were reactive to glyceraldehyde-3-phosphate-dehydrogenase of all investigated strains. This house-keeping enzyme is found to be located on the surface of several pathogens as virulence factor. Flow-cytometric analysis revealed its presence on the surface of P. blaschkeae. PMID:26484314

Structural and Phylogenetic Analysis of Laccases from Trichoderma: A Bioinformatic Approach

PubMed Central

Cázares-García, Saila Viridiana; Vázquez-Garcidueñas, Ma. Soledad; Vázquez-Marrufo, Gerardo

2013-01-01

The genus Trichoderma includes species of great biotechnological value, both for their mycoparasitic activities and for their ability to produce extracellular hydrolytic enzymes. Although activity of extracellular laccase has previously been reported in Trichoderma spp., the possible number of isoenzymes is still unknown, as are the structural and functional characteristics of both the genes and the putative proteins. In this study, the system of laccases sensu stricto in the Trichoderma species, the genomes of which are publicly available, were analyzed using bioinformatic tools. The intron/exon structure of the genes and the identification of specific motifs in the sequence of amino acids of the proteins generated in silico allow for clear differentiation between extracellular and intracellular enzymes. Phylogenetic analysis suggests that the common ancestor of the genus possessed a functional gene for each one of these enzymes, which is a characteristic preserved in T. atroviride and T. virens. This analysis also reveals that T. harzianum and T. reesei only retained the intracellular activity, whereas T. asperellum added an extracellular isoenzyme acquired through horizontal gene transfer during the mycoparasitic process. The evolutionary analysis shows that in general, extracellular laccases are subjected to purifying selection, and intracellular laccases show neutral evolution. The data provided by the present study will enable the generation of experimental approximations to better understand the physiological role of laccases in the genus Trichoderma and to increase their biotechnological potential. PMID:23383142

Proteus mirabilis fimbriae- and urease-dependent clusters assemble in an extracellular niche to initiate bladder stone formation.

PubMed

Schaffer, Jessica N; Norsworthy, Allison N; Sun, Tung-Tien; Pearson, Melanie M

2016-04-19

The catheter-associated uropathogenProteus mirabilisfrequently causes urinary stones, but little has been known about the initial stages of bladder colonization and stone formation. We found thatP. mirabilisrapidly invades the bladder urothelium, but generally fails to establish an intracellular niche. Instead, it forms extracellular clusters in the bladder lumen, which form foci of mineral deposition consistent with development of urinary stones. These clusters elicit a robust neutrophil response, and we present evidence of neutrophil extracellular trap generation during experimental urinary tract infection. We identified two virulence factors required for cluster development: urease, which is required for urolithiasis, and mannose-resistantProteus-like fimbriae. The extracellular cluster formation byP. mirabilisstands in direct contrast to uropathogenicEscherichia coli, which readily formed intracellular bacterial communities but not luminal clusters or urinary stones. We propose that extracellular clusters are a key mechanism ofP. mirabilissurvival and virulence in the bladder.

ROS Production via P2Y1-PKC-NOX2 Is Triggered by Extracellular ATP after Electrical Stimulation of Skeletal Muscle Cells.

PubMed

Díaz-Vegas, Alexis; Campos, Cristian A; Contreras-Ferrat, Ariel; Casas, Mariana; Buvinic, Sonja; Jaimovich, Enrique; Espinosa, Alejandra

2015-01-01

During exercise, skeletal muscle produces reactive oxygen species (ROS) via NADPH oxidase (NOX2) while inducing cellular adaptations associated with contractile activity. The signals involved in this mechanism are still a matter of study. ATP is released from skeletal muscle during electrical stimulation and can autocrinely signal through purinergic receptors; we searched for an influence of this signal in ROS production. The aim of this work was to characterize ROS production induced by electrical stimulation and extracellular ATP. ROS production was measured using two alternative probes; chloromethyl-2,7- dichlorodihydrofluorescein diacetate or electroporation to express the hydrogen peroxide-sensitive protein Hyper. Electrical stimulation (ES) triggered a transient ROS increase in muscle fibers which was mimicked by extracellular ATP and was prevented by both carbenoxolone and suramin; antagonists of pannexin channel and purinergic receptors respectively. In addition, transient ROS increase was prevented by apyrase, an ecto-nucleotidase. MRS2365, a P2Y1 receptor agonist, induced a large signal while UTPyS (P2Y2 agonist) elicited a much smaller signal, similar to the one seen when using ATP plus MRS2179, an antagonist of P2Y1. Protein kinase C (PKC) inhibitors also blocked ES-induced ROS production. Our results indicate that physiological levels of electrical stimulation induce ROS production in skeletal muscle cells through release of extracellular ATP and activation of P2Y1 receptors. Use of selective NOX2 and PKC inhibitors suggests that ROS production induced by ES or extracellular ATP is mediated by NOX2 activated by PKC.

Symbiodinium sp. cells produce light-induced intra- and extracellular singlet oxygen, which mediates photodamage of the photosynthetic apparatus and has the potential to interact with the animal host in coral symbiosis.

PubMed

Rehman, Ateeq Ur; Szabó, Milán; Deák, Zsuzsanna; Sass, László; Larkum, Anthony; Ralph, Peter; Vass, Imre

2016-10-01

Coral bleaching is an important environmental phenomenon, whose mechanism has not yet been clarified. The involvement of reactive oxygen species (ROS) has been implicated, but direct evidence of what species are involved, their location and their mechanisms of production remains unknown. Histidine-mediated chemical trapping and singlet oxygen sensor green (SOSG) were used to detect intra- and extracellular singlet oxygen ((1) O2 ) in Symbiodinium cultures. Inhibition of the Calvin-Benson cycle by thermal stress or high light promotes intracellular (1) O2 formation. Histidine addition, which decreases the amount of intracellular (1) O2 , provides partial protection against photosystem II photoinactivation and chlorophyll (Chl) bleaching. (1) O2 production also occurs in cell-free medium of Symbiodinium cultures, an effect that is enhanced under heat and light stress and can be attributed to the excretion of (1) O2 -sensitizing metabolites from the cells. Confocal microscopy imaging using SOSG showed most extracellular (1) O2 around the cell surface, but it is also produced across the medium distant from the cells. We demonstrate, for the first time, both intra- and extracellular (1) O2 production in Symbiodinium cultures. Intracellular (1) O2 is associated with photosystem II photodamage and pigment bleaching, whereas extracellular (1) O2 has the potential to mediate the breakdown of symbiotic interaction between zooxanthellae and their animal host during coral bleaching. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

Extracellular degradation of tetrabromobisphenol A via biogenic reactive oxygen species by a marine Pseudoalteromonas sp.

PubMed

Gu, Chen; Wang, Jing; Guo, Mengfan; Sui, Meng; Lu, Hong; Liu, Guangfei

2018-06-07

Tetrabromobisphenol A (TBBPA) has attracted considerable attention due to its ubiquitous presence in different environmental compartments worldwide. However, information on its aerobic biodegradability in coastal environments remains unknown. Here, the aerobic biodegradation of TBBPA using a Pseudoalteromonas species commonly found in the marine environment was investigated. We found that extracellular biogenic siderophore, superoxide anion radical (O 2 •- ), hydrogen peroxide (H 2 O 2 ), and hydroxyl radical ( • OH) were involved in TBBPA degradation. Upregulation of genes (nqrA and lodA) encoding Na + -translocating NADH-quinone oxidoreductase and l-lysine-ε-oxidase supported the extracellular O 2 •- and H 2 O 2 production. The underlying mechanism of TBBPA biodegradation presumably involves both O 2 •- reduction and • OH-based advanced oxidation process (AOP). Furthermore, TBBPA intermediates of tribromobisphenol A, 4-isopropylene-2,6-dibromophenol, 4-(2-hydroxyisopropyl)-2,6-dibromophenol, 2,4,6-tribromophenol (TBP), 4-hydroxybenzoic acid, and 2-bromobenzoic acid were detected in the culture medium. Debromination and β-scission pathways of TBBPA biodegradation were proposed. Additionally, membrane integrity assays revealed that the increase of intracellular catalase (CAT) activity and the extracellular polymeric substances (EPS) might account for the alleviation of oxidative damage. These findings could deepen understanding of the biodegradation mechanism of TBBPA and other related organic pollutants in coastal and artificial bioremediation systems. Copyright © 2018 Elsevier Ltd. All rights reserved.

Nanoengineered explosives

DOEpatents

Makowiecki, D.M.

1996-04-09

A complex modulated structure is described for reactive elements that have the capability of considerably more heat than organic explosives while generating a working fluid or gas. The explosive and method of fabricating same involves a plurality of very thin, stacked, multilayer structures, each composed of reactive components, such as aluminum, separated from a less reactive element, such as copper oxide, by a separator material, such as carbon. The separator material not only separates the reactive materials, but it reacts therewith when detonated to generate higher temperatures. The various layers of material, thickness of 10 to 10,000 angstroms, can be deposited by magnetron sputter deposition. The explosive detonates and combusts a high velocity generating a gas, such as CO, and high temperatures. 2 figs.

Osteopontin expression in reactive lesions of gingiva

PubMed Central

ELANAGAI, Rathinam; VEERAVARMAL, Veeran; NIRMAL, Ramdas Madhavan

2015-01-01

Reactive proliferations of the gingiva comprise lesions such as pyogenic granuloma (PG), inflammatory fibroepithelial hyperplasia (IFH), peripheral ossifying fibroma (POF), and peripheral giant cell lesion. Osteopontin (OPN) has a dual role, it promotes mineralization when it is bound to solid substrate, and on the other hand, it inhibits mineralization when it is seen in association with solution. Objectives The study aimed to evaluate the expression of osteopontin in normal gingival tissue and different types of focal reactive proliferations of gingival tissue, and its role in the development of calcification within it. Material and Methods The presence and distribution of osteopontin was assessed using immunohistochemistry in five cases of normal gingival tissue and 30 cases of focal reactive proliferations of gingiva. Results There was no expression of osteopontin in normal subjects. Few cases of pyogenic granuloma, inflammatory fibroepithelial hyperplasia, and all the cases of peripheral ossifying fibroma showed positivity for osteopontin in the inflammatory cells, stromal cells, extracellular matrix, and in the calcifications. Conclusion The expression of osteopontin in all the cases of peripheral ossifying fibroma speculates that the majority of the cases of peripheral ossifying fibroma originate from the periodontal ligament cells. The treatment modalities for peripheral ossifying fibroma should differ from other focal reactive proliferations of gingiva. PMID:25760265

Regulation of Mitochondrial Structure and Dynamics by the Cytoskeleton and Mechanical Factors.

PubMed

Bartolák-Suki, Erzsébet; Imsirovic, Jasmin; Nishibori, Yuichiro; Krishnan, Ramaswamy; Suki, Béla

2017-08-21

Mitochondria supply cells with energy in the form of ATP, guide apoptosis, and contribute to calcium buffering and reactive oxygen species production. To support these diverse functions, mitochondria form an extensive network with smaller clusters that are able to move along microtubules aided by motor proteins. Mitochondria are also associated with the actin network, which is involved in cellular responses to various mechanical factors. In this review, we discuss mitochondrial structure and function in relation to the cytoskeleton and various mechanical factors influencing cell functions. We first summarize the morphological features of mitochondria with an emphasis on fission and fusion as well as how network properties govern function. We then review the relationship between the mitochondria and the cytoskeletal structures, including mechanical interactions. We also discuss how stretch and its dynamic pattern affect mitochondrial structure and function. Finally, we present preliminary data on how extracellular matrix stiffness influences mitochondrial morphology and ATP generation. We conclude by discussing the more general role that mitochondria may play in mechanobiology and how the mechanosensitivity of mitochondria may contribute to the development of several diseases and aging.

The role of IL-1 in gout: from bench to bedside.

PubMed

So, Alexander; Dumusc, Alexandre; Nasi, Sonia

2018-01-01

The translation of our knowledge of the biology of MSU crystal-induced IL-1 secretion gives rise to new targets and therapeutic strategies in the treatment of acute gout. The NACHT, LRR and PYD domains-containing protein 3 inflammasome is key to this, and is the subject of intense research. Novel pathways that modulate inflammasome activation, reactive oxygen species generation and extracellular processing of IL-1 have been described and show promise in in vitro and animal studies. Meanwhile, blocking IL-1 by various IL-1 inhibitors has shown the validity of this concept. Patients with acute gout treated with these inhibitors showed positive clinical and biological responses. More work needs to be performed to assess the risk/benefit profile of anti-IL-1 therapies as well as to identify those who will benefit the most from this novel approach to the treatment of gout. © The Author 2018. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Susceptibility of Staphylococcus aureus biofilms to reactive discharge gases.

PubMed

Traba, Christian; Liang, Jun F

2011-08-01

Formation of bacterial biofilms at solid-liquid interfaces creates numerous problems in both industrial and biomedical sciences. In this study, the susceptibility of Staphylococcus aureus biofilms to discharge gas generated from plasma was tested. It was found that despite distinct chemical/physical properties, discharge gases from oxygen, nitrogen, and argon demonstrated very potent and almost the same anti-biofilm activity. The bacterial cells in S. aureus biofilms were killed (>99.9%) by discharge gas within minutes of exposure. Under optimal experimental conditions, no bacteria and biofilm re-growth from discharge gas treated biofilms was found. Further studies revealed that the anti-biofilm activity of the discharge gas occurred by two distinct mechanisms: (1) killing bacteria in biofilms by causing severe cell membrane damage, and (2) damaging the extracellular polymeric matrix in the architecture of the biofilm to release biofilm from the surface of the solid substratum. Information gathered from this study provides an insight into the anti-biofilm mechanisms of plasma and confirms the applications of discharge gas in the treatment of biofilms and biofilm related bacterial infections.

Regulation of Mitochondrial Structure and Dynamics by the Cytoskeleton and Mechanical Factors

PubMed Central

Bartolák-Suki, Erzsébet; Imsirovic, Jasmin; Nishibori, Yuichiro; Krishnan, Ramaswamy; Suki, Béla

2017-01-01

Mitochondria supply cells with energy in the form of ATP, guide apoptosis, and contribute to calcium buffering and reactive oxygen species production. To support these diverse functions, mitochondria form an extensive network with smaller clusters that are able to move along microtubules aided by motor proteins. Mitochondria are also associated with the actin network, which is involved in cellular responses to various mechanical factors. In this review, we discuss mitochondrial structure and function in relation to the cytoskeleton and various mechanical factors influencing cell functions. We first summarize the morphological features of mitochondria with an emphasis on fission and fusion as well as how network properties govern function. We then review the relationship between the mitochondria and the cytoskeletal structures, including mechanical interactions. We also discuss how stretch and its dynamic pattern affect mitochondrial structure and function. Finally, we present preliminary data on how extracellular matrix stiffness influences mitochondrial morphology and ATP generation. We conclude by discussing the more general role that mitochondria may play in mechanobiology and how the mechanosensitivity of mitochondria may contribute to the development of several diseases and aging. PMID:28825689

Hyaluronan – A Functional and Structural Sweet Spot in the Tissue Microenvironment

PubMed Central

Monslow, James; Govindaraju, Priya; Puré, Ellen

2015-01-01

Transition from homeostatic to reactive matrix remodeling is a fundamental adaptive tissue response to injury, inflammatory disease, fibrosis, and cancer. Alterations in architecture, physical properties, and matrix composition result in changes in biomechanical and biochemical cellular signaling. The dynamics of pericellular and extracellular matrices, including matrix protein, proteoglycan, and glycosaminoglycan modification are continually emerging as essential regulatory mechanisms underlying cellular and tissue function. Nevertheless, the impact of matrix organization on inflammation and immunity in particular and the consequent effects on tissue healing and disease outcome are arguably under-studied aspects of adaptive stress responses. Herein, we review how the predominant glycosaminoglycan hyaluronan (HA) contributes to the structure and function of the tissue microenvironment. Specifically, we examine the evidence of HA degradation and the generation of biologically active smaller HA fragments in pathological settings in vivo. We discuss how HA fragments versus nascent HA via alternate receptor-mediated signaling influence inflammatory cell recruitment and differentiation, resident cell activation, as well as tumor growth, survival, and metastasis. Finally, we discuss how HA fragmentation impacts restoration of normal tissue function and pathological outcomes in disease. PMID:26029216

Abdominal aortic aneurysm: novel mechanisms and therapies.

PubMed

Davis, Frank M; Rateri, Debra L; Daugherty, Alan

2015-11-01

Abdominal aortic aneurysm (AAA) is a pathological condition of permanent dilation that portends the potentially fatal consequence of aortic rupture. This review emphasizes recent advances in mechanistic insight into aneurysm pathogenesis and potential pharmacologic therapies that are on the horizon for AAAs. An increasing body of evidence demonstrates that genetic factors, including 3p12.3, DAB2IP, LDLr, LRP1, matrix metalloproteinase (MMP)-3, TGFBR2, and SORT1 loci, are associated with AAA development. Current human studies and animal models have shown that many leukocytes and inflammatory mediators, such as IL-1, IL-17, TGF-β, and angiotensin II, are involved in the pathogenesis of AAAs. Leukocytic infiltration into aortic media leads to smooth muscle cell depletion, generation of reactive oxygen species, and extracellular matrix fragmentation. Preclinical investigations into pharmacological therapies for AAAs have provided intriguing insight into the roles of microRNAs in regulating many pathological pathways in AAA development. Several large clinical trials are ongoing, seeking to translate preclinical findings into therapeutic options. Recent studies have identified many potential mechanisms involved in AAA pathogenesis that provide insight into the development of a medical treatment for this disease.

Effects of dielectric barrier discharge plasma on the inactivation of Zygosaccharomyces rouxii and quality of apple juice.

PubMed

Xiang, Qisen; Liu, Xiufang; Li, Junguang; Liu, Shengnan; Zhang, Hua; Bai, Yanhong

2018-07-15

This work aimed to evaluate the effects of dielectric barrier discharge (DBD) plasma on inactivation of spoilage yeast Zygosaccharomyces rouxii (Z. rouxii), in apple juice. Results showed that DBD plasma treatment at 90 W for 140 s resulted in about 5-log reduction of Z. rouxii in apple juice. The levels of extracellular nucleic acids and proteins as well as contents of H 2 O 2 and NO 2 - in yeast extract-peptone-dextrose (YPD) medium increased significantly after DBD plasma treatment at 90 W for 40-200 s. The increases in membrane permeability and generation of reactive species would likely contribute to DBD plasma-mediated inactivation of Z. rouxii. DBD plasma caused significant changes in pH, titratable acidity, and certain color parameters of apple juice, but had no effect on the contents of total soluble solids, reducing sugar, and total phenolics. This study provides key implications for the application of DBD plasma in fruit juice processing. Copyright © 2018 Elsevier Ltd. All rights reserved.

Quiescence and activation of stem and precursor cell populations in the subependymal zone of the mammalian brain are associated with distinct cellular and extracellular matrix signals

USDA-ARS?s Scientific Manuscript database

The subependymal zone (SEZ) of the lateral ventricles is one of the areas of the adult brain where new neurons are continuously generated from neural stem cells (NSCs), via rapidly dividing precursors. This neurogenic niche is a complex cellular and extracellular microenvironment, highly vascularize...

Emerging interactions between matrix components during biofilm development.

PubMed

Payne, David E; Boles, Blaise R

2016-02-01

Bacterial cells are most often found in the form of multicellular aggregates commonly referred to as biofilms. Biofilms offer their member cells several benefits, such as resistance to killing by antimicrobials and predation. During biofilm formation there is a production of extracellular substances that, upon assembly, constitute an extracellular matrix. The ability to generate a matrix encasing the microbial cells is a common feature of biofilms, but there is diversity in matrix composition and in interaction between matrix components. The different components of bacterial biofilm extracellular matrixes, known as matrix interactions, and resulting implications are discussed in this review.

Contribution of the Axon Initial Segment to Action Potentials Recorded Extracellularly.

PubMed

Teleńczuk, Maria; Brette, Romain; Destexhe, Alain; Teleńczuk, Bartosz

2018-01-01

Action potentials (APs) are electric phenomena that are recorded both intracellularly and extracellularly. APs are usually initiated in the short segment of the axon called the axon initial segment (AIS). It was recently proposed that at the onset of an AP the soma and the AIS form a dipole. We study the extracellular signature [the extracellular AP (EAP)] generated by such a dipole. First, we demonstrate the formation of the dipole and its extracellular signature in detailed morphological models of a reconstructed pyramidal neuron. Then, we study the EAP waveform and its spatial dependence in models with axonal AP initiation and contrast it with the EAP obtained in models with somatic AP initiation. We show that in the models with axonal AP initiation the dipole forms between somatodendritic compartments and the AIS, and not between soma and dendrites as in the classical models. The soma-dendrites dipole is present only in models with somatic AP initiation. Our study has consequences for interpreting extracellular recordings of single-neuron activity and determining electrophysiological neuron types, but also for better understanding the origins of the high-frequency macroscopic extracellular potentials recorded in the brain.

Spin trapping combined with quantitative mass spectrometry defines free radical redistribution within the oxidized hemoglobin:haptoglobin complex.

PubMed

Vallelian, Florence; Garcia-Rubio, Ines; Puglia, Michele; Kahraman, Abdullah; Deuel, Jeremy W; Engelsberger, Wolfgang R; Mason, Ronald P; Buehler, Paul W; Schaer, Dominik J

2015-08-01

Extracellular or free hemoglobin (Hb) accumulates during hemolysis, tissue damage, and inflammation. Heme-triggered oxidative reactions can lead to diverse structural modifications of lipids and proteins, which contribute to the propagation of tissue damage. One important target of Hb׳s peroxidase reactivity is its own globin structure. Amino acid oxidation and crosslinking events destabilize the protein and ultimately cause accumulation of proinflammatory and cytotoxic Hb degradation products. The Hb scavenger haptoglobin (Hp) attenuates oxidation-induced Hb degradation. In this study we show that in the presence of hydrogen peroxide (H2O2), Hb and the Hb:Hp complex share comparable peroxidative reactivity and free radical generation. While oxidation of both free Hb and Hb:Hp complex generates a common tyrosine-based free radical, the spin-trapping reaction with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) yields dissimilar paramagnetic products in Hb and Hb:Hp, suggesting that radicals are differently redistributed within the complex before reacting with the spin trap. With LC-MS(2) mass spectrometry we assigned multiple known and novel DMPO adduct sites. Quantification of these adducts suggested that the Hb:Hp complex formation causes extensive delocalization of accessible free radicals with drastic reduction of the major tryptophan and cysteine modifications in the β-globin chain of the Hb:Hp complex, including decreased βCys93 DMPO adduction. In contrast, the quantitative changes in DMPO adduct formation on Hb:Hp complex formation were less pronounced in the Hb α-globin chain. In contrast to earlier speculations, we found no evidence that free Hb radicals are delocalized to the Hp chain of the complex. The observation that Hb:Hp complex formation alters free radical distribution in Hb may help to better understand the structural basis for Hp as an antioxidant protein. Copyright © 2015 Elsevier Inc. All rights reserved.

Oxidation of glycosaminoglycans by free radicals and reactive oxidative species: A review of investigative methods.

PubMed

Parsons, B J

2015-05-01

Glycosaminoglycans, in particular hyaluronan (HA), and proteoglycans are components of the extracellular matrix (ECM). The ECM plays a key role in the regulation of cellular behaviour and alterations to it can modulate both the development of human diseases as well as controlling normal biochemical processes such as cell signalling and pro-inflammatory responses. For these reasons, in vitro fragmentation studies of glycosaminoglycans by free radicals and oxidative species are seen to be relevant to the understanding of in vivo studies of damage to the ECM. A wide range of investigative techniques have therefore been applied to gain insights into the relative fragmentation effects of several reactive oxidative species with the ultimate goal of determining mechanisms of fragmentation at the molecular level. These methods are reviewed here.

Yeast respond to hypotonic shock with a calcium pulse

NASA Technical Reports Server (NTRS)

Batiza, A. F.; Schulz, T.; Masson, P. H.

1996-01-01

We have used the transgenic AEQUORIN calcium reporter system to monitor the cytosolic calcium ([Ca2+]cyt) response of Saccharomyces cerevisiae to hypotonic shock. Such a shock generates an almost immediate and transient rise in [Ca2+]cyt which is eliminated by gadolinium, a blocker of stretch-activated channels. In addition, this transient rise in [Ca2+]cyt is initially insensitive to 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), an extracellular calcium chelator. However, BAPTA abruptly attenuates the maintenance of that transient rise. These data show that hypotonic shock generates a stretch-activated channel-dependent calcium pulse in yeast. They also suggest that the immediate calcium influx is primarily generated from intracellular stores, and that a sustained increase in [Ca2+]cyt depends upon extracellular calcium.

Contribution of extracellular polymeric substances from Shewanella sp. HRCR-1 biofilms to U(VI) immobilization.

PubMed

Cao, Bin; Ahmed, Bulbul; Kennedy, David W; Wang, Zheming; Shi, Liang; Marshall, Matthew J; Fredrickson, Jim K; Isern, Nancy G; Majors, Paul D; Beyenal, Haluk

2011-07-01

The goal of this study was to quantify the contribution of extracellular polymeric substances (EPS) to U(VI) immobilization by Shewanella sp. HRCR-1. Through comparison of U(VI) immobilization using cells with bound EPS (bEPS) and cells with minimal EPS, we show that (i) bEPS from Shewanella sp. HRCR-1 biofilms contribute significantly to U(VI) immobilization, especially at low initial U(VI) concentrations, through both sorption and reduction; (ii) bEPS can be considered a functional extension of the cells for U(VI) immobilization and they likely play more important roles at lower initial U(VI) concentrations; and (iii) the U(VI) reduction efficiency is dependent upon the initial U(VI) concentration and decreases at lower concentrations. To quantify the relative contributions of sorption and reduction to U(VI) immobilization by EPS fractions, we isolated loosely associated EPS (laEPS) and bEPS from Shewanella sp. HRCR-1 biofilms grown in a hollow fiber membrane biofilm reactor and tested their reactivity with U(VI). We found that, when reduced, the isolated cell-free EPS fractions could reduce U(VI). Polysaccharides in the EPS likely contributed to U(VI) sorption and dominated the reactivity of laEPS, while redox active components (e.g., outer membrane c-type cytochromes), especially in bEPS, possibly facilitated U(VI) reduction.

Contribution of Extracellular Polymeric Substances from Shewanella sp. HRCR-1 Biofilms to U(VI) Immobilization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cao, Bin; Ahmed, B.; Kennedy, David W.

2011-06-05

The goal of this study was to quantify the contribution of extracellular polymeric substances (EPS) in U(VI) immobilization by Shewanella sp. HRCR-1. Through comparison of U(VI) immobilization using cells with bound EPS (bEPS) and cells without EPS, we showed that i) bEPS from Shewanella sp. HRCR-1 biofilms contributed significantly to U(VI) immobilization, especially at low initial U(VI) concentrations, through both sorption and reduction; ii) bEPS could be considered as a functional extension of the cells for U(VI) immobilization and they likely play more important roles at initial U(VI) concentrations; and iii) U(VI) reduction efficiency was found to be dependent uponmore » initial U(VI) concentration and the efficiency decreased at lower concentrations. To quantify relative contribution of sorption and reduction in U(VI) immobilization by EPS fractions, we isolated loosely associated EPS (laEPS) and bEPS from Shewanella sp. HRCR-1 biofilms grown in a hollow fiber membrane biofilm reactor and tested their reactivity with U(V). We found that, when in reduced form, the isolated cell-free EPS fractions could reduce U(VI). Polysaccharides in the EPS likely contributed to U(VI) sorption and dominated reactivity of laEPS while redox active components (e.g., outer membrane c-type cytochromes), especially in bEPS, might facilitate U(VI) reduction.« less

Powering a burnt bridges Brownian ratchet: a model for an extracellular motor driven by proteolysis of collagen.

PubMed

Saffarian, Saveez; Qian, Hong; Collier, Ivan; Elson, Elliot; Goldberg, Gregory

2006-04-01

Biased diffusion of collagenase on collagen fibrils may represent the first observed adenosine triphosphate-independent extracellular molecular motor. The magnitude of force generated by the enzyme remains unclear. We propose a propulsion mechanism based on a burnt bridges Brownian ratchet model with a varying degree of coupling of the free energy from collagen proteolysis to the enzyme motion. When constrained by experimental observations, our model predicts 0.1 pN stall force for individual collagenase molecules. A dimer, surprisingly, can generate a force in the range of 5 pN, suggesting that the motor can be of biological significance.

Mitotic cells generate protrusive extracellular forces to divide in three-dimensional microenvironments

NASA Astrophysics Data System (ADS)

Nam, Sungmin; Chaudhuri, Ovijit

2018-06-01

During mitosis, or cell division, mammalian cells undergo extensive morphological changes, including elongation along the mitotic axis, which is perpendicular to the plane that bisects the two divided cells. Although much is known about the intracellular dynamics of mitosis, it is unclear how cells are able to divide in tissues, where the changes required for mitosis are mechanically constrained by surrounding cells and extracellular matrix. Here, by confining cells three dimensionally in hydrogels, we show that dividing cells generate substantial protrusive forces that deform their surroundings along the mitotic axis, clearing space for mitotic elongation. When forces are insufficient to create space for mitotic elongation, mitosis fails. We identify one source of protrusive force as the elongation of the interpolar spindle, an assembly of microtubules aligned with the mitotic axis. Another source of protrusive force is shown to be contraction of the cytokinetic ring, the polymeric structure that cleaves a dividing cell at its equator, which drives expansion along the mitotic axis. These findings reveal key functions for the interpolar spindle and cytokinetic ring in protrusive extracellular force generation, and explain how dividing cells overcome mechanical constraints in confining microenvironments, including some types of tumour.

Conservative Mechanisms of Extracellular Trap Formation by Annelida Eisenia andrei: Serine Protease Activity Requirement

PubMed Central

Ortmann, Weronika; Kolaczkowska, Elzbieta

2016-01-01

Formation of extracellular traps (ETs) capturing and immobilizing pathogens is now a well-established defense mechanism added to the repertoire of vertebrate phagocytes. These ETs are composed of extracellular DNA (extDNA), histones and antimicrobial proteins. Formation of mouse and human ETs depends on enzymes (i) facilitating decondensation of chromatin by citrullination of histones, and (ii) serine proteases degrading histones. In invertebrates, initial reports revealed existence of ETs composed of extDNA and histones, and here we document for the first time that also coelomocytes, immunocompetent cells of an earthworm Eisenia andrei, cast ETs which successfully trap bacteria in a reactive oxygen species (ROS)-dependent and -independent manner. Importantly, the formation of ETs was observed not only when coelomocytes were studied ex vivo, but also in vivo, directly in the earthworm coelom. These ETs were composed of extDNA, heat shock proteins (HSP27) and H3 histones. Furthermore, the formation of E. andrei ETs depended on activity of serine proteases, including elastase-like activity. Moreover, ETs interconnected and hold together aggregating coelomocytes, a processes proceeding encapsulation. In conclusion, the study confirms ET formation by earthworms, and unravels mechanisms leading to ET formation and encapsulation in invertebrates. PMID:27416067

Release of Active Peptidyl Arginine Deiminases by Neutrophils Can Explain Production of Extracellular Citrullinated Autoantigens in Rheumatoid Arthritis Synovial Fluid

PubMed Central

Spengler, Julia; Lugonja, Božo; Jimmy Ytterberg, A.; Zubarev, Roman A.; Creese, Andrew J.; Pearson, Mark J.; Grant, Melissa M.; Milward, Michael; Lundberg, Karin; Buckley, Christopher D.; Filer, Andrew; Raza, Karim; Cooper, Paul R.; Chapple, Iain L.

2015-01-01

Objective In the majority of patients with rheumatoid arthritis (RA), antibodies specifically recognize citrullinated autoantigens that are generated by peptidylarginine deiminases (PADs). Neutrophils express high levels of PAD and accumulate in the synovial fluid (SF) of RA patients during disease flares. This study was undertaken to test the hypothesis that neutrophil cell death, induced by either NETosis (extrusion of genomic DNA–protein complexes known as neutrophil extracellular traps [NETs]) or necrosis, can contribute to production of autoantigens in the inflamed joint. Methods Extracellular DNA was quantified in the SF of patients with RA, patients with osteoarthritis (OA), and patients with psoriatic arthritis (PsA). Release of PAD from neutrophils was investigated by Western blotting, mass spectrometry, immunofluorescence staining, and PAD activity assays. PAD2 and PAD4 protein expression, as well as PAD enzymatic activity, were assessed in the SF of patients with RA and those with OA. Results Extracellular DNA was detected at significantly higher levels in RA SF than in OA SF (P < 0.001) or PsA SF (P < 0.05), and its expression levels correlated with neutrophil concentrations and PAD activity in RA SF. Necrotic neutrophils released less soluble extracellular DNA compared to NETotic cells in vitro (P < 0.05). Higher PAD activity was detected in RA SF than in OA SF (P < 0.05). The citrullinated proteins PAD2 and PAD4 were found attached to NETs and also freely diffused in the supernatant. PAD enzymatic activity was detected in supernatants of neutrophils undergoing either NETosis or necrosis. Conclusion Release of active PAD isoforms into the SF by neutrophil cell death is a plausible explanation for the generation of extracellular autoantigens in RA. PMID:26245941

Reactive Power Compensating System.

DOEpatents

Williams, Timothy J.; El-Sharkawi, Mohamed A.; Venkata, Subrahmanyam S.

1985-01-04

The circuit was designed for the specific application of wind-driven induction generators. It has great potential for application in any situation where a varying reactive power load is present, such as with induction motors or generators, or for transmission network compensation.

Backside contacted field effect transistor array for extracellular signal recording.

PubMed

Ingebrandt, S; Yeung, C K; Staab, W; Zetterer, T; Offenhäusser, A

2003-04-01

A new approach to the design of field-effect transistor (FET) sensors and the use of these FETs in detecting extracellular electrophysiological recordings is reported. Backside contacts were engineered by deep reactive ion etching and a gas phase boron doping process of the holes using planar diffusion sources. The metal contacts were designed to fit on top of the bonding pads of a standard industrial 22-pin DIL (dual inline) chip carrier. To minimise contact resistance, the metal backside contacts of the chips were electroless plated with gold. The chips were mounted on top of the bonding pads using a standard flip-chip process and a fineplacer unit previously described. Rat embryonic myocytes were cultured on these new devices (effective growth area 6 x 6 mm(2)) in order to confirm their validity in electrophysiological recording. Copyright 2003 Elsevier Science B.V.

Purification and properties of extracellular polysaccharide (EPS) antigens produced by different mould species.

PubMed

Notermans, S; Wieten, G; Engel, H W; Rombouts, F M; Hoogerhout, P; van Boom, J H

1987-02-01

Extracellular polysaccharide (EPS) antigens produced by different mould species were purified and partially characterized. Purification included (NH4)2SO4 treatment, Sepharose CL-4B column chromatography and Con A-sepharose chromatography. The EPS of Penicillium digitatum, Mucor racemosus and Cladosporium cladosporioides showed high antigenic capacities. Immunologically the EPS were partially genus-specific, but cross-reactivity was observed. The EPS antigens produced by species of Penicillium, Aspergillus repens and Geotrichum candidum lost their immunological activity upon heating (100 degrees C) at pH 1.8, while the EPS antigen of M. racemosus, Rhizopus oligosporus and C. cladosporioides were stable under the same conditions. The dominant monosaccharides present in the EPS antigen were mannose, galactose and glucose. The EPS obtained from cultures of M. racemosus and R. oligosporus also contained rhamnose. In the EPS produced by Penicillium spp. and A. repens the galactose residues were determined to be immunodominant.

Ibogaine, a noncompetitive inhibitor of serotonin transport, acts by stabilizing the cytoplasm-facing state of the transporter.

PubMed

Jacobs, Miriam T; Zhang, Yuan-Wei; Campbell, Scott D; Rudnick, Gary

2007-10-05

Ibogaine, a hallucinogenic alkaloid with purported anti-addiction properties, inhibited serotonin transporter (SERT) noncompetitively by decreasing V(max) with little change in the K(m) for serotonin (5-HT). Ibogaine also inhibited binding to SERT of the cocaine analog 2beta-2-carbomethoxy-3-(4-[(125)I]iodophenyl)tropane. However, inhibition of binding was competitive, increasing the apparent K(D) without much change in B(max). Ibogaine increased the reactivity of cysteine residues positioned in the proposed cytoplasmic permeation pathway of SERT but not at nearby positions out of that pathway. In contrast, cysteines placed at positions in the extracellular permeation pathway reacted at slower rates in the presence of ibogaine. These results are consistent with the proposal that ibogaine binds to and stabilizes the state of SERT from which 5-HT dissociates to the cytoplasm, in contrast with cocaine, which stabilizes the state that binds extracellular 5-HT.

Water reactive hydrogen fuel cell power system

DOEpatents

Wallace, Andrew P; Melack, John M; Lefenfeld, Michael

2014-01-21

A water reactive hydrogen fueled power system includes devices and methods to combine reactant fuel materials and aqueous solutions to generate hydrogen. The generated hydrogen is converted in a fuel cell to provide electricity. The water reactive hydrogen fueled power system includes a fuel cell, a water feed tray, and a fuel cartridge to generate power for portable power electronics. The removable fuel cartridge is encompassed by the water feed tray and fuel cell. The water feed tray is refillable with water by a user. The water is then transferred from the water feed tray into a fuel cartridge to generate hydrogen for the fuel cell which then produces power for the user.

Water reactive hydrogen fuel cell power system

DOEpatents

Wallace, Andrew P; Melack, John M; Lefenfeld, Michael

2014-11-25

A water reactive hydrogen fueled power system includes devices and methods to combine reactant fuel materials and aqueous solutions to generate hydrogen. The generated hydrogen is converted in a fuel cell to provide electricity. The water reactive hydrogen fueled power system includes a fuel cell, a water feed tray, and a fuel cartridge to generate power for portable power electronics. The removable fuel cartridge is encompassed by the water feed tray and fuel cell. The water feed tray is refillable with water by a user. The water is then transferred from the water feed tray into the fuel cartridge to generate hydrogen for the fuel cell which then produces power for the user.

Zea mays Annexins Modulate Cytosolic Free Ca2+ and Generate a Ca2+-Permeable Conductance[W

PubMed Central

Laohavisit, Anuphon; Mortimer, Jennifer C.; Demidchik, Vadim; Coxon, Katy M.; Stancombe, Matthew A.; Macpherson, Neil; Brownlee, Colin; Hofmann, Andreas; Webb, Alex A.R.; Miedema, Henk; Battey, Nicholas H.; Davies, Julia M.

2009-01-01

Regulation of reactive oxygen species and cytosolic free calcium ([Ca2+]cyt) is central to plant function. Annexins are small proteins capable of Ca2+-dependent membrane binding or membrane insertion. They possess structural motifs that could support both peroxidase activity and calcium transport. Here, a Zea mays annexin preparation caused increases in [Ca2+]cyt when added to protoplasts of Arabidopsis thaliana roots expressing aequorin. The pharmacological profile was consistent with annexin activation (at the extracellular plasma membrane face) of Arabidopsis Ca2+-permeable nonselective cation channels. Secreted annexins could therefore modulate Ca2+ influx. As maize annexins occur in the cytosol and plasma membrane, they were incorporated at the intracellular face of lipid bilayers designed to mimic the plasma membrane. Here, they generated an instantaneously activating Ca2+-permeable conductance at mildly acidic pH that was sensitive to verapamil and Gd3+ and had a Ca2+-to-K+ permeability ratio of 0.36. These results suggest that cytosolic annexins create a Ca2+ influx pathway directly, particularly during stress responses involving acidosis. A maize annexin preparation also demonstrated in vitro peroxidase activity that appeared independent of heme association. In conclusion, this study has demonstrated that plant annexins create Ca2+-permeable transport pathways, regulate [Ca2+]cyt, and may function as peroxidases in vitro. PMID:19234085

Controlled release of drugs in electrosprayed nanoparticles for bone tissue engineering.

PubMed

Jayaraman, Praveena; Gandhimathi, Chinnasamy; Venugopal, Jayarama Reddy; Becker, David Laurence; Ramakrishna, Seeram; Srinivasan, Dinesh Kumar

2015-11-01

Generating porous topographic substrates, by mimicking the native extracellular matrix (ECM) to promote the regeneration of damaged bone tissues, is a challenging process. Generally, scaffolds developed for bone tissue regeneration support bone cell growth and induce bone-forming cells by natural proteins and growth factors. Limitations are often associated with these approaches such as improper scaffold stability, and insufficient cell adhesion, proliferation, differentiation, and mineralization with less growth factor expression. Therefore, the use of engineered nanoparticles has been rapidly increasing in bone tissue engineering (BTE) applications. The electrospray technique is advantageous over other conventional methods as it generates nanomaterials of particle sizes in the micro/nanoscale range. The size and charge of the particles are controlled by regulating the polymer solution flow rate and electric voltage. The unique properties of nanoparticles such as large surface area-to-volume ratio, small size, and higher reactivity make them promising candidates in the field of biomedical engineering. These nanomaterials are extensively used as therapeutic agents and for drug delivery, mimicking ECM, and restoring and improving the functions of damaged organs. The controlled and sustained release of encapsulated drugs, proteins, vaccines, growth factors, cells, and nucleotides from nanoparticles has been well developed in nanomedicine. This review provides an insight into the preparation of nanoparticles by electrospraying technique and illustrates the use of nanoparticles in drug delivery for promoting bone tissue regeneration. Copyright © 2015 Elsevier B.V. All rights reserved.

Antioxidative response in variegated Pelargonium zonale leaves and generation of extracellular H2O2 in (peri)vascular tissue induced by sunlight and paraquat.

PubMed

Vidović, Marija; Morina, Filis; Prokić, Ljiljana; Milić-Komić, Sonja; Živanović, Bojana; Jovanović, Sonja Veljović

2016-11-01

In this study we exposed variegated leaves of Pelargonium zonale to strong sunlight (>1100μmolm -2 s -1 of photosynthetically active radiation) with and without paraquat (Pq), with the aim to elucidate the mechanisms of H 2 O 2 regulation in green and white tissues with respect to the photosynthetically-dependent generation of reactive oxygen species (ROS). Sunlight induced marked accumulation of H 2 O 2 in the apoplast of vascular and (peri)vascular tissues only in green sectors. This effect was enhanced by the addition of Pq. In the presence of diphenyl iodide, an NADPH oxidase inhibitor, H 2 O 2 accumulation was abolished. Distinct light-induced responses were observed: in photosynthetic cells, sunlight rapidly provoked ascorbate (Asc) biosynthesis and an increase of glutathione reductase (GR) and catalase activities, while in non-photosynthetic cells, early up-regulation of soluble ascorbate peroxidase, dehydroascorbate reductase (DHAR) and GR activities was observed. Paraquat addition stimulated DHAR and GR activities in green sectors, while in white sectors activities of monodehydroascorbate reductase, DHAR and class III peroxidases, as well as Asc content rapidly increased. Differential antioxidative responses in the two tissues in the frame of their contrasting metabolisms, and the possible role of (peri)vascular H 2 O 2 in signaling were discussed. Copyright © 2016 Elsevier GmbH. All rights reserved.

DNA Protecting Activities of Nymphaea nouchali (Burm. f) Flower Extract Attenuate t-BHP-Induced Oxidative Stress Cell Death through Nrf2-Mediated Induction of Heme Oxygenase-1 Expression by Activating MAP-Kinases

PubMed Central

Ju, Mi-Kyoung

2017-01-01

This study was performed to investigate the antioxidant activities of Nymphaea nouchali flower (NNF) extract and the underlying mechanism using RAW 264.7 cells. The presence of gallic acid, catechin, epicatechin, epigallocatechin, epicatechin gallate, caffeic acid, quercetin, and apigenin in the NNF was confirmed by high-performance liquid chromatography (HPLC). The extract had a very potent capacity to scavenge numerous free radicals. NNF extract was also able to prevent DNA damage and quench cellular reactive oxygen species (ROS) generation induced by tert-Butyl hydroperoxide (t-BHP) with no signs of toxicity. The NNF extract was able to augment the expression of both primary and phase II detoxifying enzyme, resulting in combat the oxidative stress. This is accomplished by phosphorylation of mitogen-activated protein kinase (MAP kinase) (p38 kinase and extracellular signal-regulated kinase (ERK)) followed by enhancing the nuclear translocation of the nuclear factor erythroid 2-related factor 2 (Nrf2). This attenuates cellular ROS generation and confers protection from cell death. Altogether, the results of current study revealed that Nymphaea nouchali flower could be a source of natural phytochemicals that could lead to the development of new therapeutic agents for preventing oxidative stress associated diseases and attenuating disease progression. PMID:28956831

Mechanism of short-term ERK activation by electromagnetic fields at mobile phone frequencies

PubMed Central

Friedman, Joseph; Kraus, Sarah; Hauptman, Yirmi; Schiff, Yoni; Seger, Rony

2007-01-01

The exposure to non-thermal microwave electromagnetic fields generated by mobile phones affects the expression of many proteins. This effect on transcription and protein stability can be mediated by the MAPK (mitogen-activated protein kinase) cascades, which serve as central signalling pathways and govern essentially all stimulated cellular processes. Indeed, long-term exposure of cells to mobile phone irradiation results in the activation of p38 as well as the ERK (extracellular-signal-regulated kinase) MAPKs. In the present study, we have studied the immediate effect of irradiation on the MAPK cascades, and found that ERKs, but not stress-related MAPKs, are rapidly activated in response to various frequencies and intensities. Using signalling inhibitors, we delineated the mechanism that is involved in this activation. We found that the first step is mediated in the plasma membrane by NADH oxidase, which rapidly generates ROS (reactive oxygen species). These ROS then directly stimulate MMPs (matrix metalloproteinases) and allow them to cleave and release Hb-EGF [heparin-binding EGF (epidermal growth factor)]. This secreted factor activates the EGF receptor, which in turn further activates the ERK cascade. Thus this study demonstrates for the first time a detailed molecular mechanism by which electromagnetic irradiation from mobile phones induces the activation of the ERK cascade and thereby induces transcription and other cellular processes. PMID:17456048

Smooth muscle tension induces invasive remodeling of the zebrafish intestine.

PubMed

Seiler, Christoph; Davuluri, Gangarao; Abrams, Joshua; Byfield, Fitzroy J; Janmey, Paul A; Pack, Michael

2012-01-01

The signals that initiate cell invasion are not well understood, but there is increasing evidence that extracellular physical signals play an important role. Here we show that epithelial cell invasion in the intestine of zebrafish meltdown (mlt) mutants arises in response to unregulated contractile tone in the surrounding smooth muscle cell layer. Physical signaling in mlt drives formation of membrane protrusions within the epithelium that resemble invadopodia, matrix-degrading protrusions present in invasive cancer cells. Knockdown of Tks5, a Src substrate that is required for invadopodia formation in mammalian cells blocked formation of the protrusions and rescued invasion in mlt. Activation of Src-signaling induced invadopodia-like protrusions in wild type epithelial cells, however the cells did not migrate into the tissue stroma, thus indicating that the protrusions were required but not sufficient for invasion in this in vivo model. Transcriptional profiling experiments showed that genes responsive to reactive oxygen species (ROS) were upregulated in mlt larvae. ROS generators induced invadopodia-like protrusions and invasion in heterozygous mlt larvae but had no effect in wild type larvae. Co-activation of oncogenic Ras and Wnt signaling enhanced the responsiveness of mlt heterozygotes to the ROS generators. These findings present the first direct evidence that invadopodia play a role in tissue cell invasion in vivo. In addition, they identify an inducible physical signaling pathway sensitive to redox and oncogenic signaling that can drive this process.

Non-Thermal Atmospheric Pressure Plasma Preferentially Induces Apoptosis in p53-Mutated Cancer Cells by Activating ROS Stress-Response Pathways

PubMed Central

Ma, Yonghao; Ha, Chang Seung; Hwang, Seok Won; Lee, Hae June; Kim, Gyoo Cheon; Lee, Kyo-Won; Song, Kiwon

2014-01-01

Non-thermal atmospheric pressure plasma (NTAPP) is an ionized gas at room temperature and has potential as a new apoptosis-promoting cancer therapy that acts by generating reactive oxygen species (ROS). However, it is imperative to determine its selectivity and standardize the components and composition of NTAPP. Here, we designed an NTAPP-generating apparatus combined with a He gas feeding system and demonstrated its high selectivity toward p53-mutated cancer cells. We first determined the proper conditions for NTAPP exposure to selectively induce apoptosis in cancer cells. The apoptotic effect of NTAPP was greater for p53-mutated cancer cells; artificial p53 expression in p53-negative HT29 cells decreased the pro-apoptotic effect of NTAPP. We also examined extra- and intracellular ROS levels in NTAPP-treated cells to deduce the mechanism of NTAPP action. While NTAPP-mediated increases in extracellular nitric oxide (NO) did not affect cell viability, intracellular ROS increased under NTAPP exposure and induced apoptotic cell death. This effect was dose-dependently reduced following treatment with ROS scavengers. NTAPP induced apoptosis even in doxorubicin-resistant cancer cell lines, demonstrating the feasibility of NTAPP as a potent cancer therapy. Collectively, these results strongly support the potential of NTAPP as a selective anticancer treatment, especially for p53-mutated cancer cells. PMID:24759730

Microfabricated Nanotopological Surfaces for Study of Adhesion-dependent Cell mechanosensitivity**

PubMed Central

Chen, Weiqiang; Sun, Yubing

2014-01-01

Cells display high sensitivity and exhibit diverse responses to the intrinsic nanotopography of the extracellular matrix through their nanoscale cellular sensing machinery. Here, we reported a simple microfabrication method for precise control and spatial patterning of the local nanoroughness on glass surfaces using photolithography and reactive ion etching (RIE). Using RIE-generated nanorough glass surfaces, we demonstrated that local nanoroughness could provide a potent biophysical signal to regulate a diverse array of NIH/3T3 fibroblast behaviors, including cell morphology, adhesion, proliferation and migration. We further showed that cellular responses to nanotopography might be regulated by cell adhesion signaling and actin cytoskeleton remodeling. To further investigate the role of cytoskeleton contractility in nanoroughness sensing, we applied the RIE method to generate nanoroughness on the tops of an array of elastomeric poly-dimethylsiloxane (PDMS) microposts. We utilized the PDMS microposts as force sensors and demonstrated that nanoroughness could indeed regulate the cytoskeleton contractility of NIH/3T3 fibroblasts. Our results suggested that a feedback regulation and mechano-chemical integration mechanism involving adhesion signaling, actin cytoskeleton, and intracellular mechanosensory components might play an important role in regulating mechanosensitive behaviors of NIH/3T3 fibroblasts. The capability to control and further predict cellular responses to nanoroughness might suggest novel methods for developing biomaterials mimicking nanotopographic structures in vivo and suitable local cellular microenvironments for functional tissue engineering. PMID:22887768

Smooth Muscle Tension Induces Invasive Remodeling of the Zebrafish Intestine

PubMed Central

Seiler, Christoph; Davuluri, Gangarao; Abrams, Joshua; Byfield, Fitzroy J.; Janmey, Paul A.; Pack, Michael

2012-01-01

The signals that initiate cell invasion are not well understood, but there is increasing evidence that extracellular physical signals play an important role. Here we show that epithelial cell invasion in the intestine of zebrafish meltdown (mlt) mutants arises in response to unregulated contractile tone in the surrounding smooth muscle cell layer. Physical signaling in mlt drives formation of membrane protrusions within the epithelium that resemble invadopodia, matrix-degrading protrusions present in invasive cancer cells. Knockdown of Tks5, a Src substrate that is required for invadopodia formation in mammalian cells blocked formation of the protrusions and rescued invasion in mlt. Activation of Src-signaling induced invadopodia-like protrusions in wild type epithelial cells, however the cells did not migrate into the tissue stroma, thus indicating that the protrusions were required but not sufficient for invasion in this in vivo model. Transcriptional profiling experiments showed that genes responsive to reactive oxygen species (ROS) were upregulated in mlt larvae. ROS generators induced invadopodia-like protrusions and invasion in heterozygous mlt larvae but had no effect in wild type larvae. Co-activation of oncogenic Ras and Wnt signaling enhanced the responsiveness of mlt heterozygotes to the ROS generators. These findings present the first direct evidence that invadopodia play a role in tissue cell invasion in vivo. In addition, they identify an inducible physical signaling pathway sensitive to redox and oncogenic signaling that can drive this process. PMID:22973180

The Effects of Aronia melanocarpa 'Viking' Extracts in Attenuating RANKL-Induced Osteoclastic Differentiation by Inhibiting ROS Generation and c-FOS/NFATc1 Signaling.

PubMed

Ghosh, Mithun; Kim, In Sook; Lee, Young Min; Hong, Seong Min; Lee, Taek Hwan; Lim, Ji Hong; Debnath, Trishna; Lim, Beong Ou

2018-03-08

This study aimed to determine the anti-osteoclastogenic effects of extracts from Aronia melanocarpa 'Viking' (AM) and identify the underlying mechanisms in vitro. Reactive oxygen species (ROS) are signal mediators in osteoclast differentiation. AM extracts inhibited ROS production in RAW 264.7 cells in a dose-dependent manner and exhibited strong radical scavenging activity. The extracts also attenuated the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts. To attain molecular insights, the effect of the extracts on the signaling pathways induced by receptor activator of nuclear factor kappa B ligand (RANKL) were also investigated. RANKL triggers many transcription factors through the activation of mitogen-activated protein kinase (MAPK) and ROS, leading to the induction of osteoclast-specific genes. The extracts significantly suppressed RANKL-induced activation of MAPKs, such as extracellular signal-regulated kinase (ERK), c-Jun- N -terminal kinase (JNK) and p38 and consequently led to the downregulation of c-Fos and nuclear factor of activated T cells 1 (NFATc1) protein expression which ultimately suppress the activation of the osteoclast-specific genes, cathepsin K, TRAP, calcitonin receptor and integrin β₃. In conclusion, our findings suggest that AM extracts inhibited RANKL-induced osteoclast differentiation by downregulating ROS generation and inactivating JNK/ERK/p38, nuclear factor kappa B (NF-κB)-mediated c-Fos and NFATc1 signaling pathway.

Crossing the dividing surface of transition state theory. III. Once and only once. Selecting reactive trajectories

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lorquet, J. C., E-mail: jc.lorquet@ulg.ac.be

2015-09-14

The purpose of the present work is to determine initial conditions that generate reacting, recrossing-free trajectories that cross the conventional dividing surface of transition state theory (i.e., the plane in configuration space passing through a saddle point of the potential energy surface and perpendicular to the reaction coordinate) without ever returning to it. Local analytical equations of motion valid in the neighborhood of this planar surface have been derived as an expansion in Poisson brackets. We show that the mere presence of a saddle point implies that reactivity criteria can be quite simply formulated in terms of elements of thismore » series, irrespective of the shape of the potential energy function. Some of these elements are demonstrated to be equal to a sum of squares and thus to be necessarily positive, which has a profound impact on the dynamics. The method is then applied to a three-dimensional model describing an atom-diatom interaction. A particular relation between initial conditions is shown to generate a bundle of reactive trajectories that form reactive cylinders (or conduits) in phase space. This relation considerably reduces the phase space volume of initial conditions that generate recrossing-free trajectories. Loci in phase space of reactive initial conditions are presented. Reactivity is influenced by symmetry, as shown by a comparative study of collinear and bent transition states. Finally, it is argued that the rules that have been derived to generate reactive trajectories in classical mechanics are also useful to build up a reactive wave packet.« less

Pyrimidine Nucleobase Radical Reactivity in DNA and RNA.

PubMed

Greenberg, Marc M

2016-11-01

Nucleobase radicals are major products of the reactions between nucleic acids and hydroxyl radical, which is produced via the indirect effect of ionizing radiation. The nucleobase radicals also result from hydration of cation radicals that are produced via the direct effect of ionizing radiation. The role that nucleobase radicals play in strand scission has been investigated indirectly using ionizing radiation to generate them. More recently, the reactivity of nucleobase radicals resulting from formal hydrogen atom or hydroxyl radical addition to pyrimidines has been studied by independently generating the reactive intermediates via UV-photolysis of synthetic precursors. This approach has provided control over where the reactive intermediates are produced within biopolymers and facilitated studying their reactivity. The contributions to our understanding of pyrimidine nucleobase radical reactivity by this approach are summarized.

Pyrimidine nucleobase radical reactivity in DNA and RNA

NASA Astrophysics Data System (ADS)

Greenberg, Marc M.

2016-11-01

Nucleobase radicals are major products of the reactions between nucleic acids and hydroxyl radical, which is produced via the indirect effect of ionizing radiation. The nucleobase radicals also result from hydration of cation radicals that are produced via the direct effect of ionizing radiation. The role that nucleobase radicals play in strand scission has been investigated indirectly using ionizing radiation to generate them. More recently, the reactivity of nucleobase radicals resulting from formal hydrogen atom or hydroxyl radical addition to pyrimidines has been studied by independently generating the reactive intermediates via UV-photolysis of synthetic precursors. This approach has provided control over where the reactive intermediates are produced within biopolymers and facilitated studying their reactivity. The contributions to our understanding of pyrimidine nucleobase radical reactivity by this approach are summarized.

Nucleolin inhibitor GroA triggers reduction in epidermal growth factor receptor activation: Pharmacological implication for glial scarring after spinal cord injury.

PubMed

Goldshmit, Yona; Schokoroy Trangle, Sari; Afergan, Fabian; Iram, Tal; Pinkas-Kramarski, Ronit

2016-09-01

Glial scarring, formed by reactive astrocytes, is one of the major impediments for regeneration after spinal cord injury (SCI). Reactive astrocytes become hypertrophic, proliferate and secrete chondroitin sulphate proteoglycans into the extracellular matrix (ECM). Many studies have demonstrated that epidermal growth factor receptors (EGFR) can mediate astrocyte reactivity after neurotrauma. Previously we showed that there is crosstalk between nucleolin and EGFR that leads to increased EGFR activation followed by increased cell proliferation. Treatment with the nucleolin inhibitor GroA (AS1411) prevented these effects in vitro and in vivo. In this study, we hypothesized that similar interactions may mediate astrogliosis after SCI. Our results demonstrate that nucleolin and EGFR interaction may play a pivotal role in mediating astrocyte proliferation and reactivity after SCI. Moreover, we demonstrate that treatment with GroA reduces EGFR activation, astrocyte proliferation and chondroitin sulphate proteoglycans secretion, therefore promoting axonal regeneration and sprouting into the lesion site. Our results identify, for the first time, a role for the interaction between nucleolin and EGFR in astrocytes after SCI, indicating that nucleolin inhibitor GroA may be used as a novel treatment after neurotrauma. A major barrier for axonal regeneration after spinal cord injury is glial scar created by reactive and proliferating astrocytes. EGFR mediate astrocyte reactivity. We showed that inhibition of nucleolin by GroA, reduces EGFR activation, which results in attenuation of astrocyte reactivity and proliferation in vivo and in vitro. EGFR, epidermal growth factor receptor. © 2016 International Society for Neurochemistry.

Antibody Responses to Citrullinated and Noncitrullinated Antigens in the Sputum of Subjects With Rheumatoid Arthritis and Subjects at Risk for Development of Rheumatoid Arthritis.

PubMed

Demoruelle, M Kristen; Bowers, Emily; Lahey, Lauren J; Sokolove, Jeremy; Purmalek, Monica; Seto, Nickie L; Weisman, Michael H; Norris, Jill M; Kaplan, Mariana J; Holers, V Michael; Robinson, William H; Deane, Kevin D

2018-04-01

The location and mechanisms involved in the initial generation of autoantibodies to citrullinated and noncitrullinated proteins/peptides during the natural history of rheumatoid arthritis (RA) development is incompletely understood. This study sought to explore individual antibody responses to citrullinated and noncitrullinated proteins/peptides in the sputum and associations with neutrophil extracellular traps (NETs) in subjects at risk for the future development of RA. Serum and sputum samples were obtained from 41 RA-free subjects who were considered at risk for the development of RA based on familial or serologic risk factors, from 20 subjects classified as having RA, and from 22 healthy control subjects. Samples were evaluated using a bead-based array for IgG reactivity to 29 citrullinated proteins/peptides and 21 noncitrullinated proteins/peptides. Cutoff levels for antibody positivity were established in a separate control group. NET levels in the sputum were measured using sandwich enzyme-linked immunosorbent assays that quantitate DNA-myeloperoxidase and DNA-neutrophil elastase complexes. In at-risk subjects, antibody responses to the citrullinated forms of fibrinogen, apolipoprotein E, and fibronectin were highly prevalent. The most citrulline-specific antibodies in the sputum of at-risk subjects were those to fibrinogen, vimentin, and peptides of fibrinogen A and apolipoprotein A1. Patterns of sputum autoantibody positivity differed between at-risk subjects and subjects with RA. In at-risk subjects, increasing sputum NET levels significantly correlated with several citrullinated and some noncitrullinated antibody reactivities. These findings suggest that sputum antibody reactivity to particular citrullinated and noncitrullinated proteins/peptides is specific for RA and for subjects at risk of RA, and the association of these proteins/peptides with NETs may be a key feature of early RA-related autoimmunity in the lung. These results further support the hypothesis that the lung plays a role in early RA-related autoimmunity. © 2017, American College of Rheumatology.

Vitamin D Induces Increased Systolic Arterial Pressure via Vascular Reactivity and Mechanical Properties

PubMed Central

dos Santos, Priscila Portugal; Rafacho, Bruna Paola Murino; Gonçalves, Andréa de Freitas; Jaldin, Rodrigo Gibin; do Nascimento, Thiago Bruder; Silva, Marcondes Alves Barbosa; Cau, Stêfany Bruno Assis; Roscani, Meliza Goi; Azevedo, Paula Schimdt; Minicucci, Marcos Ferreira; Tostes, Rita de Cássia; Zornoff, Leonardo Antonio Memede; de Paiva, Sergio Alberto Rupp

2014-01-01

Background/Aims The aim of this study was to evaluate whether supplementation of high doses of cholecalciferol for two months in normotensive rats results in increased systolic arterial pressure and which are the mechanisms involved. Specifically, this study assesses the potential effect on cardiac output as well as the changes in aortic structure and functional properties. Methods Male Wistar rats were divided into three groups: 1) Control group (C, n = 20), with no supplementation of vitamin D, 2) VD3 (n = 19), supplemented with 3,000 IU vitamin D/kg of chow; 3) VD10 (n = 21), supplemented with 10,000 IU vitamin D/kg of chow. After two months, echocardiographic analyses, measurements of systolic arterial pressure (SAP), vascular reactivity, reactive oxygen species (ROS) generation, mechanical properties, histological analysis and metalloproteinase-2 and -9 activity were performed. Results SAP was higher in VD3 and VD10 than in C rats (p = 0.001). Echocardiographic variables were not different among groups. Responses to phenylephrine in endothelium-denuded aortas was higher in VD3 compared to the C group (p = 0.041). Vascular relaxation induced by acetylcholine (p = 0.023) and sodium nitroprusside (p = 0.005) was impaired in both supplemented groups compared to the C group and apocynin treatment reversed impaired vasodilation. Collagen volume fraction (<0.001) and MMP-2 activity (p = 0.025) was higher in VD10 group compared to the VD3 group. Elastin volume fraction was lower in VD10 than in C and yield point was lower in VD3 than in C. Conclusion Our findings support the view that vitamin D supplementation increases arterial pressure in normotensive rats and this is associated with structural and functional vascular changes, modulated by NADPH oxidase, nitric oxide, and extracellular matrix components. PMID:24921930

Ratiometric Imaging of Extracellular pH in Dental Biofilms.

PubMed

Schlafer, Sebastian; Dige, Irene

2016-03-09

The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites are not distributed evenly in dental biofilms. A complex interplay of sorption to and reaction with organic matter in the biofilm reduces the diffusion paths of solutes and creates steep gradients of reactive molecules, including organic acids, across the biofilm. Quantitative fluorescent microscopic methods, such as fluorescence life time imaging or pH ratiometry, can be employed to visualize pH in different microenvironments of dental biofilms. pH ratiometry exploits a pH-dependent shift in the fluorescent emission of pH-sensitive dyes. Calculation of the emission ratio at two different wavelengths allows determining local pH in microscopic images, irrespective of the concentration of the dye. Contrary to microelectrodes the technique allows monitoring both vertical and horizontal pH gradients in real-time without mechanically disturbing the biofilm. However, care must be taken to differentiate accurately between extra- and intracellular compartments of the biofilm. Here, the ratiometric dye, seminaphthorhodafluor-4F 5-(and-6) carboxylic acid (C-SNARF-4) is employed to monitor extracellular pH in in vivo grown dental biofilms of unknown species composition. Upon exposure to glucose the dye is up-concentrated inside all bacterial cells in the biofilms; it is thus used both as a universal bacterial stain and as a marker of extracellular pH. After confocal microscopic image acquisition, the bacterial biomass is removed from all pictures using digital image analysis software, which permits to exclusively calculate extracellular pH. pH ratiometry with the ratiometric dye is well-suited to study extracellular pH in thin biofilms of up to 75 µm thickness, but is limited to the pH range between 4.5 and 7.0.

High-Temperature Induced Changes of Extracellular Metabolites in Pleurotus ostreatus and Their Positive Effects on the Growth of Trichoderma asperellum.

PubMed

Qiu, Zhiheng; Wu, Xiangli; Zhang, Jinxia; Huang, Chenyang

2018-01-01

Pleurotus ostreatus is a widely cultivated edible fungus in China. Green mold disease of P. ostreatus which can seriously affect yield is a common disease during cultivation. It occurs mostly after P. ostreatus mycelia have been subjected to high temperatures. However, little information is available on the relationship between high temperature and green mold disease. The aim of this study is to prove that extracellular metabolites of P. ostreatus affected by high temperature can promote the growth of Trichoderma asperellum . After P. ostreatus mycelia was subjected to high temperature, the extracellular fluid of P. ostreatus showed a higher promoting effect on mycelial growth and conidial germination of T. asperellum . The thiobarbituric acid reactive substance (TBARS) content reached the maximum after 48 h at 36°C. A comprehensive metabolite profiling strategy involving gas chromatography-mass spectrometry (GC/MS) combined with liquid chromatography-mass spectrometry (LC/MS) was used to analyze the changes of extracellular metabolites in response to high temperature. A total of 141 differential metabolites were identified, including 84.4% up-regulated and 15.6% down-regulated. Exogenous metabolites whose concentrations were increased after high temperature were randomly selected, and nearly all of them were able to promote the mycelial growth and conidial germination of T. asperellum . The combination of all selected exogenous metabolites also has the promotion effects on the mycelial growth and conidial germination of T. asperellum in a given concentration range in vitro . Overall, these results provide a first view that high temperature affects the extracellular metabolites of P. ostreatus , and the extensive change in metabolites promotes T. asperellum growth.

ROS Production via P2Y1-PKC-NOX2 Is Triggered by Extracellular ATP after Electrical Stimulation of Skeletal Muscle Cells

PubMed Central

Díaz-Vegas, Alexis; Campos, Cristian A.; Contreras-Ferrat, Ariel; Casas, Mariana; Buvinic, Sonja; Jaimovich, Enrique; Espinosa, Alejandra

2015-01-01

During exercise, skeletal muscle produces reactive oxygen species (ROS) via NADPH oxidase (NOX2) while inducing cellular adaptations associated with contractile activity. The signals involved in this mechanism are still a matter of study. ATP is released from skeletal muscle during electrical stimulation and can autocrinely signal through purinergic receptors; we searched for an influence of this signal in ROS production. The aim of this work was to characterize ROS production induced by electrical stimulation and extracellular ATP. ROS production was measured using two alternative probes; chloromethyl-2,7- dichlorodihydrofluorescein diacetate or electroporation to express the hydrogen peroxide-sensitive protein Hyper. Electrical stimulation (ES) triggered a transient ROS increase in muscle fibers which was mimicked by extracellular ATP and was prevented by both carbenoxolone and suramin; antagonists of pannexin channel and purinergic receptors respectively. In addition, transient ROS increase was prevented by apyrase, an ecto-nucleotidase. MRS2365, a P2Y1 receptor agonist, induced a large signal while UTPyS (P2Y2 agonist) elicited a much smaller signal, similar to the one seen when using ATP plus MRS2179, an antagonist of P2Y1. Protein kinase C (PKC) inhibitors also blocked ES-induced ROS production. Our results indicate that physiological levels of electrical stimulation induce ROS production in skeletal muscle cells through release of extracellular ATP and activation of P2Y1 receptors. Use of selective NOX2 and PKC inhibitors suggests that ROS production induced by ES or extracellular ATP is mediated by NOX2 activated by PKC. PMID:26053483

A Bioelectrochemical Approach to Characterize Extracellular Electron Transfer by Synechocystis sp. PCC6803

PubMed Central

Cereda, Angelo; Hitchcock, Andrew; Symes, Mark D.; Cronin, Leroy; Bibby, Thomas S.; Jones, Anne K.

2014-01-01

Biophotovoltaic devices employ photosynthetic organisms at the anode of a microbial fuel cell to generate electrical power. Although a range of cyanobacteria and algae have been shown to generate photocurrent in devices of a multitude of architectures, mechanistic understanding of extracellular electron transfer by phototrophs remains minimal. Here we describe a mediatorless bioelectrochemical device to measure the electrogenic output of a planktonically grown cyanobacterium, Synechocystis sp. PCC6803. Light dependent production of current is measured, and its magnitude is shown to scale with microbial cell concentration and light intensity. Bioelectrochemical characterization of a Synechocystis mutant lacking Photosystem II demonstrates conclusively that production of the majority of photocurrent requires a functional water splitting aparatus and electrons are likely ultimately derived from water. This shows the potential of the device to rapidly and quantitatively characterize photocurrent production by genetically modified strains, an approach that can be used in future studies to delineate the mechanisms of cyanobacterial extracellular electron transport. PMID:24637387

Sustained generation of nitric oxide and control of mycobacterial infection requires argininosuccinate synthase 1

PubMed Central

Qualls, Joseph E.; Subramanian, Chitra; Rafi, Wasiulla; Smith, Amber M.; Balouzian, Liza; DeFreitas, Ashley A.; Shirey, Kari Ann; Reutterer, Benjamin; Kernbauer, Elisabeth; Stockinger, Silvia; Decker, Thomas; Miyairi, Isao; Vogel, Stefanie N.; Salgame, Padmini; Rock, Charles O.; Murray, Peter J.

2012-01-01

Nitric oxide (NO) defends against intracellular pathogens but its synthesis must be regulated due to cell and tissue toxicity. During infection, macrophages import extracellular arginine to synthesize NO, generating the byproduct citrulline. Accumulated intracellular citrulline is thought to fuel arginine synthesis catalyzed by argininosuccinate synthase (Ass1) and argininosuccinate lyase (Asl), which would lead to abundant NO production. Instead, we find that citrulline is exported from macrophages during early stages of NO production with < 2% retained for recycling via the Ass1-Asl pathway. Later, extracellular arginine is depleted, and Ass1 expression allows macrophages to synthesize arginine from imported citrulline to sustain NO output. Ass1-deficient macrophages fail to salvage citrulline in arginine-scarce conditions, leading to their inability to control mycobacteria infection. Thus, extracellular arginine fuels rapid NO production in activated macrophages, and citrulline recycling via Ass1 and Asl is a fail-safe system that sustains optimum NO production. PMID:22980328

Neutrophil extracellular traps enriched in oxidized mitochondrial DNA are interferogenic and contribute to lupus-like disease

PubMed Central

Lood, Christian; Blanco, Luz P.; Purmalek, Monica M.; Carmona-Rivera, Carmelo; De Ravin, Suk S.; Smith, Carolyne K.; Malech, Harry L.; Ledbetter, Jeffrey A.; Elkon, Keith B.; Kaplan, Mariana J.

2015-01-01

Neutrophil extracellular traps (NETs) are implicated in autoimmunity but how they are generated and their roles in sterile inflammation remain unclear. Ribonucleoprotein immune complexes, inducers of NETosis, require mitochondrial ROS for maximal NET stimulation. During this process, mitochondria become hypopolarized and translocate to the cell surface. Extracellular release of oxidized mitochondrial DNA is proinflammatory in vitro and, when injected into mice, stimulates type-I interferon (IFN) signaling through a pathway dependent on the DNA sensor, STING. Mitochondrial ROS is also necessary for spontaneous NETosis of low-density granulocytes from individuals with systemic lupus erythematosus (SLE). This was also observed in individuals with chronic granulomatous disease (CGD), which lack NADPH-oxidase activity, but still develop autoimmunity and type I-IFN signatures. Mitochondrial ROS inhibition in vivo reduces disease severity and type-I IFN responses in a mouse model of lupus. These findings highlight a role for mitochondria in the generation not only of NETs but also of pro-inflammatory oxidized mitochondrial DNA in autoimmune diseases. PMID:26779811

Identification of the Single Immunodominant Region of the Native Human CC Chemokine Receptor 6 Recognized by Mouse Monoclonal Antibodies.

PubMed

Dorgham, Karim; Dejou, Cécile; Piesse, Christophe; Gorochov, Guy; Pène, Jérôme; Yssel, Hans

2016-01-01

Chemokines and their receptors play an important role in cell trafficking and recruitment. The CCR6 chemokine receptor, selectively expressed on leukocyte populations, has been shown to play a deleterious role in the pathogenesis of various chronic inflammatory diseases and, as such, may constitute a prime target in the development of immunotherapeutic treatment. However, to date no neutralizing mouse monoclonal antibodies (mAbs) specific for this chemokine receptor have been reported, whereas information on small molecules capable of interfering with the interaction of CCR6 and its ligands is scant. Here, we report the failure to generate neutralizing mouse mAbs specific for human (hu)CCR6. Immunization of mice with peptides mimicking extracellular domains, potentially involved in CCR6 function, failed to induce Abs reactive with the native receptor. Although the use of NIH-3T3 cells expressing huCCR6 resulted in the isolation of mAbs specific for this receptor, they were not able to block the interaction between huCCR6 and huCCL20. Investigation of the anti-huCCR6 mAbs generated in the present study, as well as those commercially available, show that all mAbs invariably recognize a unique, non-neutralizing, immunodominant region in the first part of its N-terminal domain. Together, these results indicate that the generation of potential neutralizing anti-huCCR6 mAbs in the mouse is unlikely to succeed and that alternative techniques, such as the use of other animal species for immunization, might constitute a better approach to generate such a potentially therapeutic tool for the treatment of inflammatory disease.

Promyelocytic extracellular chromatin exacerbates coagulation and fibrinolysis in acute promyelocytic leukemia

PubMed Central

Cao, Muhua; Li, Tao; He, Zhangxiu; Wang, Lixiu; Yang, Xiaoyan; Kou, Yan; Zou, Lili; Dong, Xue; Novakovic, Valerie A.; Bi, Yayan; Kou, Junjie; Yu, Bo; Fang, Shaohong; Wang, Jinghua; Zhou, Jin

2017-01-01

Despite routine treatment of unselected acute promyelocytic leukemia (APL) with all-trans-retinoic acid (ATRA), early death because of hemorrhage remains unacceptably common, and the mechanism underlying this complication remains elusive. We have recently demonstrated that APL cells undergo a novel cell death program, termed ETosis, which involves release of extracellular chromatin. However, the role of promyelocytic extracellular chromatin in APL-associated coagulation remains unclear. Our objectives were to identify the novel role of ATRA-promoted extracellular chromatin in inducing a hypercoagulable and hyperfibrinolytic state in APL and to evaluate its interaction with fibrin and endothelial cells (ECs). Results from a series of coagulation assays have shown that promyelocytic extracellular chromatin increases thrombin and plasmin generation, causes a shortening of plasma clotting time of APL cells, and increases fibrin formation. DNase I but not anti-tissue factor antibody could inhibit these effects. Immunofluorescence staining showed that promyelocytic extracellular chromatin and phosphatidylserine on APL cells provide platforms for fibrin deposition and render clots more resistant to fibrinolysis. Additionally, coincubation assays revealed that promyelocytic extracellular chromatin is cytotoxic to ECs, converting them to a procoagulant phenotype. This cytotoxity was blocked by DNase I by 20% or activated protein C by 31%. Our current results thus delineate the pathogenic role of promyelocytic extracellular chromatin in APL coagulopathy. Furthermore, the remaining coagulation disturbance in high-risk APL patients after ATRA administration may be treatable by intrinsic pathway inhibition via accelerating extracellular chromatin degradation. PMID:28053193

Endogenous Production of Extracellular Adenosine by Trabecular Meshwork Cells: Potential Role in Outflow Regulation

PubMed Central

Wu, Jing; Li, Guorong; Luna, Coralia; Spasojevic, Ivan; Epstein, David L.; Gonzalez, Pedro

2012-01-01

Purpose. To investigate the mechanisms for endogenous production of extracellular adenosine in trabecular meshwork (TM) cells and evaluate its physiological relevance to the regulation of aqueous humor outflow facility. Methods. Extra-cellular levels of adenosine monophosphate (AMP) and adenosine in porcine trabecular meshwork (PTM) cells treated with adenosine triphosphate (ATP), AMP, cAMP or forskolin with or without specific inhibitors of phosphodiesterases (IBMX) and CD73 (AMPCP) were determined by high-pressure liquid chromatography fluorometry. Extracellular adenosine was also evaluated in cell cultures subjected to cyclic mechanical stress (CMS) (20% stretching; 1 Hz) and after disruption of lipid rafts with methyl-β-cyclodextrin. Expression of CD39 and CD73 in porcine TM cells and tissue were examined by Q-PCR and Western blot. The effect of inhibition of CD73 on outflow facility was evaluated in perfused living mouse eyes. Results. PTM cells generated extracellular adenosine from extracellular ATP and AMP but not from extracellular cAMP. Increased intracellular cAMP mediated by forskolin led to a significant increase in extracellular adenosine production that was not prevented by IBMX. Inhibition of CD73 resulted, in all cases, in a significant decrease in extracellular adenosine. CMS induced a significant activation of extracellular adenosine production. Inhibition of CD73 activity with AMPCP in living mouse eyes resulted in a significant decrease in outflow facility. Conclusions. These results support the concept that the extracellular adenosine pathway might play an important role in the homeostatic regulation of outflow resistance in the TM, and suggest a novel mechanism by which pathologic alteration of the TM, such as increased tissue rigidity, could lead to abnormal elevation of IOP in glaucoma. PMID:22997289

Proteus mirabilis fimbriae- and urease-dependent clusters assemble in an extracellular niche to initiate bladder stone formation

PubMed Central

Schaffer, Jessica N.; Norsworthy, Allison N.; Sun, Tung-Tien

2016-01-01

The catheter-associated uropathogen Proteus mirabilis frequently causes urinary stones, but little has been known about the initial stages of bladder colonization and stone formation. We found that P. mirabilis rapidly invades the bladder urothelium, but generally fails to establish an intracellular niche. Instead, it forms extracellular clusters in the bladder lumen, which form foci of mineral deposition consistent with development of urinary stones. These clusters elicit a robust neutrophil response, and we present evidence of neutrophil extracellular trap generation during experimental urinary tract infection. We identified two virulence factors required for cluster development: urease, which is required for urolithiasis, and mannose-resistant Proteus-like fimbriae. The extracellular cluster formation by P. mirabilis stands in direct contrast to uropathogenic Escherichia coli, which readily formed intracellular bacterial communities but not luminal clusters or urinary stones. We propose that extracellular clusters are a key mechanism of P. mirabilis survival and virulence in the bladder. PMID:27044107

Extracellular signals that define distinct and coexisting cell fates in Bacillus subtilis.

PubMed

López, Daniel; Kolter, Roberto

2010-03-01

The soil-dwelling bacterium Bacillus subtilis differentiates into distinct subpopulations of specialized cells that coexist within highly structured communities. The coordination and interplay between these cell types requires extensive extracellular communication driven mostly by sensing self-generated secreted signals. These extracellular signals activate a set of sensor kinases, which respond by phosphorylating three major regulatory proteins, Spo0A, DegU and ComA. Each phosphorylated regulator triggers a specific differentiation program while at the same time repressing other differentiation programs. This allows a cell to differentiate in response to a specific cue, even in the presence of other, possibly conflicting, signals. The sensor kinases involved respond to an eclectic group of extracellular signals, such as quorum-sensing molecules, natural products, temperature, pH or scarcity of nutrients. This article reviews the cascades of cell differentiation pathways that are triggered by sensing extracellular signals. We also present a tentative developmental model in which the diverse cell types sequentially differentiate to achieve the proper development of the bacterial community.

Total OH reactivity in a mediterranean forest of downy oaks

NASA Astrophysics Data System (ADS)

Zannoni, Nora; Gros, Valerie; Sarda, Roland; Lanza, Matteo; Bonsang, Bernard; Kalogridis, Cerise; Preunkert, Suzanne; Legrand, Michel; Jambert, Corinne; Boissard, Christophe; Lathiere, Juliette

2015-04-01

Forests emit large quantities of reactive molecules which can affect the concentration of the most important oxidizing agent in the atmosphere, the hydroxyl radical OH. There are still many unknowns on how biogenic compounds interact with the atmosphere. Among those, we still lack to fully understand the species that can potentially influence the atmospheric oxidative capacity and thus the OH cleansing effect over several forested areas. We conducted total OH reactivity measurements during spring 2014 inside and above the canopy height of a forest dominated at 80% by downy oaks in the Mediterranean basin (Observatoire Haute Provence site, France). Downy oak trees are capable to emit almost exclusively isoprene (~99%), the most abundant volatile organic compound and among the most reactive towards the OH radical. We measured the total OH reactivity with the Comparative Reactivity Method together with atmospheric concentrations of the primary compounds emitted by the forest, main secondary species generated from the oxidation of isoprene, and main atmospheric constituents. We then compared the OH reactivity inferred by measured compounds and their oxidation rate coefficients with the measured total OH reactivity. This approach permits to identify the presence of any primary emitted biogenic compound, unknown before and relevant for OH oxidation; or any secondary generated compound whose associated chemical mechanism is not well established. Our results show higher OH reactivity inside the canopy, with peaks up to 78 s-1, when isoprene concentration reached ~20 ppb due to temperature and PAR increase. Such high level of OH reactivity has only been observed in the tropics so far. Furthermore, our measured total OH reactivity closes the total amount of reactive species present in this specific forest, suggesting that we quantified precisely both the primary emitted species as well as the secondary generated products.

Age-dependent changes in inflammation and extracellular matrix in bovine oviduct epithelial cells during the post-ovulatory phase.

PubMed

Tanaka, Hazuki; Ohtsu, Ayaka; Shiratsuki, Shogo; Kawahara-Miki, Ryoka; Iwata, Hisataka; Kuwayama, Takehito; Shirasuna, Koumei

2016-09-01

The mammalian oviduct is an essential site for sperm storage, the transport of gametes, fertilization, and embryo development-functions that are aided by cytokines secreted from oviduct epithelial cells (OECs). Aging leads to cellular and organ dysfunction, with infertility associated with advanced maternal age. Few studies have investigated age-dependent changes in the oviduct as a possible cause of infertility, so we compared OECs from young (30-50 months) versus aged (more than 120 months) cattle. Next-generation sequencing was first used to identify age-related differences in gene expression. Several proinflammatory-related genes (including IL1B, IL1A, IL17C, IL8, S100A8, S100A9, and TNFA) were activated in OECs from aged (more than 120 months) compare to young (30-50 months) individuals, whereas genes associated with extracellular matrix-related factors (COLs, POSTN, BGN, and LUM) were down-regulation in aged OECs. Indeed, IL1 B and IL8 abundance was higher in aged OECs than in young OECs. Young OECs also tended to proliferate faster, and the revolution frequency of young, ciliated OECs was higher than that of their aged counterparts. In contrast, aged OECs possessed more F-actin, an actin cytoskeleton marker associated with reduced elasticity, and contained high levels of reactive oxygen species, which are mediators of inflammation and senescence. These different functional characteristics of bovine OECs during the post-ovulatory phase support the emerging concept of "inflammaging," that is, age-dependent inflammation. Mol. Reprod. Dev. 83: 815-826, 2016 © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Extracellular NGFR Spacers Allow Efficient Tracking and Enrichment of Fully Functional CAR-T Cells Co-Expressing a Suicide Gene

PubMed Central

Casucci, Monica; Falcone, Laura; Camisa, Barbara; Norelli, Margherita; Porcellini, Simona; Stornaiuolo, Anna; Ciceri, Fabio; Traversari, Catia; Bordignon, Claudio; Bonini, Chiara; Bondanza, Attilio

2018-01-01

Chimeric antigen receptor (CAR)-T cell immunotherapy is at the forefront of innovative cancer therapeutics. However, lack of standardization of cellular products within the same clinical trial and lack of harmonization between different trials have hindered the clear identification of efficacy and safety determinants that should be unveiled in order to advance the field. With the aim of facilitating the isolation and in vivo tracking of CAR-T cells, we here propose the inclusion within the CAR molecule of a novel extracellular spacer based on the low-affinity nerve-growth-factor receptor (NGFR). We screened four different spacer designs using as target antigen the CD44 isoform variant 6 (CD44v6). We successfully generated NGFR-spaced CD44v6 CAR-T cells that could be efficiently enriched with clinical-grade immuno-magnetic beads without negative consequences on subsequent expansion, immuno-phenotype, in vitro antitumor reactivity, and conditional ablation when co-expressing a suicide gene. Most importantly, these cells could be tracked with anti-NGFR monoclonal antibodies in NSG mice, where they expanded, persisted, and exerted potent antitumor effects against both high leukemia and myeloma burdens. Similar results were obtained with NGFR-enriched CAR-T cells specific for CD19 or CEA, suggesting the universality of this strategy. In conclusion, we have demonstrated that the incorporation of the NGFR marker gene within the CAR sequence allows for a single molecule to simultaneously work as a therapeutic and selection/tracking gene. Looking ahead, NGFR spacer enrichment might allow good manufacturing procedures-manufacturing of standardized CAR-T cell products with high therapeutic potential, which could be harmonized in different clinical trials and used in combination with a suicide gene for future application in the allogeneic setting. PMID:29619024

Quinone Reduction by the Na+-Translocating NADH Dehydrogenase Promotes Extracellular Superoxide Production in Vibrio cholerae▿ †

PubMed Central

Lin, Po-Chi; Türk, Karin; Häse, Claudia C.; Fritz, Günter; Steuber, Julia

2007-01-01

The pathogenicity of Vibrio cholerae is influenced by sodium ions which are actively extruded from the cell by the Na+-translocating NADH:quinone oxidoreductase (Na+-NQR). To study the function of the Na+-NQR in the respiratory chain of V. cholerae, we examined the formation of organic radicals and superoxide in a wild-type strain and a mutant strain lacking the Na+-NQR. Upon reduction with NADH, an organic radical was detected in native membranes by electron paramagnetic resonance spectroscopy which was assigned to ubisemiquinones generated by the Na+-NQR. The radical concentration increased from 0.2 mM at 0.08 mM Na+ to 0.4 mM at 14.7 mM Na+, indicating that the concentration of the coupling cation influences the redox state of the quinone pool in V. cholerae membranes. During respiration, V. cholerae cells produced extracellular superoxide with a specific activity of 10.2 nmol min−1 mg−1 in the wild type compared to 3.1 nmol min−1 mg−1 in the NQR deletion strain. Raising the Na+ concentration from 0.1 to 5 mM increased the rate of superoxide formation in the wild-type V. cholerae strain by at least 70%. Rates of respiratory H2O2 formation by wild-type V. cholerae cells (30.9 nmol min−1 mg−1) were threefold higher than rates observed with the mutant strain lacking the Na+-NQR (9.7 nmol min−1 mg−1). Our study shows that environmental Na+ could stimulate ubisemiquinone formation by the Na+-NQR and hereby enhance the production of reactive oxygen species formed during the autoxidation of reduced quinones. PMID:17322313

Characterization of N-glycosylation sites on the extracellular domain of NOX1/NADPH oxidase.

PubMed

Matsumoto, Misaki; Katsuyama, Masato; Iwata, Kazumi; Ibi, Masakazu; Zhang, Jia; Zhu, Kai; Nauseef, William M; Yabe-Nishimura, Chihiro

2014-03-01

Extensive evidence demonstrates the pathophysiological importance of NOX1, the catalytic subunit of superoxide-generating enzyme NADPH oxidase, as a source of reactive oxygen species in nonphagocytic cells. However, the biochemical properties of NOX1 have not been extensively characterized due to a lack of specific immunological tools. We used a newly raised NOX1 polyclonal antibody to investigate posttranslational modifications of NOX1 overexpressed in cultured cells and in the colon, where endogenous NOX1 is highly expressed. Immunoblots of lysates from cells expressing NOX1 revealed a doublet of 56 and 60kDa accompanied by a broad band of 60-90kDa. Based on differential sensitivity to glycosidases, the doublet was identified as two high-mannose-type glycoforms of NOX1, whereas the broad band represented NOX1 with complex-type N-linked oligosaccharides. Deglycosylated NOX1 migrated at ~53kDa and N-glycosylation was demonstrated in NOX1 derived from both rat and human. Site-directed mutagenesis identified N-glycosylation sites at Asn(161) and Asn(241) on the extracellular loop of mouse NOX1. Elimination of N-glycosylation on NOX1 did not affect its electron transferase activity, protein stability, targeting to the cell surface, or localization in F-actin-positive membrane protrusions. Taken together, these data identify the two specific sites of N-linked glycosylation of murine NOX1 and demonstrate that they are not required for normal enzyme activity, protein stability, and membrane trafficking. As is true for NOX2, the contribution of glycosylation in NOX1 to its biologic function(s) merits further study. Copyright © 2013 Elsevier Inc. All rights reserved.

Heme as a danger molecule in pathogen recognition.

PubMed

Wegiel, Barbara; Hauser, Carl J; Otterbein, Leo E

2015-12-01

Appropriate control of redox mechanisms are critical for and effective innate immune response, which employs multiple cell types, receptors and molecules that recognize danger signals when they reach the host. Recognition of pathogen-associated pattern molecules (PAMPs) is a fundamental host survival mechanism for efficient elimination of invading pathogens and resolution of the infection and inflammation. In addition to PAMPs, eukaryotic cells contain a plethora of intracellular molecules that are normally secured within the confines of the plasma membrane, but if liberated and encountered in the extracellular milieu can provoke rapid cell activation. These are known as Alarmins or Danger-Associated Molecular Patterns (DAMPs) and can be released actively by cells or passively as a result of sterile cellular injury after trauma, ischemia, or toxin-induced cell rupture. Both PAMPs and DAMPs are recognized by a series of cognate receptors that increase the generation of free radicals and activate specific signaling pathways that result in regulation of a variety of stress response, redox sensitive genes. Multiple mediators released, as cells die include, but are not limited to ATP, hydrogen peroxide, heme, formyl peptides, DNA or mitochondria provide the second signal to amplify immune responses. In this review, we will focus on how sterile and infective stimuli activate the stress response gene heme oxygenase-1 (Hmox1, HO-1), a master gene critical to an appropriate host response that is now recognized as one with enormous therapeutic potential. HO-1 gene expression is regulated in large part by redox-sensitive proteins including but not limited to nrf2. Both PAMPs and DAMPs increase the activation of nrf2 and HO-1. Heme is a powerful pro-oxidant and as such should be qualified as a DAMP. With its degradation by HO-1a molecule of carbon monoxide (CO) is generated that in turn serves as a bioactive signaling molecule. PAMPs such as bacterial endotoxin activate HO-1, and the CO that is generated diffuses into the extracellular milieu where it interacts with bacteria, altering their behavior to increase production of ATP, which then functions as a second signal danger molecule. This two-hit cycle scenario results in efficient and effective activation of host leukocytes to attack and clear bacteria in part via enhanced reactive oxygen species generation. We discuss this intimate communication that occurs between host and bacteria and how these molecules serve as critical regulators of the acute inflammatory response, the overall redox status of the cell, and survival of the host. Copyright © 2015 Elsevier Inc. All rights reserved.

A Model of Extracellular Enzymes in Free-Living Microbes: Which Strategy Pays Off?

PubMed Central

Thygesen, Uffe H.; Riemann, Lasse; Stedmon, Colin A.

2015-01-01

An initial modeling approach was applied to analyze how a single, nonmotile, free-living, heterotrophic bacterial cell may optimize the deployment of its extracellular enzymes. Free-living cells live in a dilute and complex substrate field, and to gain enough substrate, their extracellular enzymes must be utilized efficiently. The model revealed that surface-attached and free enzymes generate unique enzyme and substrate fields, and each deployment strategy has distinctive advantages. For a solitary cell, surface-attached enzymes are suggested to be the most cost-efficient strategy. This strategy entails potential substrates being reduced to very low concentrations. Free enzymes, on the other hand, generate a radically different substrate field, which suggests significant benefits for the strategy if free cells engage in social foraging or experience high substrate concentrations. Swimming has a slight positive effect for the attached-enzyme strategy, while the effect is negative for the free-enzyme strategy. The results of this study suggest that specific dissolved organic compounds in the ocean likely persist below a threshold concentration impervious to biological utilization. This could help explain the persistence and apparent refractory state of oceanic dissolved organic matter (DOM). Microbial extracellular enzyme strategies, therefore, have important implications for larger-scale processes, such as shaping the role of DOM in ocean carbon sequestration. PMID:26253668

Mechanisms of Resistance to Photodynamic Therapy

PubMed Central

Casas, Adriana; Di Venosa, Gabriela; Hasan, Tayyaba; Batlle, Alcira

2013-01-01

Photodynamic therapy (PDT) involves the administration of a photosensitizer (PS) followed by illumination with visible light, leading to generation of reactive oxygen species. The mechanisms of resistance to PDT ascribed to the PS may be shared with the general mechanisms of drug resistance, and are related to altered drug uptake and efflux rates or altered intracellular trafficking. As a second step, an increased inactivation of oxygen reactive species is also associated to PDT resistance via antioxidant detoxifying enzymes and activation of heat shock proteins. Induction of stress response genes also occurs after PDT, resulting in modulation of proliferation, cell detachment and inducing survival pathways among other multiple extracellular signalling events. In addition, an increased repair of induced damage to proteins, membranes and occasionally to DNA may happen. PDT-induced tissue hypoxia as a result of vascular damage and photochemical oxygen consumption may also contribute to the appearance of resistant cells. The structure of the PS is believed to be a key point in the development of resistance, being probably related to its particular subcellular localization. Although most of the features have already been described for chemoresistance, in many cases, no cross-resistance between PDT and chemotherapy has been reported. These findings are in line with the enhancement of PDT efficacy by combination with chemotherapy. The study of cross resistance in cells with developed resistance against a particular PS challenged against other PS is also highly complex and comprises different mechanisms. In this review we will classify the different features observed in PDT resistance, leading to a comparison with the mechanisms most commonly found in chemo resistant cells. PMID:21568910

40 CFR 98.90 - Definition of the source category.

Code of Federal Regulations, 2012 CFR

2012-07-01

... uses plasma-generated fluorine atoms and other reactive fluorine-containing fragments, that chemically... thin films are cleaned periodically using plasma-generated fluorine atoms and other reactive fluorine-containing fragments. (3) Any electronics production process in which wafers are cleaned using plasma...

40 CFR 98.90 - Definition of the source category.

Code of Federal Regulations, 2011 CFR

2011-07-01

... uses plasma-generated fluorine atoms and other reactive fluorine-containing fragments, that chemically... thin films are cleaned periodically using plasma-generated fluorine atoms and other reactive fluorine-containing fragments. (3) Any electronics production process in which wafers are cleaned using plasma...

40 CFR 98.90 - Definition of the source category.

Code of Federal Regulations, 2013 CFR

2013-07-01

... uses plasma-generated fluorine atoms and other reactive fluorine-containing fragments, that chemically... thin films are cleaned periodically using plasma-generated fluorine atoms and other reactive fluorine-containing fragments. (3) Any electronics production process in which wafers are cleaned using plasma...

40 CFR 98.90 - Definition of the source category.

Code of Federal Regulations, 2014 CFR

2014-07-01

... uses plasma-generated fluorine atoms and other reactive fluorine-containing fragments, that chemically... thin films are cleaned periodically using plasma-generated fluorine atoms and other reactive fluorine-containing fragments. (3) Any electronics production process in which wafers are cleaned using plasma...

In vitro genotoxicity of asbestos substitutes induced by coupled stimulation of dissolved high-valence ions and oxide radicals.

PubMed

Huo, Tingting; Dong, Faqin; Deng, Jianjun; Zhang, Qingbi; Ye, Wei; Zhang, Wei; Wang, Pingping; Sun, Dongping

2017-08-01

The wide use of asbestos and its substitutes has given rise to studies on their possible harmful effects on human health and environment. However, their toxic effects remain unclear. The present study was aimed to disclose the coupled effects of dissolved high-valence ions and oxide radicals using the in vitro cytotoxicity and genotoxicity of chrysotile (CA), nano-SiO 2 (NS), ceramic fiber (CF), glass fiber (GF), and rock wool (RW) on Chinese hamster lung cells V79. All samples induced cell mortality correlated well with the chemical SiO 2 content of asbestos substitutes and the amount of dissolved Si. Alkali or alkaline earth metal elements relieved mortality of V79 cells; Al 2 O 3 reinforced toxicity of materials. Asbestos substitutes generated lasting, increasing amount of acellular ·OH which formed at the fiber surface at sites with loose/unsaturated bonds, as well as by catalytic reaction through dissolved iron. Accumulated mechanical and radical stimulation induced the intracellular reactive oxygen species (ROS) elevation, morphology change, and deviating trans-membrane ion flux. The cellular ROS appeared as NS > GF > CF ≈ CA > RW, consistent with cell mortality rather than with acellular ·OH generation. Chromosomal and DNA lesions in V79 cells were not directly associated with the cellular ROS, while influenced by dissolved high-valence irons in the co-culture medium. In conclusion, ions from short-time dissolution of dust samples and the generation of extracellular ·OH presented combined effects in the elevation of intracellular ROS, which further synergistically induced cytotoxicity and genotoxicity.

Efficacy of simple short-term in vitro assays for predicting the potential of metal oxide nanoparticles to cause pulmonary inflammation.

PubMed

Lu, Senlin; Duffin, Rodger; Poland, Craig; Daly, Paul; Murphy, Fiona; Drost, Ellen; Macnee, William; Stone, Vicki; Donaldson, Ken

2009-02-01

There has been concern regarding risks from inhalation exposure to nanoparticles (NPs). The large number of particles requiring testing means that alternative approaches to animal testing are needed. We set out to determine whether short-term in vitro assays that assess intrinsic oxidative stress potential and membrane-damaging potency of a panel of metal oxide NPs can be used to predict their inflammogenic potency. For a panel of metal oxide NPs, we investigated intrinsic free radical generation, oxidative activity in an extracellular environment, cytotoxicity to lung epithelial cells, hemolysis, and inflammation potency in rat lungs. All exposures were carried out at equal surface area doses. Only nickel oxide (NiO) and alumina 2 caused significant lung inflammation when instilled into rat lungs at equal surface area, suggesting that these two had extra surface reactivity. We observed significant free radical generation with 4 of 13 metal oxides, only one of which was inflammogenic. Only 3 of 13 were significantly hemolytic, two of which were inflammogenic. Potency in generating free radicals in vitro did not predict inflammation, whereas alumina 2 had no free radical activity but was inflammogenic. The hemolysis assay was correct in predicting the proinflammatory potential of 12 of 13 of the particles examined. Using a battery of simple in vitro tests, it is possible to predict the inflammogenicity of metal oxide NPs, although some false-positive results are likely. More research using a larger panel is needed to confirm the efficacy and generality of this approach for metal oxide NPs.

77 FR 11109 - Reactive Power Resources; Notice of Technical Conference

Federal Register 2010, 2011, 2012, 2013, 2014

2012-02-24

... options for and cost of installing reactive power equipment at the time of interconnection as well as... DEPARTMENT OF ENERGY Federal Energy Regulatory Commission [Docket No. AD12-10-000] Reactive Power... the need for reactive power capability among newly interconnecting asynchronous generators and raises...

CYP3A-Mediated Generation of Aldehyde and Hydrazine in Atazanavir MetabolismS⃞

PubMed Central

Li, Feng; Lu, Jie; Wang, Laiyou

2011-01-01

Atazanavir (ATV) is an antiretroviral drug of the protease inhibitor class. Multiple adverse effects of ATV have been reported in clinical practice, such as jaundice, nausea, abdominal pain, and headache. The exact mechanisms of ATV-related adverse effects are unknown. It is generally accepted that a predominant pathway of drug-induced toxicity is through the generation of reactive metabolites. Our current study was designed to explore reactive metabolites of ATV. We used a metabolomic approach to profile ATV metabolism in mice and human liver microsomes. We identified 5 known and 13 novel ATV metabolites. Three potential reactive metabolites were detected and characterized for the first time: one aromatic aldehyde, one α-hydroxyaldehyde, and one hydrazine. These potential reactive metabolites were primarily generated by CYP3A. Our results provide a clue for studies on ATV-related adverse effects from the aspect of metabolic activation. Further studies are suggested to illustrate the impact of these potential reactive metabolites on ATV-related adverse effects. PMID:21148252

Mechanism of superoxide and hydrogen peroxide generation by human electron-transfer flavoprotein and pathological variants.

PubMed

Rodrigues, João V; Gomes, Cláudio M

2012-07-01

Reactive oxygen species production by mitochondrial enzymes plays a fundamental role both in cellular signaling and in the progression of dysfunctional states. However, sources of reactive oxygen species and the mechanisms by which enzymes produce these reactive species still remain elusive. We characterized the generation of reactive oxygen species by purified human electron-transfer flavoprotein (ETF), a mitochondrial enzyme that has a central role in the metabolism of lipids, amino acids, and choline. The results showed that ETF produces significant amounts of both superoxide and hydrogen peroxide in the presence of its partner enzyme medium-chain acyl-CoA dehydrogenase (MCAD). ETF-mediated production of reactive oxygen species is partially inhibited at high MCAD/ETF ratios, whereas it is enhanced at high ionic strength. Determination of the reduction potentials of ETF showed that thermodynamic properties of the FAD cofactor are changed upon formation of a complex between ETF and MCAD, supporting the notion that protein:protein interactions modulate the reactivity of the protein with dioxygen. Two pathogenic ETF variants were also studied to determine which factors modulate the reactivity toward molecular oxygen and promote reactive oxygen species production. The results obtained show that destabilized conformations and defective protein:protein interactions increase the ability of ETF to generate reactive oxygen species. A possible role for these processes in mitochondrial dysfunction in metabolic disorders of fatty acid β-oxidation is discussed. Copyright © 2012 Elsevier Inc. All rights reserved.

Reactive power generation in high speed induction machines by continuously occurring space-transients

NASA Astrophysics Data System (ADS)

Laithwaite, E. R.; Kuznetsov, S. B.

1980-09-01

A new technique of continuously generating reactive power from the stator of a brushless induction machine is conceived and tested on a 10-kw linear machine and on 35 and 150 rotary cage motors. An auxiliary magnetic wave traveling at rotor speed is artificially created by the space-transient attributable to the asymmetrical stator winding. At least two distinct windings of different pole-pitch must be incorporated. This rotor wave drifts in and out of phase repeatedly with the stator MMF wave proper and the resulting modulation of the airgap flux is used to generate reactive VA apart from that required for magnetization or leakage flux. The VAR generation effect increases with machine size, and leading power factor operation of the entire machine is viable for large industrial motors and power system induction generators.

ARSENIC SPECIES CAUSE RELEASE OF IRON FROM FERRITIN GENERATING REACTIVE OXYGEN SPECIES

EPA Science Inventory

ARSENIC SPECIES CAUSE RELEASE OF IRON FROM FERRITIN GENERATING REACTIVE OXYGEN SPECIES

Arsenic-associated cancer (lung, bladder, skin, liver, kidney) remains a significant world- wide public health problem (e.g., Taiwan, Chile, Bangladesh, India, China and Thailand). Rece...

Chemical stability and in chemico reactivity of 24 fragrance ingredients of concern for skin sensitization risk assessment.

PubMed

Avonto, Cristina; Wang, Mei; Chittiboyina, Amar G; Vukmanovic, Stanislav; Khan, Ikhlas A

2018-02-01

Twenty-four pure fragrance ingredients have been identified as potential concern for skin sensitization. Several of these compounds are chemically unstable and convert into reactive species upon exposure to air or light. In the present work, a systematic investigation of the correlation between chemical stability and reactivity has been undertaken. The compounds were subjected to forced photodegradation for three months and the chemical changes were studied with GC-MS. At the end of the stability study, two-thirds of the samples were found to be unstable. The generation of chemically reactive species was investigated using the in chemico HTS-DCYA assay. Eleven and fourteen compounds were chemically reactive before and after three months, respectively. A significant increase in reactivity upon degradation was found for isoeugenol, linalool, limonene, lyral, citronellol and geraniol; in the same conditions, the reactivity of hydroxycitronellal decreased. The non-reactive compounds α-isomethyl ionone, benzyl alcohol, amyl cinnamal and farnesol became reactive after photo-oxidative degradation. Overall, forced degradation resulted in four non-reactive fragrance compounds to display in chemico thiol reactivity, while ten out of 24 compounds remained inactive. Chemical degradation does not necessarily occur with generation of reactive species. Non-chemical activation may be involved for the 10 stable unreactive compounds. Copyright © 2017 Elsevier Ltd. All rights reserved.

The P2X7 Receptor in Inflammatory Diseases: Angel or Demon?

PubMed Central

Savio, Luiz E. B.; de Andrade Mello, Paola; da Silva, Cleide Gonçalves; Coutinho-Silva, Robson

2018-01-01

Under physiological conditions, adenosine triphosphate (ATP) is present at low levels in the extracellular milieu, being massively released by stressed or dying cells. Once outside the cells, ATP and related nucleotides/nucleoside generated by ectonucleotidases mediate a high evolutionary conserved signaling system: the purinergic signaling, which is involved in a variety of pathological conditions, including inflammatory diseases. Extracellular ATP has been considered an endogenous adjuvant that can initiate inflammation by acting as a danger signal through the activation of purinergic type 2 receptors—P2 receptors (P2Y G-protein coupled receptors and P2X ligand-gated ion channels). Among the P2 receptors, the P2X7 receptor is the most extensively studied from an immunological perspective, being involved in both innate and adaptive immune responses. P2X7 receptor activation induces large-scale ATP release via its intrinsic ability to form a membrane pore or in association with pannexin hemichannels, boosting purinergic signaling. ATP acting via P2X7 receptor is the second signal to the inflammasome activation, inducing both maturation and release of pro-inflammatory cytokines, such as IL-1β and IL-18, and the production of reactive nitrogen and oxygen species. Furthermore, the P2X7 receptor is involved in caspases activation, as well as in apoptosis induction. During adaptive immune response, P2X7 receptor modulates the balance between the generation of T helper type 17 (Th17) and T regulatory (Treg) lymphocytes. Therefore, this receptor is involved in several inflammatory pathological conditions. In infectious diseases and cancer, P2X7 receptor can have different and contrasting effects, being an angel or a demon depending on its level of activation, cell studied, type of pathogen, and severity of infection. In neuroinflammatory and neurodegenerative diseases, P2X7 upregulation and function appears to contribute to disease progression. In this review, we deeply discuss P2X7 receptor dual function and its pharmacological modulation in the context of different pathologies, and we also highlight the P2X7 receptor as a potential target to treat inflammatory related diseases. PMID:29467654

A spectral element method with adaptive segmentation for accurately simulating extracellular electrical stimulation of neurons.

PubMed

Eiber, Calvin D; Dokos, Socrates; Lovell, Nigel H; Suaning, Gregg J

2017-05-01

The capacity to quickly and accurately simulate extracellular stimulation of neurons is essential to the design of next-generation neural prostheses. Existing platforms for simulating neurons are largely based on finite-difference techniques; due to the complex geometries involved, the more powerful spectral or differential quadrature techniques cannot be applied directly. This paper presents a mathematical basis for the application of a spectral element method to the problem of simulating the extracellular stimulation of retinal neurons, which is readily extensible to neural fibers of any kind. The activating function formalism is extended to arbitrary neuron geometries, and a segmentation method to guarantee an appropriate choice of collocation points is presented. Differential quadrature may then be applied to efficiently solve the resulting cable equations. The capacity for this model to simulate action potentials propagating through branching structures and to predict minimum extracellular stimulation thresholds for individual neurons is demonstrated. The presented model is validated against published values for extracellular stimulation threshold and conduction velocity for realistic physiological parameter values. This model suggests that convoluted axon geometries are more readily activated by extracellular stimulation than linear axon geometries, which may have ramifications for the design of neural prostheses.

Reactive uptake of HOCl to laboratory generated sea salt particles and nascent sea-spray aerosol

NASA Astrophysics Data System (ADS)

Campbell, N. R.; Ryder, O. S.; Bertram, T. H.

2013-12-01

Field observations suggest that the reactive uptake of HOCl on marine aerosol particles is an important source of chlorine radicals, particularly under low NOx conditions. However to date, laboratory measurements disagree on the magnitude of the reactive uptake coefficient for HOCl by a factor of 5 (γ(HOCl) ranges between 0.0004 and 0.0018), and there are no measurements of γ(HOCl) on nascent sea-spray aerosol. Here, we present measurements of the reactive uptake of HOCl to laboratory generated sodium chloride and sea-spray aerosol particles generated in a novel Marine Aerosol Reference Tank (MART), coupled to an entrained aerosol flow reactor and Chemical Ionization Mass Spectrometer (CIMS). Measurements of γ(HOCl) retrieved here are compared against those in the literature, and the role of organic coatings on nascent sea-spray aerosol is explored.

Matrix elasticity regulates the optimal cardiac myocyte shape for contractility

PubMed Central

McCain, Megan L.; Yuan, Hongyan; Pasqualini, Francesco S.; Campbell, Patrick H.

2014-01-01

Concentric hypertrophy is characterized by ventricular wall thickening, fibrosis, and decreased myocyte length-to-width aspect ratio. Ventricular thickening is considered compensatory because it reduces wall stress, but the functional consequences of cell shape remodeling in this pathological setting are unknown. We hypothesized that decreases in myocyte aspect ratio allow myocytes to maximize contractility when the extracellular matrix becomes stiffer due to conditions such as fibrosis. To test this, we engineered neonatal rat ventricular myocytes into rectangles mimicking the 2-D profiles of healthy and hypertrophied myocytes on hydrogels with moderate (13 kPa) and high (90 kPa) elastic moduli. Actin alignment was unaffected by matrix elasticity, but sarcomere content was typically higher on stiff gels. Microtubule polymerization was higher on stiff gels, implying increased intracellular elastic modulus. On moderate gels, myocytes with moderate aspect ratios (∼7:1) generated the most peak systolic work compared with other cell shapes. However, on stiffer gels, low aspect ratios (∼2:1) generated the most peak systolic work. To compare the relative contributions of intracellular vs. extracellular elasticity to contractility, we developed an analytical model and used our experimental data to fit unknown parameters. Our model predicted that matrix elasticity dominates over intracellular elasticity, suggesting that the extracellular matrix may potentially be a more effective therapeutic target than microtubules. Our data and model suggest that myocytes with lower aspect ratios have a functional advantage when the elasticity of the extracellular matrix decreases due to conditions such as fibrosis, highlighting the role of the extracellular matrix in cardiac disease. PMID:24682394

Extracellular ATP is Differentially Metabolized on Papillary Thyroid Carcinoma Cells Surface in Comparison to Normal Cells.

PubMed

Bertoni, Ana Paula Santin; de Campos, Rafael Paschoal; Tsao, Marisa; Braganhol, Elizandra; Furlanetto, Tania Weber; Wink, Márcia Rosângela

2018-02-17

The incidence of differentiated thyroid cancer has been increasing. Nevertheless, its molecular mechanisms are not well understood. In recent years, extracellular nucleotides and nucleosides have emerged as important modulators of tumor microenvironment. Extracellular ATP is mainly hydrolyzed by NTPDase1/CD39 and NTPDase2/CD39L1, generating AMP, which is hydrolyzed by ecto-5'-nucleotidase (CD73) to adenosine, a possible promoter of tumor growth and metastasis. There are no studies evaluating the expression and functionality of these ectonucleotidases on normal or tumor-derived thyroid cells. Thus, we investigated the ability of thyroid cancer cells to hydrolyze extracellular ATP generating adenosine, and the expression of ecto-enzymes, as compared to normal cells. We found that normal thyroid derived cells presented a higher ability to hydrolyze ATP and higher mRNA levels for ENTDP1-2, when compared to papillary thyroid carcinoma (PTC) derived cells, which had a higher ability to hydrolyze AMP and expressed CD73 mRNA and protein at higher levels. In addition, adenosine induced an increase in proliferation and migration in PTC derived cells, whose effect was blocked by APCP, a non-hydrolysable ADP analogue, which is an inhibitor of CD73. Taken together, these results showed that thyroid follicular cells have a functional purinergic signaling. The higher expression of CD73 in PTC derived cells might favor the accumulation of extracellular adenosine in the tumor microenvironment, which could promote tumor progression. Therefore, as already shown for other tumors, the purinergic signaling should be considered a potential target for thyroid cancer management and treatment.

Mechanical Control of Myotendinous Junction Formation and Tendon Differentiation during Development.

PubMed

Valdivia, Mauricio; Vega-Macaya, Franco; Olguín, Patricio

2017-01-01

The development of the musculoskeletal system is a great model to study the interplay between chemical and mechanical inter-tissue signaling in cell adhesion, tissue morphogenesis and differentiation. In both vertebrates and invertebrates (e.g., Drosophila melanogaster ) the formation of muscle-tendon interaction generates mechanical forces which are required for myotendinous junction maturation and tissue differentiation. In addition, these forces must be withstood by muscles and tendons in order to prevent detachment from each other, deformation or even losing their integrity. Extracellular matrix remodeling at the myotendinous junction is key to resist mechanical load generated by muscle contraction. Recent evidences in vertebrates indicate that mechanical forces generated during junction formation regulate chemical signaling leading to extracellular matrix remodeling, however, the mechanotransduction mechanisms associated to this response remains elusive. In addition to extracellular matrix remodeling, the ability of Drosophila tendon-cells to bear mechanical load depends on rearrangement of tendon cell cytoskeleton, thus studying the molecular mechanisms involved in this process is critical to understand the contribution of mechanical forces to the development of the musculoskeletal system. Here, we review recent findings regarding the role of chemical and mechanical signaling in myotendinous junction formation and tendon differentiation, and discuss molecular mechanisms of mechanotransduction that may allow tendon cells to withstand mechanical load during development of the musculoskeletal system.

Detection of abnormal extracellular matrix in the interstitium of regenerating renal tubules.

PubMed

Minuth, Will W; Denk, Lucia

2014-12-15

Stem/progenitor cells are promising candidates for the regeneration of parenchyma in acute and chronic renal failure. However, recent data exhibit that survival of stem/progenitor cells after implantation in diseased renal parenchyma is restricted. To elaborate basic parameters improving survival, cell seeding was simulated under advanced in vitro conditions. After isolation, renal stem/progenitor cells were mounted in a polyester interstitium for perfusion culture. During generation of tubules, chemically defined CO2 Independent Medium or Leibovitz's L-15 Medium was applied. Specimens were then fixed for transmission electron microscopy to analyze morphological features in generated tubules. Fixation in conventional glutaraldehyde (GA) solution shows development of tubules each exhibiting a polarized epithelium, an intact basal lamina and an inconspicuous interstitium. In contrast, special fixation of specimens in GA solution containing cupromeronic blue, ruthenium red or tannic acid unveils previously not visible extracellular matrix. Control experiments elucidate that a comparable extracellular matrix is not present in the interstitium of the matured kidney. Thus, generation of renal tubules in combination with advanced fixation of specimens for electron microscopy demonstrates that development of abnormal features in the newly developed interstitium has to be considered, when repair of renal parenchyma is performed by implantation of stem/progenitor cells.

A mechanism for hot-spot generation in a reactive two-dimensional sheared viscous layer

NASA Astrophysics Data System (ADS)

Timms, Robert; Purvis, Richard; Curtis, John P.

2018-05-01

A two-dimensional model for the non-uniform melting of a thin sheared viscous layer is developed. An asymptotic solution is presented for both a non-reactive and a reactive material. It is shown that the melt front is linearly stable to small perturbations in the non-reactive case, but becomes linearly unstable upon introduction of an Arrhenius source term to model the chemical reaction. Results demonstrate that non-uniform melting acts as a mechanism to generate hot spots that are found to be sufficient to reduce the time to ignition when compared with the corresponding one-dimensional model of melting.

Generation of memory B cells and their reactivation.

PubMed

Inoue, Takeshi; Moran, Imogen; Shinnakasu, Ryo; Phan, Tri Giang; Kurosaki, Tomohiro

2018-05-01

The successful establishment of humoral memory response depends on at least two layers of defense. Pre-existing protective antibodies secreted by long-lived plasma cells act as a first line of defense against reinfection ("constitutive humoral memory"). Previously, a second line of defense in which pathogen-experienced memory B cells are rapidly reactivated to produce antibodies ("reactive humoral memory"), was considered as simply a back-up system for the first line (particularly for re-infection with homologous viruses). However, in the case of re-infection with similar but different strains of viruses, or in response to viral escape mutants, the reactive humoral memory plays a crucial role. Here, we review recent progress in our understanding of how memory B cells are generated in the pre-GC stage and during the GC reaction, and how these memory B cells are robustly reactivated with the help of memory Tfh cells to generate the secondary antibody response. In addition, we discuss how these advances may be relevant to the quest for a vaccine that can induce broadly reactive antibodies against influenza and HIV. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Reactive oxygen species generation and signaling in plants

PubMed Central

Tripathy, Baishnab Charan; Oelmüller, Ralf

2012-01-01

The introduction of molecular oxygen into the atmosphere was accompanied by the generation of reactive oxygen species (ROS) as side products of many biochemical reactions. ROS are permanently generated in plastids, peroxisomes, mitochiondria, the cytosol and the apoplast. Imbalance between ROS generation and safe detoxification generates oxidative stress and the accumulating ROS are harmful for the plants. On the other hand, specific ROS function as signaling molecules and activate signal transduction processes in response to various stresses. Here, we summarize the generation of ROS in the different cellular compartments and the signaling processes which are induced by ROS. PMID:23072988

Striatal dopamine neurotransmission: regulation of release and uptake

PubMed Central

Sulzer, David; Cragg, Stephanie J.; Rice, Margaret E.

2016-01-01

Dopamine (DA) transmission is governed by processes that regulate release from axonal boutons in the forebrain and the somatodendritic compartment in midbrain, and by clearance by the DA transporter, diffusion, and extracellular metabolism. We review how axonal DA release is regulated by neuronal activity and by autoreceptors and heteroreceptors, and address how quantal release events are regulated in size and frequency. In brain regions densely innervated by DA axons, DA clearance is due predominantly to uptake by the DA transporter, whereas in cortex, midbrain, and other regions with relatively sparse DA inputs, the norepinephrine transporter and diffusion are involved. We discuss the role of DA uptake in restricting the sphere of influence of DA and in temporal accumulation of extracellular DA levels upon successive action potentials. The tonic discharge activity of DA neurons may be translated into a tonic extracellular DA level, whereas their bursting activity can generate discrete extracellular DA transients. PMID:27141430

Tubulointerstitial nephritis antigen: an extracellular matrix protein that selectively regulates tubulogenesis vs. glomerulogenesis during mammalian renal development.

PubMed

Kanwar, Y S; Kumar, A; Yang, Q; Tian, Y; Wada, J; Kashihara, N; Wallner, E I

1999-09-28

Tubulointerstitial nephritis antigen (TIN-ag) is an extracellular matrix protein and is expressed in the renal tubular basement membranes. Its role in metanephric development was investigated. TIN-ag cDNA, isolated from the newborn mouse library, had an ORF of 1,425 nucleotides, a putative signal sequence, and an ATP/GTP-binding site. The translated sequence had approximately 80% identity with rabbit TIN-ag. Among various tissues, TIN-ag mRNA was primarily expressed in the newborn kidney. In the embryonic metanephros, TIN-ag expression was confined to the distal convolution or pole of the S-shaped body, the segment of the nascent nephron that is the progenitor of renal tubules. Treatment with TIN-ag antisense oligodeoxynucleotide induced dysmorphogenesis of the embryonic metanephroi, malformation of the S-shaped body, and a decrease in the tubular population, whereas the glomeruli were unaffected. Treatment also led to a decrease of TIN-Ag mRNA, de novo synthesis of TIN-ag protein, and its antibody reactivity. The mRNA expression of glomerular epithelial protein 1 (a marker for renal podocytes), anti-heparan-sulfate-proteoglycan antibody reactivity, and wheat germ agglutinin lectin staining of the metanephros were unaffected. The anti-TIN-ag antibody treatment also caused deformation of the S-shaped body and a reduction in the tubular population, whereas the glomeruli were unchanged. The data suggest that the TIN-ag, unlike other basement membrane proteins, selectively regulates tubulogenesis, whereas glomerulogenesis is largely unaffected.

Use of cephalosporins in patients with immediate penicillin hypersensitivity: cross-reactivity revisited.

PubMed

Lee, Q U

2014-10-01

A 10% cross-reactivity rate is commonly cited between penicillins and cephalosporins. However, this figure originated from studies in the 1960s and 1970s which included first-generation cephalosporins with similar side-chains to penicillins. Cephalosporins were frequently contaminated by trace amount of penicillins at that time. The side-chain hypothesis for beta-lactam hypersensitivity is supported by abundant scientific evidence. Newer generations of cephalosporins possess side-chains that are dissimilar to those of penicillins, leading to low cross-reactivity. In the assessment of cross-reactivity between penicillins and cephalosporins, one has to take into account the background beta-lactam hypersensitivity, which occurs in up to 10% of patients. Cross-reactivity based on skin testing or in-vitro test occurs in up to 50% and 69% of cases, respectively. Clinical reactivity and drug challenge test suggest an average cross-reactivity rate of only 4.3%. For third- and fourth-generation cephalosporins, the rate is probably less than 1%. Recent international guidelines are in keeping with a low cross-reactivity rate. Despite that, the medical community in Hong Kong remains unnecessarily skeptical. Use of cephalosporins in patients with penicillin hypersensitivity begins with detailed history and physical examination. Clinicians can choose a cephalosporin with a different side-chain. Skin test for penicillin is not predictive of cephalosporin hypersensitivity, while cephalosporin skin test is not sensitive. Drug provocation test by experienced personnel remains the best way to exclude or confirm the diagnosis of drug hypersensitivity and to find a safe alternative for future use. A personalised approach to cross-reactivity is advocated.

Differential recognition of terminal extracellular Plasmodium falciparum VAR2CSA domains by sera from multigravid, malaria-exposed Malian women.

PubMed

Travassos, Mark A; Coulibaly, Drissa; Bailey, Jason A; Niangaly, Amadou; Adams, Matthew; Nyunt, Myaing M; Ouattara, Amed; Lyke, Kirsten E; Laurens, Matthew B; Pablo, Jozelyn; Jasinskas, Algis; Nakajima, Rie; Berry, Andrea A; Takala-Harrison, Shannon; Kone, Abdoulaye K; Kouriba, Bourema; Rowe, J Alexandra; Doumbo, Ogobara K; Thera, Mahamadou A; Laufer, Miriam K; Felgner, Philip L; Plowe, Christopher V

2015-06-01

The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family mediates parasite sequestration in small capillaries through tissue-specific cytoadherence. The best characterized of these proteins is VAR2CSA, which is expressed on the surface of infected erythrocytes that bind to chondroitin sulfate in the placental matrix. Antibodies to VAR2CSA prevent placental cytoadherence and protect against placental malaria. The size and complexity of the VAR2CSA protein pose challenges for vaccine development, but smaller constitutive domains may be suitable for subunit vaccine development. A protein microarray was printed to include five overlapping fragments of the 3D7 VAR2CSA extracellular region. Malian women with a history of at least one pregnancy had antibody recognition of four of these fragments and had stronger reactivity against the two distal fragments than did nulliparous women, children, and men from Mali, suggesting that the C-terminal extracellular VAR2CSA domains are a potential focus of protective immunity. With carefully chosen sera from longitudinal studies of pregnant women, this approach has the potential to identify seroreactive VAR2CSA domains associated with protective immunity against pregnancy-associated malaria. © The American Society of Tropical Medicine and Hygiene.

Role of RpoS in virulence and stress tolerance of the plant pathogen Erwinia carotovora subsp. carotovora.

PubMed

Andersson, R A; Kõiv, V; Norman-Setterblad, C; Pirhonen, M

1999-12-01

The plant-pathogenic bacterium Erwinia carotovora subsp. carotovora causes plant disease mainly through a number of extracellular plant-cell-wall-degrading enzymes. In this study, the ability of an rpoS mutant of the Er. carotovora subsp. carotovora strain SCC3193 to infect plants and withstand environmental stress was characterized. This mutant was found to be sensitive to osmotic and oxidative stresses in vitro and to be deficient in glycogen accumulation. The production of extracellular enzymes in vitro was similar in the mutant and in the wild-type strains. However, the rpoS mutant caused more severe symptoms than the wild-type strain on tobacco plants and also produced more extracellular enzymes in planta, but did not grow to higher cell density in planta compared to the wild-type strain. When tested on plants with reduced catalase activities, which show higher levels of reactive oxygen species, the rpoS mutant was found to cause lower symptom levels and to have impaired growth. In addition, the mutant was unable to compete with the wild-type strain in planta and in vitro. These results suggest that a functional rpoS gene is needed mainly for survival in a competitive environment and during stress conditions, and not for effective infection of plants.

Levels of intra- and extracellular heat shock protein 60 in Kawasaki disease patients treated with intravenous immunoglobulin.

PubMed

Yin Ji, Xu; Kang, Mi-Ran; Choi, Jong-Sung; Jeon, Hak-Soo; Han, Heon-Seok; Kim, Ji-Yoon; Son, Bo-Ra; Lee, Young-Min; Hahn, Youn-Soo

2007-09-01

Immune reactivity to autologous heat shock protein 60 (HSP60) has been reported to be associated with a favorable prognosis in autoimmune diseases. To provide a clue for the possible role of HSP60 in Kawasaki disease (KD), we investigated the levels of intra- and extracellular HSP60 in the course of KD. In KD patients, autologous HSP60 was abundantly expressed in CD11c(+) cells during the acute phase and subsequently decreased during the subacute phase. Most of HSP60-expressing CD11c(+) cells observed in the acute phase was composed of CD11c(low) cells instead of CD11c(high) cells, which were dominant in the subacute phase. In contrast, circulating HSP60 levels were higher in the subacute phase than those in the acute phase, reflecting higher level of HSP60 exposure to the immune system of patients during recovery. These changes in the levels of intra- and extracellular HSP60 were not observed in patients with other febrile diseases. The observed features of HSP60 expression in patients with KD are in favor of a role for autologous HSP60 as a regulator for control of inflammation, rather than a proinflammatory mediator in KD.

Method for generating a highly reactive plasma for exhaust gas after treatment and enhanced catalyst reactivity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Whealton, John H.; Hanson, Gregory R.; Storey, John M.

This patent application describes a method and apparatus of exhaust gas remediation that enhance the reactivity of the material catalysts found within catalytic converters of cars, trucks, and power stations.

A Novel α2/α4 Subtype-selective Positive Allosteric Modulator of Nicotinic Acetylcholine Receptors Acting from the C-tail of an α Subunit*

PubMed Central

Wang, Jingyi; Kuryatov, Alexander; Jin, Zhuang; Norleans, Jack; Kamenecka, Theodore M.; Kenny, Paul J.; Lindstrom, Jon

2015-01-01

Positive allosteric modulators (PAMs) of nicotinic acetylcholine receptors (nAChR) are important therapeutic candidates as well as valuable research tools. We identified a novel type II PAM, (R)-7-bromo-N-(piperidin-3-yl)benzo[b]thiophene-2-carboxamide (Br-PBTC), which both increases activation and reactivates desensitized nAChRs. This compound increases acetylcholine-evoked responses of α2* and α4* nAChRs but is without effect on α3* or α6* nAChRs (* indicates the presence of other nAChR subunits). Br-BPTC acts from the C-terminal extracellular sequences of α4 subunits, which is also a PAM site for steroid hormone estrogens such as 17β-estradiol. Br-PBTC is much more potent than estrogens. Like 17β-estradiol, the non-steroid Br-PBTC only requires one α4 subunit to potentiate nAChR function, and its potentiation is stronger with more α4 subunits. This feature enables Br-BPTC to potentiate activation of (α4β2)(α6β2)β3 but not (α6β2)2β3 nAChRs. Therefore, this compound is potentially useful in vivo for determining functions of different α6* nAChR subtypes. Besides activation, Br-BPTC affects desensitization of nAChRs induced by sustained exposure to agonists. After minutes of exposure to agonists, Br-PBTC reactivated short term desensitized nAChRs that have at least two α4 subunits but not those with only one. Three α4 subunits were required for Br-BPTC to reactivate long term desensitized nAChRs. These data suggest that higher PAM occupancy promotes channel opening more efficiently and overcomes short and long term desensitization. This C-terminal extracellular domain could be a target for developing subtype or state-selective drugs for nAChRs. PMID:26432642

Sensitive luminescent reporter viruses reveal appreciable release of hepatitis C virus NS5A protein into the extracellular environment.

PubMed

Eyre, Nicholas S; Aloia, Amanda L; Joyce, Michael A; Chulanetra, Monrat; Tyrrell, D Lorne; Beard, Michael R

2017-07-01

The HCV NS5A protein is essential for viral RNA replication and virus particle assembly. To study the viral replication cycle and NS5A biology we generated an infectious HCV construct with a NanoLuciferase (NLuc) insertion within NS5A. Surprisingly, beyond its utility as a sensitive reporter of cytoplasmic viral RNA replication, we also observed strong luminescence in cell culture fluids. Further analysis using assembly-defective viruses and subgenomic replicons revealed that infectious virus production was not required for extracellular NS5A-NLuc activity but was associated with enrichment of extracellular NS5A-NLuc in intermediate-density fractions similar to those of exosomes and virus particles. Additionally, BRET analysis indicated that intracellular and extracellular forms of NS5A may adopt differing conformations. Importantly, infection studies using a human liver chimeric mouse model confirmed robust infection in vivo and ready detection of NLuc activity in serum. We hypothesise that the presence of NS5A in extracellular fluids contributes to HCV pathogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

The Serotonin Transporter Undergoes Constitutive Internalization and Is Primarily Sorted to Late Endosomes and Lysosomal Degradation*

PubMed Central

Rahbek-Clemmensen, Troels; Bay, Tina; Eriksen, Jacob; Gether, Ulrik; Jørgensen, Trine Nygaard

2014-01-01

The serotonin transporter (SERT) plays a critical role in regulating serotonin signaling by mediating reuptake of serotonin from the extracellular space. The molecular and cellular mechanisms controlling SERT levels in the membrane remain poorly understood. To study trafficking of the surface resident SERT, two functional epitope-tagged variants were generated. Fusion of a FLAG-tagged one-transmembrane segment protein Tac to the SERT N terminus generated a transporter with an extracellular epitope suited for trafficking studies (TacSERT). Likewise, a construct with an extracellular antibody epitope was generated by introducing an HA (hemagglutinin) tag in the extracellular loop 2 of SERT (HA-SERT). By using TacSERT and HA-SERT in antibody-based internalization assays, we show that SERT undergoes constitutive internalization in a dynamin-dependent manner. Confocal images of constitutively internalized SERT demonstrated that SERT primarily co-localized with the late endosomal/lysosomal marker Rab7, whereas little co-localization was observed with the Rab11, a marker of the “long loop” recycling pathway. This sorting pattern was distinct from that of a prototypical recycling membrane protein, the β2-adrenergic receptor. Furthermore, internalized SERT co-localized with the lysosomal marker LysoTracker and not with transferrin. The sorting pattern was further confirmed by visualizing internalization of SERT using the fluorescent cocaine analog JHC1-64 and by reversible and pulse-chase biotinylation assays showing evidence for lysosomal degradation of the internalized transporter. Finally, we found that SERT internalized in response to stimulation with 12-myristate 13-acetate co-localized primarily with Rab7- and LysoTracker-positive compartments. We conclude that SERT is constitutively internalized and that the internalized transporter is sorted mainly to degradation. PMID:24973209

Quantifying intracellular rates of glycolytic and oxidative ATP production and consumption using extracellular flux measurements

PubMed Central

Mookerjee, Shona A.; Gerencser, Akos A.; Nicholls, David G.; Brand, Martin D.

2017-01-01

Partitioning of ATP generation between glycolysis and oxidative phosphorylation is central to cellular bioenergetics but cumbersome to measure. We describe here how rates of ATP generation by each pathway can be calculated from simultaneous measurements of extracellular acidification and oxygen consumption. We update theoretical maximum ATP yields by mitochondria and cells catabolizing different substrates. Mitochondrial P/O ratios (mol of ATP generated per mol of [O] consumed) are 2.73 for oxidation of pyruvate plus malate and 1.64 for oxidation of succinate. Complete oxidation of glucose by cells yields up to 33.45 ATP/glucose with a maximum P/O of 2.79. We introduce novel indices to quantify bioenergetic phenotypes. The glycolytic index reports the proportion of ATP production from glycolysis and identifies cells as primarily glycolytic (glycolytic index > 50%) or primarily oxidative. The Warburg effect is a chronic increase in glycolytic index, quantified by the Warburg index. Additional indices quantify the acute flexibility of ATP supply. The Crabtree index and Pasteur index quantify the responses of oxidative and glycolytic ATP production to alterations in glycolysis and oxidative reactions, respectively; the supply flexibility index quantifies overall flexibility of ATP supply; and the bioenergetic capacity quantifies the maximum rate of total ATP production. We illustrate the determination of these indices using C2C12 myoblasts. Measurement of ATP use revealed no significant preference for glycolytic or oxidative ATP by specific ATP consumers. Overall, we demonstrate how extracellular fluxes quantitatively reflect intracellular ATP turnover and cellular bioenergetics. We provide a simple spreadsheet to calculate glycolytic and oxidative ATP production rates from raw extracellular acidification and respiration data. PMID:28270511

Adaptive Q–V Scheme for the Voltage Control of a DFIG-Based Wind Power Plant

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Jinho; Seok, Jul-Ki; Muljadi, Eduard

Wind generators within a wind power plant (WPP) will produce different amounts of active power because of the wake effect, and therefore, they have different reactive power capabilities. This paper proposes an adaptive reactive power to the voltage (Q-V) scheme for the voltage control of a doubly fed induction generator (DFIG)-based WPP. In the proposed scheme, the WPP controller uses a voltage control mode and sends a voltage error signal to each DFIG. The DFIG controller also employs a voltage control mode utilizing the adaptive Q-V characteristics depending on the reactive power capability such that a DFIG with a largermore » reactive power capability will inject more reactive power to ensure fast voltage recovery. Test results indicate that the proposed scheme can recover the voltage within a short time, even for a grid fault with a small short-circuit ratio, by making use of the available reactive power of a WPP and differentiating the reactive power injection in proportion to the reactive power capability. This will, therefore, help to reduce the additional reactive power and ensure fast voltage recovery.« less

Ecto-Fc MS identifies ligand-receptor interactions through extracellular domain Fc fusion protein baits and shotgun proteomic analysis

PubMed Central

Savas, Jeffrey N.; De Wit, Joris; Comoletti, Davide; Zemla, Roland; Ghosh, Anirvan

2015-01-01

Ligand-receptor interactions represent essential biological triggers which regulate many diverse and important cellular processes. We have developed a discovery-based proteomic biochemical protocol which couples affinity purification with multidimensional liquid chromatographic tandem mass spectrometry (LCLC-MS/MS) and bioinformatic analysis. Compared to previous approaches, our analysis increases sensitivity, shortens analysis duration, and boosts comprehensiveness. In this protocol, receptor extracellular domains are fused with the Fc region of IgG to generate fusion proteins that are purified from transfected HEK293T cells. These “ecto-Fcs” are coupled to protein A beads and serve as baits for binding assays with prey proteins extracted from rodent brain. After capture, the affinity purified proteins are digested into peptides and comprehensively analyzed by LCLC-MS/MS with ion trap mass spectrometers. In four working days, this protocol can generate shortlists of candidate ligand-receptor protein-protein interactions. Our “Ecto-Fc MS” approach outperforms antibody-based approaches and provides a reproducible and robust framework to identify extracellular ligand – receptor interactions. PMID:25101821

Cell Surface Translocation of Annexin A2 Facilitates Glutamate-induced Extracellular Proteolysis*

PubMed Central

Valapala, Mallika; Maji, Sayantan; Borejdo, Julian; Vishwanatha, Jamboor K.

2014-01-01

Glutamate-induced elevation in intracellular Ca2+ has been implicated in excitotoxic cell death. Neurons respond to increased glutamate levels by activating an extracellular proteolytic cascade involving the components of the plasmin-plasminogen system. AnxA2 is a Ca2+-dependent phospholipid binding protein and serves as an extracellular proteolytic center by recruiting the tissue plasminogen activator and plasminogen and mediating the localized generation of plasmin. Ratiometric Ca2+ imaging and time-lapse confocal microscopy demonstrated glutamate-induced Ca2+ influx. We showed that glutamate translocated both endogenous and AnxA2-GFP to the cell surface in a process dependent on the activity of the NMDA receptor. Glutamate-induced translocation of AnxA2 is dependent on the phosphorylation of tyrosine 23 at the N terminus, and mutation of tyrosine 23 to a non-phosphomimetic variant inhibits the translocation process. The cell surface-translocated AnxA2 forms an active plasmin-generating complex, and this activity can be neutralized by a hexapeptide directed against the N terminus. These results suggest an involvement of AnxA2 in potentiating glutamate-induced cell death processes. PMID:24742684

Extracellular metabolic energetics can promote cancer progression.

PubMed

Loo, Jia Min; Scherl, Alexis; Nguyen, Alexander; Man, Fung Ying; Weinberg, Ethan; Zeng, Zhaoshi; Saltz, Leonard; Paty, Philip B; Tavazoie, Sohail F

2015-01-29

Colorectal cancer primarily metastasizes to the liver and globally kills over 600,000 people annually. By functionally screening 661 microRNAs (miRNAs) in parallel during liver colonization, we have identified miR-551a and miR-483 as robust endogenous suppressors of liver colonization and metastasis. These miRNAs convergently target creatine kinase, brain-type (CKB), which phosphorylates the metabolite creatine, to generate phosphocreatine. CKB is released into the extracellular space by metastatic cells encountering hepatic hypoxia and catalyzes production of phosphocreatine, which is imported through the SLC6A8 transporter and used to generate ATP—fueling metastatic survival. Combinatorial therapeutic viral delivery of miR-551a and miR-483-5p through single-dose adeno-associated viral (AAV) delivery significantly suppressed colon cancer metastasis, as did CKB inhibition with a small-molecule inhibitor. Importantly, human liver metastases express higher CKB and SLC6A8 levels and reduced miR-551a/miR-483 levels relative to primary tumors. We identify the extracellular space as an important compartment for malignant energetic catalysis and therapeutic targeting. Copyright © 2015 Elsevier Inc. All rights reserved.

A model of extracellular enzymes in free-living microbes: which strategy pays off?

PubMed

Traving, Sachia J; Thygesen, Uffe H; Riemann, Lasse; Stedmon, Colin A

2015-11-01

An initial modeling approach was applied to analyze how a single, nonmotile, free-living, heterotrophic bacterial cell may optimize the deployment of its extracellular enzymes. Free-living cells live in a dilute and complex substrate field, and to gain enough substrate, their extracellular enzymes must be utilized efficiently. The model revealed that surface-attached and free enzymes generate unique enzyme and substrate fields, and each deployment strategy has distinctive advantages. For a solitary cell, surface-attached enzymes are suggested to be the most cost-efficient strategy. This strategy entails potential substrates being reduced to very low concentrations. Free enzymes, on the other hand, generate a radically different substrate field, which suggests significant benefits for the strategy if free cells engage in social foraging or experience high substrate concentrations. Swimming has a slight positive effect for the attached-enzyme strategy, while the effect is negative for the free-enzyme strategy. The results of this study suggest that specific dissolved organic compounds in the ocean likely persist below a threshold concentration impervious to biological utilization. This could help explain the persistence and apparent refractory state of oceanic dissolved organic matter (DOM). Microbial extracellular enzyme strategies, therefore, have important implications for larger-scale processes, such as shaping the role of DOM in ocean carbon sequestration. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Reactive power planning under high penetration of wind energy using Benders decomposition

DOE PAGES

Xu, Yan; Wei, Yanli; Fang, Xin; ...

2015-11-05

This study addresses the optimal allocation of reactive power volt-ampere reactive (VAR) sources under the paradigm of high penetration of wind energy. Reactive power planning (RPP) in this particular condition involves a high level of uncertainty because of wind power characteristic. To properly model wind generation uncertainty, a multi-scenario framework optimal power flow that considers the voltage stability constraint under the worst wind scenario and transmission N 1 contingency is developed. The objective of RPP in this study is to minimise the total cost including the VAR investment cost and the expected generation cost. Therefore RPP under this condition ismore » modelled as a two-stage stochastic programming problem to optimise the VAR location and size in one stage, then to minimise the fuel cost in the other stage, and eventually, to find the global optimal RPP results iteratively. Benders decomposition is used to solve this model with an upper level problem (master problem) for VAR allocation optimisation and a lower problem (sub-problem) for generation cost minimisation. Impact of the potential reactive power support from doubly-fed induction generator (DFIG) is also analysed. Lastly, case studies on the IEEE 14-bus and 118-bus systems are provided to verify the proposed method.« less

Cold atmospheric plasma as a potential tool for multiple myeloma treatment.

PubMed

Xu, Dehui; Xu, Yujing; Cui, Qingjie; Liu, Dingxin; Liu, Zhijie; Wang, Xiaohua; Yang, Yanjie; Feng, Miaojuan; Liang, Rong; Chen, Hailan; Ye, Kai; Kong, Michael G

2018-04-06

Multiple myeloma (MM) is a fatal and incurable hematological malignancy thus new therapy need to be developed. Cold atmospheric plasma, a new technology that could generate various active species, could efficiently induce various tumor cells apoptosis. More details about the interaction of plasma and tumor cells need to be addressed before the application of gas plasma in clinical cancer treatment. In this study, we demonstrate that He+O 2 plasma could efficiently induce myeloma cell apoptosis through the activation of CD95 and downstream caspase cascades. Extracellular and intracellular reactive oxygen species (ROS) accumulation is essential for CD95-mediated cell apoptosis in response to plasma treatment. Furthermore, p53 is shown to be a key transcription factor in activating CD95 and caspase cascades. More importantly, we demonstrate that CD95 expression is higher in tumor cells than in normal cells in both MM cell lines and MM clinical samples, which suggests that CD95 could be a favorable target for plasma treatment as it could selectively inactivate myeloma tumor cells. Our results illustrate the molecular details of plasma induced myeloma cell apoptosis and it shows that gas plasma could be a potential tool for myeloma therapy in the future.

In vitro antimicrobial effects and mechanism of atmospheric-pressure He/O2 plasma jet on Staphylococcus aureus biofilm

NASA Astrophysics Data System (ADS)

Xu, Zimu; Shen, Jie; Cheng, Cheng; Hu, Shuheng; Lan, Yan; Chu, Paul K.

2017-03-01

The antimicrobial effects and associated mechanism of inactivation of Staphylococcus aureus (S. aureus) NCTC-8325 biofilms induced by a He/O2 atmospheric-pressure plasma jet (APPJ) are investigated in vitro. According to CFU (colony forming units) counting and the resazurin-based assay, the 10 min He/O2 (0.5%) APPJ treatment produces the optimal inactivation efficacy (>5 log10 ml-1) against the S. aureus biofilm and 5% of the bacteria enter a viable but non-culturable (VBNC) state. Meanwhile, 94% of the bacteria suffer from membrane damage according to SYTO 9/PI counterstaining. Scanning electron microscopy (SEM) reveals that plasma exposure erodes the extracellular polymeric substances (EPS) and then the cellular structure. The H2DCFDA-stained biofilms show larger concentrations of intracellular reactive oxygen species (ROS) in membrane-intact bacteria with increasing plasma dose. The admixture of oxygen in the working gas highly contributes to the deactivation efficacy of the APPJ against S. aureus and the plasma-induced endogenous ROS may work together with the discharge-generated ROS to continuously damage the bacterial membrane structure leading to deactivation of the biofilm microbes.

Abdominal Aortic Aneurysm: Novel Mechanisms and Therapies

PubMed Central

Davis, Frank M.; Rateri, Debra L.; Daugherty, Alan

2015-01-01

Purpose of review Abdominal aortic aneurysm (AAA) is a pathological condition of permanent dilation that portends the potentially fatal consequence of aortic rupture. This review emphasizes recent advances in mechanistic insight into aneurysm pathogenesis and potential pharmacologic therapies that are on the horizon for AAAs. Recent Findings An increasing body of evidence demonstrates that genetic factors, including 3p12.3, DAB2IP, LDLr, LRP1, MMP3, TGFβR2 and SORT1 loci, are associated with AAA development. Current human studies and animal models have shown that many leukocytes and inflammatory mediators, such as IL-1, IL-17, TGF-β and angiotensin II, are involved in the pathogenesis of AAAs. Leukocytic infiltration into aortic media leads to smooth muscle cell depletion, generation of reactive oxygen species, and extracellular matrix fragmentation. Recent preclinical investigations into pharmacological therapies for AAAs have provided intriguing insight for roles of microRNAs to regulate many pathological pathways in AAA development. Several large clinical trials are ongoing seeking to translate preclinical findings into therapeutic options. Summary Recent studies have identified many potential mechanisms involved in AAA pathogenesis that provide insight for the development of a medical treatment for this disease. PMID:26352243

Parallel Aspects of the Microenvironment in Cancer and Autoimmune Disease

PubMed Central

Rahat, Michal A.

2016-01-01

Cancer and autoimmune diseases are fundamentally different pathological conditions. In cancer, the immune response is suppressed and unable to eradicate the transformed self-cells, while in autoimmune diseases it is hyperactivated against a self-antigen, leading to tissue injury. Yet, mechanistically, similarities in the triggering of the immune responses can be observed. In this review, we highlight some parallel aspects of the microenvironment in cancer and autoimmune diseases, especially hypoxia, and the role of macrophages, neutrophils, and their interaction. Macrophages, owing to their plastic mode of activation, can generate a pro- or antitumoral microenvironment. Similarly, in autoimmune diseases, macrophages tip the Th1/Th2 balance via various effector cytokines. The contribution of neutrophils, an additional plastic innate immune cell population, to the microenvironment and disease progression is recently gaining more prominence in both cancer and autoimmune diseases, as they can secrete cytokines, chemokines, and reactive oxygen species (ROS), as well as acquire an enhanced ability to produce neutrophil extracellular traps (NETs) that are now considered important initiators of autoimmune diseases. Understanding the contribution of macrophages and neutrophils to the cancerous or autoimmune microenvironment, as well as the role their interaction and cooperation play, may help identify new targets and improve therapeutic strategies. PMID:26997761

Cold atmospheric plasma as a potential tool for multiple myeloma treatment

PubMed Central

Cui, Qingjie; Liu, Dingxin; Liu, Zhijie; Wang, Xiaohua; Yang, Yanjie; Feng, Miaojuan; Liang, Rong; Chen, Hailan; Ye, Kai; Kong, Michael G.

2018-01-01

Multiple myeloma (MM) is a fatal and incurable hematological malignancy thus new therapy need to be developed. Cold atmospheric plasma, a new technology that could generate various active species, could efficiently induce various tumor cells apoptosis. More details about the interaction of plasma and tumor cells need to be addressed before the application of gas plasma in clinical cancer treatment. In this study, we demonstrate that He+O2 plasma could efficiently induce myeloma cell apoptosis through the activation of CD95 and downstream caspase cascades. Extracellular and intracellular reactive oxygen species (ROS) accumulation is essential for CD95-mediated cell apoptosis in response to plasma treatment. Furthermore, p53 is shown to be a key transcription factor in activating CD95 and caspase cascades. More importantly, we demonstrate that CD95 expression is higher in tumor cells than in normal cells in both MM cell lines and MM clinical samples, which suggests that CD95 could be a favorable target for plasma treatment as it could selectively inactivate myeloma tumor cells. Our results illustrate the molecular details of plasma induced myeloma cell apoptosis and it shows that gas plasma could be a potential tool for myeloma therapy in the future. PMID:29719586

Effect of magnesium oxide nanoparticles on microbial diversity and removal performance of sequencing batch reactor.

PubMed

Ma, Bingrui; Yu, Naling; Han, Yuetong; Gao, Mengchun; Wang, Sen; Li, Shanshan; Guo, Liang; She, Zonglian; Zhao, Yangguo; Jin, Chunji; Gao, Feng

2018-06-13

The performance, microbial enzymatic activity and microbial community of a sequencing batch reactor (SBR) have been explored under magnesium oxide nanoparticles (MgO NPs) stress. The NH 4 + -N removal efficiency kept relatively stable during the whole operational process. The MgO NPs at 30-60 mg/L slightly restrained the removal of chemical oxygen demand (COD), and the presence of MgO NPs also affected the denitrification and phosphorus removal. The specific oxygen uptake rate, nitrifying and denitrifying rates, phosphorus removal rate, and microbial enzymatic activities distinctly varied with the increase of MgO NPs concentration. The appearance of MgO NPs promoted more reactive oxygen species generation and lactate dehydrogenase leakage from activated sludge, suggesting that MgO NPs had obvious toxicity to activated sludge in the SBR. The protein and polysaccharide contents of extracellular polymeric substances from activated sludge increased with the increase of MgO NPs concentration. The microbial richness and diversity at different MgO NPs concentrations obviously varied at the phylum, class and genus levels due to the biological toxicity of MgO NPs. Copyright © 2018 Elsevier Ltd. All rights reserved.

Control of Reactive Species Generated by Low-frequency Biased Nanosecond Pulse Discharge in Atmospheric Pressure Plasma Effluent

NASA Astrophysics Data System (ADS)

Takashima, Keisuke; Kaneko, Toshiro

2016-09-01

The control of hydroxyl radical and the other gas phase species generation in the ejected gas through air plasma (air plasma effluent) has been experimentally studied, which is a key to extend the range of plasma treatment. Nanosecond pulse discharge is known to produce high reduced electric field (E/N) discharge that leads to efficient generation of the reactive species than conventional low frequency discharge, while the charge-voltage cycle in the low frequency discharge is known to be well-controlled. In this study, the nanosecond pulse discharge biased with AC low frequency high voltage is used to take advantages of these discharges, which allows us to modulate the reactive species composition in the air plasma effluent. The utilization of the gas-liquid interface and the liquid phase chemical reactions between the modulated long-lived reactive species delivered from the air plasma effluent could realize efficient liquid phase chemical reactions leading to short-lived reactive species production far from the air plasma, which is crucial for some plasma agricultural applications.

Can direct extracellular electron transfer occur in the absence of outer membrane cytochromes in Desulfovibrio vulgaris?

DOE Office of Scientific and Technical Information (OSTI.GOV)

Elias, Dwayne A; Zane, Mr. Grant M.; Auer, Dr. Manfred

2010-01-01

Extracellular electron transfer has been investigated over several decades via forms of soluble electron transfer proteins that are exported for extracellular reoxidation. More recently, several organisms have been shown to reduce extracellular metals via the direct transfer of electron through appendages; also known as nanowires. They have been reported most predominantly in Shewanella and Geobacter. While the relevancy and composition of these structures in each genus has been debated, both possess outer membrane cytochrome complexes that could theoretically come into direct contact with solid phase oxidized metals. Members of the genus Desulfovibrio apparently have no such cytochromes although similar appendagesmore » are present, are electrically conductive, and are different from flagella. Upon U(VI)-reduction, the structures in Desulfovibrio become coated with U(IV). Deletion of flagellar genes did not alter soluble or amorphous Fe(III) or U(VI) reduction, or appendage appearance. Removal of the chromosomal pilA gene hampered amorphous Fe(III)-reduction by ca. 25%, but cells lacking the native plasmid, pDV1, reduced soluble Fe(III) and U(VI) at ca. 50% of the wild type rate while amorphous Fe(III)-reduction slowed to ca. 20% of the wild type rate. Appendages were present in all deletions as well as pDV1, except pilA. Gene complementation restored all activities and morphologies to wild type levels. This suggests that pilA encodes the structural component, whereas genes within pDV1 may provide the reactive members. How such appendages function without outer membrane cytochromes is under investigation.« less

Formation and Detoxification of Reactive Oxygen Species

ERIC Educational Resources Information Center

Kuciel, Radoslawa; Mazurkiewicz, Aleksandra

2004-01-01

A model of reactive oxygen species metabolism is proposed as a laboratory exercise for students. The superoxide ion in this model is generated during the reaction of oxidation of xanthine, catalyzed by xanthine oxidase. The effect of catalase, superoxide dismutase, and allopurinol on superoxide ion generation and removal in this system is also…

LFPy: a tool for biophysical simulation of extracellular potentials generated by detailed model neurons.

PubMed

Lindén, Henrik; Hagen, Espen; Lęski, Szymon; Norheim, Eivind S; Pettersen, Klas H; Einevoll, Gaute T

2013-01-01

Electrical extracellular recordings, i.e., recordings of the electrical potentials in the extracellular medium between cells, have been a main work-horse in electrophysiology for almost a century. The high-frequency part of the signal (≳500 Hz), i.e., the multi-unit activity (MUA), contains information about the firing of action potentials in surrounding neurons, while the low-frequency part, the local field potential (LFP), contains information about how these neurons integrate synaptic inputs. As the recorded extracellular signals arise from multiple neural processes, their interpretation is typically ambiguous and difficult. Fortunately, a precise biophysical modeling scheme linking activity at the cellular level and the recorded signal has been established: the extracellular potential can be calculated as a weighted sum of all transmembrane currents in all cells located in the vicinity of the electrode. This computational scheme can considerably aid the modeling and analysis of MUA and LFP signals. Here, we describe LFPy, an open source Python package for numerical simulations of extracellular potentials. LFPy consists of a set of easy-to-use classes for defining cells, synapses and recording electrodes as Python objects, implementing this biophysical modeling scheme. It runs on top of the widely used NEURON simulation environment, which allows for flexible usage of both new and existing cell models. Further, calculation of extracellular potentials using the line-source-method is efficiently implemented. We describe the theoretical framework underlying the extracellular potential calculations and illustrate by examples how LFPy can be used both for simulating LFPs, i.e., synaptic contributions from single cells as well a populations of cells, and MUAs, i.e., extracellular signatures of action potentials.

An Overview of the Chemistry and Biology of Reactive Aldehydes

PubMed Central

Fritz, Kristofer S.; Petersen, Dennis R.

2012-01-01

The non-enzymatic free radical generation of reactive aldehydes is known to contribute to diseases of sustained oxidative stress including rheumatoid arthritis, atherosclerosis, neurodegenerative and a number of liver diseases. At the same time, the accumulation of lipid electrophiles has been demonstrated to play a role in cell signaling events through modification of proteins critical for cellular homeostasis. Given the broad scope of reactivity profiles and the ability to modify numerous proteomic and genomic processes, new emphasis is being placed on a systems-based analysis of the consequences of electrophilic adduction. This review focuses on the generation and chemical reactivity of lipid-derived aldehydes with a special focus on the homeostatic responses to electrophilic stress. PMID:22750507

Functional Characterization of Resting and Adenovirus-Induced Reactive Astrocytes in Three-Dimensional Culture

PubMed Central

Woo, Junsung; Im, Sun-Kyoung; Chun, Heejung; Jung, Soon-Young; Oh, Soo-Jin; Choi, Nakwon

2017-01-01

Brain is a rich environment where neurons and glia interact with neighboring cells as well as extracellular matrix in three-dimensional (3D) space. Astrocytes, which are the most abundant cells in the mammalian brain, reside in 3D space and extend highly branched processes that form microdomains and contact synapses. It has been suggested that astrocytes cultured in 3D might be maintained in a less reactive state as compared to those growing in a traditional, two-dimensional (2D) monolayer culture. However, the functional characterization of the astrocytes in 3D culture has been lacking. Here we cocultured neurons and astrocytes in 3D and examined the morphological, molecular biological, and electrophysiological properties of the 3D-cultured hippocampal astrocytes. In our 3D neuron-astrocyte coculture, astrocytes showed a typical morphology of a small soma with many branches and exhibited a unique membrane property of passive conductance, more closely resembling their native in vivo counterparts. Moreover, we also induced reactive astrocytosis in culture by infecting with high-titer adenovirus to mimic pathophysiological conditions in vivo. Adenoviral infection induced morphological changes in astrocytes, increased passive conductance, and increased GABA content as well as tonic GABA release, which are characteristics of reactive gliosis. Together, our study presents a powerful in vitro model resembling both physiological and pathophysiological conditions in vivo, and thereby provides a versatile experimental tool for studying various neurological diseases that accompany reactive astrocytes. PMID:28680301

Lysyl Hydroxylase 2 Is Secreted by Tumor Cells and Can Modify Collagen in the Extracellular Space.

PubMed

Chen, Yulong; Guo, Houfu; Terajima, Masahiko; Banerjee, Priyam; Liu, Xin; Yu, Jiang; Momin, Amin A; Katayama, Hiroyuki; Hanash, Samir M; Burns, Alan R; Fields, Gregg B; Yamauchi, Mitsuo; Kurie, Jonathan M

2016-12-09

Lysyl hydroxylase 2 (LH2) catalyzes the hydroxylation of lysine residues in the telopeptides of fibrillar collagens, which leads to the formation of stable collagen cross-links. Recently we reported that LH2 enhances the metastatic propensity of lung cancer by increasing the amount of stable hydroxylysine aldehyde-derived collagen cross-links (HLCCs), which generate a stiffer tumor stroma (Chen, Y., et al. (2015) J. Clin. Invest. 125, 125, 1147-1162). It is generally accepted that LH2 modifies procollagen α chains on the endoplasmic reticulum before the formation of triple helical procollagen molecules. Herein, we report that LH2 is also secreted and modifies collagen in the extracellular space. Analyses of lung cancer cell lines demonstrated that LH2 is present in the cell lysates and the conditioned media in a dimeric, active form in both compartments. LH2 co-localized with collagen fibrils in the extracellular space in human lung cancer specimens and in orthotopic lung tumors generated by injection of a LH2-expressing human lung cancer cell line into nude mice. LH2 depletion in MC3T3 osteoblastic cells impaired the formation of HLCCs, resulting in an increase in the unmodified lysine aldehyde-derived collagen cross-link (LCC), and the addition of recombinant LH2 to the media of LH2-deficient MC3T3 cells was sufficient to rescue HLCC formation in the extracellular matrix. The finding that LH2 modifies collagen in the extracellular space challenges the current view that LH2 functions solely on the endoplasmic reticulum and could also have important implications for cancer biology. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Lysyl Hydroxylase 2 Is Secreted by Tumor Cells and Can Modify Collagen in the Extracellular Space*

PubMed Central

Chen, Yulong; Guo, Houfu; Terajima, Masahiko; Banerjee, Priyam; Liu, Xin; Yu, Jiang; Momin, Amin A.; Katayama, Hiroyuki; Hanash, Samir M.; Burns, Alan R.; Fields, Gregg B.; Yamauchi, Mitsuo; Kurie, Jonathan M.

2016-01-01

Lysyl hydroxylase 2 (LH2) catalyzes the hydroxylation of lysine residues in the telopeptides of fibrillar collagens, which leads to the formation of stable collagen cross-links. Recently we reported that LH2 enhances the metastatic propensity of lung cancer by increasing the amount of stable hydroxylysine aldehyde-derived collagen cross-links (HLCCs), which generate a stiffer tumor stroma (Chen, Y., et al. (2015) J. Clin. Invest. 125, 125, 1147–1162). It is generally accepted that LH2 modifies procollagen α chains on the endoplasmic reticulum before the formation of triple helical procollagen molecules. Herein, we report that LH2 is also secreted and modifies collagen in the extracellular space. Analyses of lung cancer cell lines demonstrated that LH2 is present in the cell lysates and the conditioned media in a dimeric, active form in both compartments. LH2 co-localized with collagen fibrils in the extracellular space in human lung cancer specimens and in orthotopic lung tumors generated by injection of a LH2-expressing human lung cancer cell line into nude mice. LH2 depletion in MC3T3 osteoblastic cells impaired the formation of HLCCs, resulting in an increase in the unmodified lysine aldehyde-derived collagen cross-link (LCC), and the addition of recombinant LH2 to the media of LH2-deficient MC3T3 cells was sufficient to rescue HLCC formation in the extracellular matrix. The finding that LH2 modifies collagen in the extracellular space challenges the current view that LH2 functions solely on the endoplasmic reticulum and could also have important implications for cancer biology. PMID:27803159

Pharmacologic inhibition of lactate production prevents myofibroblast differentiation.

PubMed

Kottmann, Robert Matthew; Trawick, Emma; Judge, Jennifer L; Wahl, Lindsay A; Epa, Amali P; Owens, Kristina M; Thatcher, Thomas H; Phipps, Richard P; Sime, Patricia J

2015-12-01

Myofibroblasts are one of the primary cell types responsible for the accumulation of extracellular matrix in fibrosing diseases, and targeting myofibroblast differentiation is an important therapeutic strategy for the treatment of pulmonary fibrosis. Transforming growth factor-β (TGF-β) has been shown to be an important inducer of myofibroblast differentiation. We previously demonstrated that lactate dehydrogenase and its metabolic product lactic acid are important mediators of myofibroblast differentiation, via acid-induced activation of latent TGF-β. Here we explore whether pharmacologic inhibition of LDH activity can prevent TGF-β-induced myofibroblast differentiation. Primary human lung fibroblasts from healthy patients and those with pulmonary fibrosis were treated with TGF-β and or gossypol, an LDH inhibitor. Protein and RNA were analyzed for markers of myofibroblast differentiation and extracellular matrix generation. Gossypol inhibited TGF-β-induced expression of the myofibroblast marker α-smooth muscle actin (α-SMA) in a dose-dependent manner in both healthy and fibrotic human lung fibroblasts. Gossypol also inhibited expression of collagen 1, collagen 3, and fibronectin. Gossypol inhibited LDH activity, the generation of extracellular lactic acid, and the rate of extracellular acidification in a dose-dependent manner. Furthermore, gossypol inhibited TGF-β bioactivity in a dose-dependent manner. Concurrent treatment with an LDH siRNA increased the ability of gossypol to inhibit TGF-β-induced myofibroblast differentiation. Gossypol inhibits TGF-β-induced myofibroblast differentiation through inhibition of LDH, inhibition of extracellular accumulation of lactic acid, and inhibition of TGF-β bioactivity. These data support the hypothesis that pharmacologic inhibition of LDH may play an important role in the treatment of pulmonary fibrosis. Copyright © 2015 the American Physiological Society.

Immunization of A4galt-deficient mice with glycosphingolipids from renal cell cancers resulted in the generation of anti-sulfoglycolipid monoclonal antibodies.

PubMed

Ando, Reiko; Tokuda, Noriyo; Yamamoto, Tokunori; Ikeda, Kazutaka; Hashimoto, Noboru; Taguchi, Ryo; Fan, Xiaoen; Furukawa, Keiko; Niimura, Yukio; Suzuki, Akemi; Goto, Momokazu; Furukawa, Koichi

2016-04-01

In this study, we immunized Gb3/CD77 synthase gene (A4galt) knockout (KO) mice with glycosphingolipids (GSLs) extracted from 3 renal cell cancer (RCC) cell lines to raise monoclonal antibodies (mAbs) reactive with globo-series GSLs specifically expressed in RCCs. Although a number of mAbs reactive with globo-series GSLs were generated, they reacted with both RCC cell lines and normal kidney cells. When we analyzed recognized antigens by mAbs that were specifically reactive with RCC, but not with normal kidney cells at least on the cell surface, many of them turned out to be reactive with sulfoglycolipids. Eight out of 11 RCC-specific mAbs were reactive with SM2 alone, and the other 3 mAbs were more broadly reactive with sulfated glycolipids, i.e. SM3 and SM4 as well as SM2. In the immunohistochemistry, these anti-sulfoglycolipids mAbs showed RCC-specific reaction, with no or minimal reaction with adjacent normal tissues. Thus, immunization of A4galt KO mice with RCC-derived GSLs resulted in the generation of anti sulfated GSL mAbs, and these mAbs may be applicable for the therapeutics for RCC patients.

Protective Effects of Resveratrol against UVA-Induced Damage in ARPE19 Cells

PubMed Central

Chan, Chi-Ming; Huang, Cheng-Hua; Li, Hsin-Ju; Hsiao, Chien-Yu; Su, Ching-Chieh; Lee, Pei-Lan; Hung, Chi-Feng

2015-01-01

Ultraviolet radiation, especially UVA, can penetrate the lens, reach the retina, and induce oxidative stress to retinal pigment epithelial (RPE) cells. Even though it is weakly absorbed by protein and DNA, it may trigger the production of reactive oxygen species (ROS) and generate oxidative injury; oxidative injury to the retinal pigment epithelium has been implicated to play a contributory role in age-related macular degeneration (AMD). Studies showed that resveratrol, an abundant and active component of red grapes, can protect several cell types from oxidative stress. In this study, adult RPE cells being treated with different concentrations of resveratrol were used to evaluate the protective effect of resveratrol on RPE cells against UVA-induced damage. Cell viability assay showed that resveratrol reduced the UVA-induced decrease in RPE cell viability. Through flow cytometry analysis, we found that the generation of intracellular H2O2 induced by UVA irradiation in RPE cells could be suppressed by resveratrol in a concentration-dependent manner. Results of Western blot analysis demonstrated that resveratrol lowered the activation of UVA-induced extracellular signal-regulated kinase, c-jun-NH2 terminal kinase and p38 kinase in RPE cells. In addition, there was also a reduction in UVA-induced cyclooxygenase-2 (COX-2) expression in RPE cells pretreated with resveratrol. Our observations suggest that resveratrol is effective in preventing RPE cells from being damaged by UVA radiation, and is worth considering for further development as a chemoprotective agent for the prevention of early AMD. PMID:25775159

Protective effects of resveratrol against UVA-induced damage in ARPE19 cells.

PubMed

Chan, Chi-Ming; Huang, Cheng-Hua; Li, Hsin-Ju; Hsiao, Chien-Yu; Su, Ching-Chieh; Lee, Pei-Lan; Hung, Chi-Feng

2015-03-12

Ultraviolet radiation, especially UVA, can penetrate the lens, reach the retina, and induce oxidative stress to retinal pigment epithelial (RPE) cells. Even though it is weakly absorbed by protein and DNA, it may trigger the production of reactive oxygen species (ROS) and generate oxidative injury; oxidative injury to the retinal pigment epithelium has been implicated to play a contributory role in age-related macular degeneration (AMD). Studies showed that resveratrol, an abundant and active component of red grapes, can protect several cell types from oxidative stress. In this study, adult RPE cells being treated with different concentrations of resveratrol were used to evaluate the protective effect of resveratrol on RPE cells against UVA-induced damage. Cell viability assay showed that resveratrol reduced the UVA-induced decrease in RPE cell viability. Through flow cytometry analysis, we found that the generation of intracellular H2O2 induced by UVA irradiation in RPE cells could be suppressed by resveratrol in a concentration-dependent manner. Results of Western blot analysis demonstrated that resveratrol lowered the activation of UVA-induced extracellular signal-regulated kinase, c-jun-NH2 terminal kinase and p38 kinase in RPE cells. In addition, there was also a reduction in UVA-induced cyclooxygenase-2 (COX-2) expression in RPE cells pretreated with resveratrol. Our observations suggest that resveratrol is effective in preventing RPE cells from being damaged by UVA radiation, and is worth considering for further development as a chemoprotective agent for the prevention of early AMD.

SIRT3 activator Honokiol attenuates β-Amyloid by modulating amyloidogenic pathway

PubMed Central

Lynd, Tyler; Briggs, Gwyneth; Adamek, Danielle; Jones, Ellery; Heiner, Jake; Majrashi, Mohammed; Moore, Timothy; Amin, Rajesh; Suppiramaniam, Vishnu

2018-01-01

Honokiol (poly-phenolic lignan from Magnolia grandiflora) is a Sirtuin-3 (SIRT3) activator which exhibit antioxidant activity and augment mitochondrial functions in several experimental models. Modern evidence suggests the critical role of SIRT3 in the progression of several metabolic and neurodegenerative diseases. Amyloid beta (Aβ), the precursor to extracellular senile plaques, accumulates in the brains of patients with Alzheimer’s disease (AD) and is related to the development of cognitive impairment and neuronal cell death. Aβ is generated from amyloid-β precursor protein (APP) through sequential cleavages, first by β-secretase and then by γ-secretase. Drugs modulating this pathway are believed to be one of the most promising strategies for AD treatment. In the present study, we found that Honokiol significantly enhanced SIRT3 expression, reduced reactive oxygen species generation and lipid peroxidation, enhanced antioxidant activities, and mitochondrial function thereby reducing Aβ and sAPPβ levels in Chinese Hamster Ovarian (CHO) cells (carrying the amyloid precursor protein-APP and Presenilin PS1 mutation). Mechanistic studies revealed that Honokiol affects neither protein levels of APP nor α-secretase activity. In contrast, Honokiol increased the expression of AMPK, CREB, and PGC-1α, thereby inhibiting β-secretase activity leading to reduced Aβ levels. These results suggest that Honokiol is an activator of SIRT3 capable of improving antioxidant activity, mitochondrial energy regulation, while decreasing Aβ, thereby indicating it to be a lead compound for AD drug development. PMID:29324783

Redox Signaling and Bioenergetics Influence Lung Cancer Cell Line Sensitivity to the Isoflavone ME-344

PubMed Central

Manevich, Yefim; Reyes, Leticia; Britten, Carolyn D.; Townsend, Danyelle M.

2016-01-01

ME-344 [(3R,4S)-3,4-bis(4-hydroxyphenyl)-8-methyl-3,4-dihydro-2H-chromen-7-ol] is a second-generation derivative natural product isoflavone presently under clinical development. ME-344 effects were compared in lung cancer cell lines that are either intrinsically sensitive or resistant to the drug and in primary immortalized human lung embryonic fibroblasts (IHLEF). Cytotoxicity at low micromolar concentrations occurred only in sensitive cell lines, causing redox stress, decreased mitochondrial ATP production, and subsequent disruption of mitochondrial function. In a dose-dependent manner the drug caused instantaneous and pronounced inhibition of oxygen consumption rates (OCR) in drug-sensitive cells (quantitatively significantly less in drug-resistant cells). This was consistent with targeting of mitochondria by ME-344, with specific effects on the respiratory chain (resistance correlated with higher glycolytic indexes). OCR inhibition did not occur in primary IHLEF. ME-344 increased extracellular acidification rates in drug-resistant cells (significantly less in drug-sensitive cells), implying that ME-344 targets mitochondrial proton pumps. Only in drug-sensitive cells did ME-344 dose-dependently increase the intracellular generation of reactive oxygen species and cause oxidation of total (mainly glutathione) and protein thiols and the concomitant immediate increases in NADPH levels. We conclude that ME-344 causes complex, redox-specific, and mitochondria-targeted effects in lung cancer cells, which differ in extent from normal cells, correlate with drug sensitivity, and provide indications of a beneficial in vitro therapeutic index. PMID:27255112

Evaluation of Extracellular Endosulfatase HSULF-2 in Breast Cancer Cell WNT Signaling and Its Inhibition by a Sulfate Ester Derivative of Synthetic Antioxidant

DTIC Science & Technology

2010-08-01

Breast Cancer, Balb/c nude mice, hydrogen peroxide, extracellular matrix, sulfatase, extracellular sulfatase, aryl sulfatase, PBN, nitrones , phenyl...previously made in studying the anti‐cancer activity of the PBN  nitrones  has helped us generate new novel and testable  ideas for future research in...cancer development appears to be still in question.    Anti‐cancer activity of the PBN‐ nitrones . PBN refers to α‐phenyl‐tert‐butylnitrone. Our

Inflammatory Stroke Extracellular Vesicles Induce Macrophage Activation.

PubMed

Couch, Yvonne; Akbar, Naveed; Davis, Simon; Fischer, Roman; Dickens, Alex M; Neuhaus, Ain A; Burgess, Annette I; Rothwell, Peter M; Buchan, Alastair M

2017-08-01

Extracellular vesicles (EVs) are protein-lipid complexes released from cells, as well as actively exocytosed, as part of normal physiology, but also during pathological processes such as those occurring during a stroke. Our aim was to determine the inflammatory potential of stroke EVs. EVs were quantified and analyzed in the sera of patients after an acute stroke (<24 hours; OXVASC [Oxford Vascular Study]). Isolated EV fractions were subjected to untargeted proteomic analysis by liquid chromatography mass-spectrometry/mass-spectrometry and then applied to macrophages in culture to investigate inflammatory gene expression. EV number, but not size, is significantly increased in stroke patients when compared to age-matched controls. Proteomic analysis reveals an overall increase in acute phase proteins, including C-reactive protein. EV fractions applied to monocyte-differentiated macrophage cultures induced inflammatory gene expression. Together these data show that EVs from stroke patients are proinflammatory in nature and are capable of inducing inflammation in immune cells. © 2017 American Heart Association, Inc.

Delivery of Therapeutic Proteins via Extracellular Vesicles: Review and Potential Treatments for Parkinson's Disease, Glioma, and Schwannoma.

PubMed

Hall, Justin; Prabhakar, Shilpa; Balaj, Leonora; Lai, Charles P; Cerione, Richard A; Breakefield, Xandra O

2016-04-01

Extracellular vesicles present an attractive delivery vehicle for therapeutic proteins. They intrinsically contain many proteins which can provide information to other cells. Advantages include reduced immune reactivity, especially if derived from the same host, stability in biologic fluids, and ability to target uptake. Those from mesenchymal stem cells appear to be intrinsically therapeutic, while those from cancer cells promote tumor progression. Therapeutic proteins can be loaded into vesicles by overexpression in the donor cell, with oligomerization and membrane sequences increasing their loading. Examples of protein delivery for therapeutic benefit in pre-clinical models include delivery of: catalase for Parkinson's disease to reduce oxidative stress and thus help neurons to survive; prodrug activating enzymes which can convert a prodrug which crosses the blood-brain barrier into a toxic chemotherapeutic drug for schwannomas and gliomas; and the apoptosis-inducing enzyme, caspase-1 under a Schwann cell specific promoter for schwannoma. This therapeutic delivery strategy is novel and being explored for a number of diseases.

Are soluble factors relevant for polymorphonuclear leukocyte dysregulation in septicemia?

PubMed Central

Wenisch, C; Graninger, W

1995-01-01

Polymorphonuclear leukocytes (PMNs) of twelve patients with gram-negative septicemia exhibited a decreased capacity to phagocytize Escherichia coli and generate reactive oxygen products which normalized within 7 days of treatment. Ex vivo exchange of plasma from age-, sex-, and blood-group-identical normal controls resulted in an increase of both phagocytic capacity and reactive oxygen intermediate generation in PMNs of septicemic patients and transiently reduced phagocytosis and reactive oxygen intermediate production in PMNs of normal controls. These results suggest that extrinsic factors are crucial for PMN function. PMID:7697538

Reactive oxygen species contribute to arsenic-induced EZH2 phosphorylation in human bronchial epithelial cells and lung cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Lingzhi; Qiu, Ping; Chen, Bailing

Our previous studies suggested that arsenic is able to induce serine 21 phosphorylation of the EZH2 protein through activation of JNK, STAT3, and Akt signaling pathways in the bronchial epithelial cell line, BEAS-2B. In the present report, we further demonstrated that reactive oxygen species (ROS) were involved in the arsenic-induced protein kinase activation that leads to EZH2 phosphorylation. Several lines of evidence supported this notion. First, the pretreatment of the cells with N-acetyl-L-cysteine (NAC), a potent antioxidant, abolishes arsenic-induced EZH2 phosphorylation along with the inhibition of JNK, STAT3, and Akt. Second, H{sub 2}O{sub 2}, the most important form of ROSmore » in the cells in response to extracellular stress signals, can induce phosphorylation of the EZH2 protein and the activation of JNK, STAT3, and Akt. By ectopic expression of the myc-tagged EZH2, we additionally identified direct interaction and phosphorylation of the EZH2 protein by Akt in response to arsenic and H{sub 2}O{sub 2}. Furthermore, both arsenic and H{sub 2}O{sub 2} were able to induce the translocation of ectopically expressed or endogenous EZH2 from nucleus to cytoplasm. In summary, the data presented in this report indicate that oxidative stress due to ROS generation plays an important role in the arsenic-induced EZH2 phosphorylation. - Highlights:: • Arsenic (As{sup 3+}) induces EZH phosphorylation. • JNK, STAT3, and Akt contribute to EZH2 phosphorylation. • Oxidative stress is involved in As{sup 3+}-induced EZH2 phosphorylation. • As{sup 3+} induces direct interaction of Akt and EZH2. • Phosphorylated EZH2 localized in cytoplasm.« less

Vimentin is an endogenous ligand for the pattern recognition receptor Dectin-1.

PubMed

Thiagarajan, Praveena S; Yakubenko, Valentin P; Elsori, Deena H; Yadav, Satya P; Willard, Belinda; Tan, Carmela D; Rodriguez, E René; Febbraio, Maria; Cathcart, Martha K

2013-08-01

Atherosclerosis is a chronic inflammatory disorder of cholesterol deposition in monocyte-derived macrophages (MDM) within the arterial wall leading to impingement on the lumen of the vessel. In atherosclerotic lesions, MDM are the primary source of NADPH oxidase-derived superoxide anion (O₂⁻) inducing low-density lipoprotein (LDL) oxidation leading to their unregulated uptake of oxidized LDL and foam cell formation. We recently discovered that zymosan potently activates monocyte NADPH oxidase via the non-toll pattern recognition receptor (PRR), Dectin-1. Other PRRs bind endogenous human ligands, yet no such ligands have been identified for Dectin-1. Our hypothesis was that inflammation generates endogenous ligands for Dectin-1 that activate O₂⁻ production and thereby contributes to atherogenesis. Human: anti-zymosan antibodies were used to identify similar, cross-reactive epitopes in human atherosclerotic tissue extracts. Immunoblot analysis revealed consistent antibody reactive protein bands on one- and two-dimensional gel electrophoreses. Vimentin was identified by mass spectrometry in the immunoreactive bands across different tissue samples. Direct binding of vimentin to Dectin-1 was observed using BIACORE. Further data revealed that vimentin induces O₂⁻ production by human monocytes. Analysis of human atherosclerotic lesions revealed that vimentin was detected extracellularly in the necrotic core and in areas of active inflammation. Vimentin also co-localized with Dectin-1 in macrophage-rich regions where O₂⁻ is produced. We conclude that vimentin is an endogenous, activating ligand for Dectin-1. Its presence in areas of artery wall inflammation and O₂⁻ production suggests that vimentin activates Dectin-1 and contributes to the oxidation of lipids and cholesterol accumulation in atherosclerosis.

Analytical interference of HBOC-201 (Hemopure, a synthetic hemoglobin-based oxygen carrier) on four common clinical chemistry platforms.

PubMed

Korte, Erik A; Pozzi, Nicole; Wardrip, Nina; Ayyoubi, M Tayyeb; Jortani, Saeed A

2018-07-01

There are 13 million blood transfusions each year in the US. Limitations in the donor pool, storage capabilities, mass casualties, access in remote locations and reactivity of donors all limit the availability of transfusable blood products to patients. HBOC-201 (Hemopure®) is a second-generation glutaraldehyde-polymer of bovine hemoglobin, which can serve as an "oxygen bridge" to maintain oxygen carrying capacity while transfusion products are unavailable. Hemopure presents the advantages of extended shelf life, ambient storage, and limited reactive potential, but its extracellular location can also cause significant interference in modern laboratory analyzers similar to severe hemolysis. Observed error in 26 commonly measured analytes was determined on 4 different analytical platforms in plasma from a patient therapeutically transfused Hemopure as well as donor blood spiked with Hemopure at a level equivalent to the therapeutic loading dose (10% v/v). Significant negative error ratios >50% of the total allowable error (>0.5tAE) were reported in 23/104 assays (22.1%), positive bias of >0.5tAE in 26/104 assays (25.0%), and acceptable bias between -0.5tAE and 0.5tAE error ratio was reported in 44/104 (42.3%). Analysis failed in the presence of Hemopure in 11/104 (10.6%). Observed error is further subdivided by platform, wavelength, dilution and reaction method. Administration of Hemopure (or other hemoglobin-based oxygen carriers) presents a challenge to laboratorians tasked with analyzing patient specimens. We provide laboratorians with a reference to evaluate patient samples, select optimal analytical platforms for specific analytes, and predict possible bias beyond the 4 analytical platforms included in this study. Copyright © 2018 Elsevier B.V. All rights reserved.

Modulation of IgE-dependent COX-2 gene expression by reactive oxygen species in human neutrophils.

PubMed

Vega, Antonio; Chacón, Pedro; Alba, Gonzalo; El Bekay, Rajaa; Martín-Nieto, José; Sobrino, Francisco

2006-07-01

Cyclooxygenase (COX) is a key enzyme in prostaglandin (PG) synthesis. Up-regulation of its COX-2 isoform is responsible for the increased PG release, taking place under inflammatory conditions, and also, is thought to be involved in allergic and inflammatory diseases. In the present work, we demonstrate that COX-2 expression becomes highly induced by anti-immunoglobulin E (IgE) antibodies and by antigens in human neutrophils from allergic patients. This induction was detected at mRNA and protein levels and was accompanied by a concomitant PGE(2) and thromboxane A(2) release. We also show evidence that inhibitors of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, such as 4-(2-aminoethyl)benzenesulphonyl fluoride and 4-hydroxy-3-methoxyaceto-phenone, completely cancelled anti-IgE-induced COX-2 protein up-regulation, suggesting that this process is mediated by reactive oxygen species (ROS) derived from NADPH oxidase activity. Moreover, the mitogen-activated protein kinases (MAPKs), p38 and extracellular signal-regulated kinase, and also, the transcription factor, nuclear factor (NF)-kappaB, are involved in the up-regulation of COX-2 expression, as specific chemical inhibitors of these two kinases, such as SB203580 and PD098059, and of the NF-kappaB pathway, such as N(alpha)-benzyloxycarbonyl-l-leucyl-l-leucyl-l-leucinal, abolished IgE-dependent COX-2 induction. Evidence is also presented, using Fe(2)(+)/Cu(2)(+) ions, that hydroxyl radicals generated from hydrogen peroxide through Fenton reactions could constitute candidate modulators able to directly trigger anti-IgE-elicited COX-2 expression through MAPK and NF-kappaB pathways. Present results underscore a new role for ROS as second messengers in the modulation of COX-2 expression by human neutrophils in allergic conditions.

Pancreatic Cancer-Derived Exosomes Cause Paraneoplastic β-cell Dysfunction.

PubMed

Javeed, Naureen; Sagar, Gunisha; Dutta, Shamit K; Smyrk, Thomas C; Lau, Julie S; Bhattacharya, Santanu; Truty, Mark; Petersen, Gloria M; Kaufman, Randal J; Chari, Suresh T; Mukhopadhyay, Debabrata

2015-04-01

Pancreatic cancer frequently causes diabetes. We recently proposed adrenomedullin as a candidate mediator of pancreatic β-cell dysfunction in pancreatic cancer. How pancreatic cancer-derived adrenomedullin reaches β cells remote from the cancer to induce β-cell dysfunction is unknown. We tested a novel hypothesis that pancreatic cancer sheds adrenomedullin-containing exosomes into circulation, which are transported to β cells and impair insulin secretion. We characterized exosomes from conditioned media of pancreatic cancer cell lines (n = 5) and portal/peripheral venous blood of patients with pancreatic cancer (n = 20). Western blot analysis showed the presence of adrenomedullin in pancreatic cancer-exosomes. We determined the effect of adrenomedullin-containing pancreatic cancer exosomes on insulin secretion from INS-1 β cells and human islets, and demonstrated the mechanism of exosome internalization into β cells. We studied the interaction between β-cell adrenomedullin receptors and adrenomedullin present in pancreatic cancer-exosomes. In addition, the effect of adrenomedullin on endoplasmic reticulum (ER) stress response genes and reactive oxygen/nitrogen species generation in β cells was shown. Exosomes were found to be the predominant extracellular vesicles secreted by pancreatic cancer into culture media and patient plasma. Pancreatic cancer-exosomes contained adrenomedullin and CA19-9, readily entered β cells through caveolin-mediated endocytosis or macropinocytosis, and inhibited insulin secretion. Adrenomedullin in pancreatic cancer exosomes interacted with its receptor on β cells. Adrenomedullin receptor blockade abrogated the inhibitory effect of exosomes on insulin secretion. β cells exposed to adrenomedullin or pancreatic cancer exosomes showed upregulation of ER stress genes and increased reactive oxygen/nitrogen species. Pancreatic cancer causes paraneoplastic β-cell dysfunction by shedding adrenomedullin(+)/CA19-9(+) exosomes into circulation that inhibit insulin secretion, likely through adrenomedullin-induced ER stress and failure of the unfolded protein response. ©2014 American Association for Cancer Research.

Systematic Analysis of γ-Aminobutyric Acid (GABA) Metabolism and Function in the Social Amoeba Dictyostelium discoideum*

PubMed Central

Wu, Yuantai; Janetopoulos, Chris

2013-01-01

While GABA has been suggested to regulate spore encapsulation in the social amoeba Dictyostelium discoideum, the metabolic profile and other potential functions of GABA during development remain unclear. In this study, we investigated the homeostasis of GABA metabolism by disrupting genes related to GABA metabolism and signaling. Extracellular levels of GABA are tightly regulated during early development, and GABA is generated by the glutamate decarboxylase, GadB, during growth and in early development. However, overexpression of the prespore-specific homologue, GadA, in the presence of GadB reduces production of extracellular GABA. Perturbation of extracellular GABA levels delays the process of aggregation. Cytosolic GABA is degraded by the GABA transaminase, GabT, in the mitochondria. Disruption of a putative vesicular GABA transporter (vGAT) homologue DdvGAT reduces secreted GABA. We identified the GABAB receptor-like family member GrlB as the major GABA receptor during early development, and either disruption or overexpression of GrlB delays aggregation. This delay is likely the result of an abolished pre-starvation response and late expression of several “early” developmental genes. Distinct genes are employed for GABA generation during sporulation. During sporulation, GadA alone is required for generating GABA and DdvGAT is likely responsible for GABA secretion. GrlE but not GrlB is the GABA receptor during late development. PMID:23548898

Thermo-responsive cell culture carriers based on poly(vinyl methyl ether)—the effect of biomolecular ligands to balance cell adhesion and stimulated detachment

NASA Astrophysics Data System (ADS)

Teichmann, Juliane; Nitschke, Mirko; Pette, Dagmar; Valtink, Monika; Gramm, Stefan; Härtel, Frauke V.; Noll, Thomas; Funk, Richard H. W.; Engelmann, Katrin; Werner, Carsten

2015-08-01

Two established material systems for thermally stimulated detachment of adherent cells were combined in a cross-linked polymer blend to merge favorable properties. Through this approach poly(N-isopropylacrylamide) (PNiPAAm) with its superior switching characteristic was paired with a poly(vinyl methyl ether)-based composition that allows adjusting physico-chemical and biomolecular properties in a wide range. Beyond pure PNiPAAm, the proposed thermo-responsive coating provides thickness, stiffness and swelling behavior, as well as an apposite density of reactive sites for biomolecular functionalization, as effective tuning parameters to meet specific requirements of a particular cell type regarding initial adhesion and ease of detachment. To illustrate the strength of this approach, the novel cell culture carrier was applied to generate transplantable sheets of human corneal endothelial cells (HCEC). Sheets were grown, detached, and transferred onto planar targets. Cell morphology, viability and functionality were analyzed by immunocytochemistry and determination of transepithelial electrical resistance (TEER) before and after sheet detachment and transfer. HCEC layers showed regular morphology with appropriate TEER. Cells were positive for function-associated marker proteins ZO-1, Na+/K+-ATPase, and paxillin, and extracellular matrix proteins fibronectin, laminin and collagen type IV before and after transfer. Sheet detachment and transfer did not impair cell viability. Subsequently, a potential application in ophthalmology was demonstrated by transplantation onto de-endothelialized porcine corneas in vitro. The novel thermo-responsive cell culture carrier facilitates the generation and transfer of functional HCEC sheets. This paves the way to generate tissue engineered human corneal endothelium as an alternative transplant source for endothelial keratoplasty.

Citral, A Monoterpene Protect Against High Glucose Induced Oxidative Injury in HepG2 Cell In Vitro-An Experimental Study.

PubMed

Subramaniyan, Sri Devi; Natarajan, Ashok Kumar

2017-08-01

Diabetes mellitus, a major metabolic disorder associated with hyperglycaemia is one of the leading cause of death in many developed countries. However, use of natural phytochemicals have been proved to have a protective effect against oxidative damage. To investigate the effect of citral, a monoterpene on high glucose induced cytotoxicity and oxidative stress in human hepatocellular liver carcinoma (Hep G2) cell line. Cells were treated with 50 mM concentration of glucose for 24 hours incubation following citral (30 μM) was added to confluent HepG2 cells. Cell viability, Reactive Oxygen Species (ROS) generation, DNA damage, lipid peroxidation, antioxidants and Mitogen Activated Protein Kinases (MAPKs) signaling were assessed in citral and/or high glucose induced HepG2 cells. Cells treated with glucose (50 mM), resulted in increased cytotoxicity, ROS generation, DNA damage, lipid peroxidation and depletion of enzymatic and non enzymatic antioxidants. In contrast, treatment with citral (30 μM) significantly decreased cell cytotoxicity, ROS generation, DNA damage, lipid peroxidation and increased antioxidants enzymes in high glucose induced HepG2 cells. In addition, the present study highlighted that high glucose treated cells showed increased expression of Extracellular Signal Regulated Protein Kinase-1 (ERK-1), c-Jun N-terminal Kinase (JNK) and p38 in HepG2 cells. On the other hand treatment with citral significantly suppressed the expression of ERK-1, JNK and p38 in high glucose induced HepG2 cells. Citral protects against high glucose induced oxidative stress through inhibiting ROS activated MAPK signaling pathway in HepG2 cells.

Characterization of two new putative adhesins of Leptospira interrogans.

PubMed

Figueredo, Jupciana M; Siqueira, Gabriela H; de Souza, Gisele O; Heinemann, Marcos B; Vasconcellos, Silvio A; Chapola, Erica G B; Nascimento, Ana L T O

2017-01-01

We here report the characterization of two novel proteins encoded by the genes LIC11122 and LIC12287, identified in the genome sequences of Leptospira interrogans, annotated, respectively, as a putative sigma factor and a hypothetical protein. The CDSs LIC11122 and LIC12287 have signal peptide SPII and SPI and are predicted to be located mainly at the cytoplasmic membrane of the bacteria. The genes were cloned and the proteins expressed using Escherichia coli. Proteinase K digestion showed that both proteins are surface exposed. Evaluation of interaction of recombinant proteins with extracellular matrix components revealed that they are laminin binding and they were called Lsa19 (LIC11122) and Lsa14 (LIC12287), for Leptospiral-surface adhesin of 19 and 14 kDa, respectively. The bindings were dose-dependent on protein concentration, reaching saturation, fulfilling the ligand-binding criteria. Reactivity of the recombinant proteins with leptospirosis human sera has shown that Lsa19 and, to a lesser extent, Lsa14, are recognized by antibodies, suggesting that, most probably, Lsa19 is expressed during infection. The proteins interact with plasminogen and generate plasmin in the presence of urokinase-type plasminogen activator. Plasmin generation in Leptospira has been associated with tissue penetration and immune evasion strategies. The presence of a sigma factor on the cell surface playing a secondary role, probably mediating host -pathogen interaction, suggests that LIC11122 is a moonlighting protein candidate. Although the biological significance of these putative adhesins will require the generation of mutants, our data suggest that Lsa19 is a potential candidate for future evaluation of its role in adhesion/colonization activities during L. interrogans infection.

Mitochondrial-derived hydrogen peroxide inhibits relaxation of bovine coronary arterial smooth muscle to hypoxia through stimulation of ERK MAP kinase.

PubMed

Gao, Qun; Zhao, Xiangmin; Ahmad, Mansoor; Wolin, Michael S

2009-12-01

Mitochondrial reactive oxygen species (ROS) are potentially important in vascular oxygen-sensing mechanisms because hypoxia appears to be a stimulus for mitochondrial ROS generation; however, scavenging of endogenous ROS does not alter relaxation of endothelium-denuded bovine coronary arteries (BCA) to hypoxia. The purpose of this study was to investigate the influence of increasing mitochondrial ROS on the relaxation of BCA to hypoxia. Increasing mitochondrial superoxide with inhibitors of electron transport (10 microM rotenone and antimycin) and by opening mitochondrial ATP-dependent K+ channels with 100 microM diazoxide were observed in this study to attenuate relaxation of BCA precontracted with 30 mM KCl to hypoxia by 68-76% and 38%, respectively. This effect of rotenone is not prevented by inhibiting NADPH oxidase (Nox) activation or scavenging superoxide with Peg-SOD; however, it is reversed 85% and 26% by increasing the consumption of intracellular peroxide by 0.1 mM ebselen and 32.5 U/ml Peg-catalase. Because inhibition of extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase (10 microM PD-98059), but not src kinase or rho kinase, also reverses the effects of rotenone by 69%, the peroxide-elicited force-enhancing effects of ERK appear to be attenuating the response to hypoxia. Rotenone increased the phosphorylation of ERK (by 163%). Activation of ERK in BCA with 0.1 mM peroxide or endogenous peroxide generated by stimulating Nox2 with a stretch treatment or contraction with 100 nM U-46619 also attenuated relaxation to hypoxia. Thus coronary arterial relaxation to hypoxia may be attenuated by pathophysiological conditions associated with increased peroxide generation by mitochondria or other sources that stimulate ERK.

The myeloperoxidase-derived oxidant HOSCN inhibits protein tyrosine phosphatases and modulates cell signalling via the mitogen-activated protein kinase (MAPK) pathway in macrophages

PubMed Central

Lane, Amanda E.; Tan, Joanne T. M.; Hawkins, Clare L.; Heather, Alison K.; Davies, Michael J.

2010-01-01

MPO (myeloperoxidase) catalyses the oxidation of chloride, bromide and thiocyanate by hydrogen peroxide to HOCl (hypochlorous acid), HOBr (hypobromous acid) and HOSCN (hypothiocyanous acid) respectively. Specificity constants indicate that SCN− is a major substrate for MPO. HOSCN is also a major oxidant generated by other peroxidases including salivary, gastric and eosinophil peroxidases. While HOCl and HOBr are powerful oxidizing agents, HOSCN is a less reactive, but more specific, oxidant which targets thiols and especially low pKa species. In the present study we show that HOSCN targets cysteine residues present in PTPs (protein tyrosine phosphatases) with this resulting in a loss of PTP activity for the isolated enzyme, in cell lysates and intact J774A.1 macrophage-like cells. Inhibition also occurs with MPO-generated HOCl and HOBr, but is more marked with MPO-generated HOSCN, particularly at longer incubation times. This inhibition is reversed by dithiothreitol, particularly at early time points, consistent with the reversible oxidation of the active site cysteine residue to give either a cysteine–SCN adduct or a sulfenic acid. Inhibition of PTP activity is associated with increased phosphorylation of p38a and ERK2 (extracellular-signal-regulated kinase 2) as detected by Western blot analysis and phosphoprotein arrays, and results in altered MAPK (mitogen-activated protein kinase) signalling. These data indicate that the highly selective targeting of some protein thiols by HOSCN can result in perturbation of cellular phosphorylation and altered cell signalling. These changes occur with (patho)physiological concentrations of SCN− ions, and implicate HOSCN as an important mediator of inflammation-induced oxidative damage, particularly in smokers who have elevated plasma levels of SCN−. PMID:20528774

Thermo-responsive cell culture carriers based on poly(vinyl methyl ether)-the effect of biomolecular ligands to balance cell adhesion and stimulated detachment.

PubMed

Teichmann, Juliane; Nitschke, Mirko; Pette, Dagmar; Valtink, Monika; Gramm, Stefan; Härtel, Frauke V; Noll, Thomas; Funk, Richard H W; Engelmann, Katrin; Werner, Carsten

2015-08-01

Two established material systems for thermally stimulated detachment of adherent cells were combined in a cross-linked polymer blend to merge favorable properties. Through this approach poly( N -isopropylacrylamide) (PNiPAAm) with its superior switching characteristic was paired with a poly(vinyl methyl ether)-based composition that allows adjusting physico-chemical and biomolecular properties in a wide range. Beyond pure PNiPAAm, the proposed thermo-responsive coating provides thickness, stiffness and swelling behavior, as well as an apposite density of reactive sites for biomolecular functionalization, as effective tuning parameters to meet specific requirements of a particular cell type regarding initial adhesion and ease of detachment. To illustrate the strength of this approach, the novel cell culture carrier was applied to generate transplantable sheets of human corneal endothelial cells (HCEC). Sheets were grown, detached, and transferred onto planar targets. Cell morphology, viability and functionality were analyzed by immunocytochemistry and determination of transepithelial electrical resistance (TEER) before and after sheet detachment and transfer. HCEC layers showed regular morphology with appropriate TEER. Cells were positive for function-associated marker proteins ZO-1, Na + /K + -ATPase, and paxillin, and extracellular matrix proteins fibronectin, laminin and collagen type IV before and after transfer. Sheet detachment and transfer did not impair cell viability. Subsequently, a potential application in ophthalmology was demonstrated by transplantation onto de-endothelialized porcine corneas in vitro . The novel thermo-responsive cell culture carrier facilitates the generation and transfer of functional HCEC sheets. This paves the way to generate tissue engineered human corneal endothelium as an alternative transplant source for endothelial keratoplasty.

Thermo-responsive cell culture carriers based on poly(vinyl methyl ether)—the effect of biomolecular ligands to balance cell adhesion and stimulated detachment

PubMed Central

Teichmann, Juliane; Nitschke, Mirko; Pette, Dagmar; Valtink, Monika; Gramm, Stefan; Härtel, Frauke V; Noll, Thomas; Funk, Richard H W; Engelmann, Katrin; Werner, Carsten

2015-01-01

Two established material systems for thermally stimulated detachment of adherent cells were combined in a cross-linked polymer blend to merge favorable properties. Through this approach poly(N-isopropylacrylamide) (PNiPAAm) with its superior switching characteristic was paired with a poly(vinyl methyl ether)-based composition that allows adjusting physico-chemical and biomolecular properties in a wide range. Beyond pure PNiPAAm, the proposed thermo-responsive coating provides thickness, stiffness and swelling behavior, as well as an apposite density of reactive sites for biomolecular functionalization, as effective tuning parameters to meet specific requirements of a particular cell type regarding initial adhesion and ease of detachment. To illustrate the strength of this approach, the novel cell culture carrier was applied to generate transplantable sheets of human corneal endothelial cells (HCEC). Sheets were grown, detached, and transferred onto planar targets. Cell morphology, viability and functionality were analyzed by immunocytochemistry and determination of transepithelial electrical resistance (TEER) before and after sheet detachment and transfer. HCEC layers showed regular morphology with appropriate TEER. Cells were positive for function-associated marker proteins ZO-1, Na+/K+-ATPase, and paxillin, and extracellular matrix proteins fibronectin, laminin and collagen type IV before and after transfer. Sheet detachment and transfer did not impair cell viability. Subsequently, a potential application in ophthalmology was demonstrated by transplantation onto de-endothelialized porcine corneas in vitro. The novel thermo-responsive cell culture carrier facilitates the generation and transfer of functional HCEC sheets. This paves the way to generate tissue engineered human corneal endothelium as an alternative transplant source for endothelial keratoplasty. PMID:27877823

Stereoselectivity in catalytic reactions: CO oxidation on Pd(100) by rotationally aligned O2 molecules

NASA Astrophysics Data System (ADS)

Vattuone, L.; Gerbi, A.; Savio, L.; Cappelletti, D.; Pirani, F.; Rocca, M.

2010-05-01

We report on stereodynamical effects in heterogeneous catalytic reactions as measured by molecular beam-surface experiments. Specifically for CO oxidation on Pd(100) we find that the rotational alignment of the incoming O2 at low (Θ = 0.04 ML) and at intermediate (ΘCO = 0.17 ML) CO pre-coverage, causes a higher reactivity of molecules in high and in low helicity states, respectively (corresponding to helicoptering and cartwheeling motion of O2). In first approximation, at low CO pre-coverage the difference in reactivity is determined by the different location of the O atoms generated in the dissociation process by the different parent molecules, while at intermediate CO pre-coverage the reactivity is influenced also by the different ability of cartwheeling and helicoptering O2 to penetrate through the CO adlayer. In accord with this the total amount of CO2 produced is always largest for helicopters which generate supersurface O atoms at least in the low CO pre-coverage limit. A deeper inspection of the data indicates, however, that the dynamics is more complex, two different pathways being present for the reaction with O generated by helicopters and one for O generated by cartwheels. Moreover, cartwheels generated oxygen influences the reactivity of subsequently arriving helicopters.

Moderate extracellular acidification inhibits capsaicin-induced cell death through regulating calcium mobilization, NF-{kappa}B translocation and ROS production in synoviocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hu, Fen; Yang, Shuang; Zhao, Dan

Highlights: Black-Right-Pointing-Pointer Moderate extracellular acidification regulates intracellular Ca{sup 2+} mobilization. Black-Right-Pointing-Pointer Moderate acidification activates NF-{kappa}B nuclear translocation in synoviocytes. Black-Right-Pointing-Pointer Moderate acidification depresses the ROS production induced by capsaicin. Black-Right-Pointing-Pointer Moderate acidification inhibits capsaicin-caused synoviocyte death. -- Abstract: We previously show the expression of transient receptor potential vanilloid 1 (TRPV1) in primary synoviocytes from collagen-induced arthritis (CIA) rats. Capsaicin and lowered extracellular pH from 7.4 to 5.5 induce cell death through TRPV1-mediated Ca{sup 2+} entry and reactive oxygen species (ROS) production. However, under the pathological condition in rheumatoid arthritis, the synovial fluid is acidified to a moderate level (about pHmore » 6.8). In the present study, we examined the effects of pH 6.8 on the TRPV1-mediated cell death. Our finding is different or even opposite from what was observed at pH 5.5. We found that the moderate extracellular acidification (from pH 7.4 to 6.8) inhibited the capsaicin-induced Ca{sup 2+} entry through attenuating the activity of TRPV1. In the mean time, it triggered a phospholipse C (PLC)-related Ca{sup 2+} release from intracellular stores. The nuclear translocation of NF-{kappa}B was found at pH 6.8, and this also depends on PLC activation. Moreover, the capsaicin-evoked massive ROS production and cell death were depressed at pH 6.8, both of which are dependent on the activation of PLC and NF-{kappa}B. Taken together, these results suggested that the moderate extracellular acidification inhibited the capsaicin-induced synoviocyte death through regulating Ca{sup 2+} mobilization, activating NF-{kappa}B nuclear translocation and depressing ROS production.« less

A Novel Approach to Teach the Generation of Bioelectrical Potentials from a Descriptive and Quantitative Perspective

ERIC Educational Resources Information Center

Rodriguez-Falces, Javier

2013-01-01

In electrophysiology studies, it is becoming increasingly common to explain experimental observations using both descriptive methods and quantitative approaches. However, some electrophysiological phenomena, such as the generation of extracellular potentials that results from the propagation of the excitation source along the muscle fiber, are…

High efficiency H6 single-phase transformerless grid-tied PV inverter with proposed modulation for reactive power generation

NASA Astrophysics Data System (ADS)

Almasoudi, Fahad M.; Alatawi, Khaled S.; Matin, Mohammad

2017-08-01

Implementation of transformerless inverters in PV grid-tied system offer great benefits such as high efficiency, light weight, low cost, etc. Most of the proposed transformerless inverters in literature are verified for only real power application. Currently, international standards such as VDE-AR-N 4105 has demanded that PV grid-tied inverters should have the ability of controlling a specific amount of reactive power. Generation of reactive power cannot be accomplished in single phase transformerless inverter topologies because the existing modulation techniques are not adopted for a freewheeling path in the negative power region. This paper enhances a previous high efficiency proposed H6 trnasformerless inverter with SiC MOSFETs and demonstrates new operating modes for the generation of reactive power. A proposed pulse width modulation (PWM) technique is applied to achieve bidirectional current flow through freewheeling state. A comparison of the proposed H6 transformerless inverter using SiC MOSFETs and Si MOSFTEs is presented in terms of power losses and efficiency. The results show that reactive power control is attained without adding any additional active devices or modification to the inverter structure. Also, the proposed modulation maintains a constant common mode voltage (CM) during every operating mode and has low leakage current. The performance of the proposed system verifies its effectiveness in the next generation PV system.

Exploiting algal NADPH oxidase for biophotovoltaic energy

DOE PAGES

Anderson, Alexander; Laohavisit, Anuphon; Blaby, Ian K.; ...

2015-01-29

Photosynthetic microbes exhibit light-dependent electron export across the cell membrane, which can generate electricity in biological photovoltaic (BPV) devices. How electrons are exported remains to be determined; the identification of mechanisms would help selection or generation of photosynthetic microbes capable of enhanced electrical output. We show that plasma membrane NADPH oxidase activity is a significant component of light-dependent generation of electricity by the unicellular green alga Chlamydomonas reinhardtii. NADPH oxidases export electrons across the plasma membrane to form superoxide anion from oxygen. The C. reinhardtii mutant lacking the NADPH oxidase encoded by RBO1 is impaired in both extracellular superoxide anionmore » production and current generation in a BPV device. Complementation with the wild-type gene restores both capacities, demonstrating the role of the enzyme in electron export. Monitoring light-dependent extracellular superoxide production with a colorimetric assay is shown to be an effective way of screening for electrogenic potential of candidate algal strains. Furthermore, the results show that algal NADPH oxidases are important for superoxide anion production and open avenues for optimizing the biological component of these devices.« less

AMELIORATION OF ACID MINE DRAINAGE USING REACTIVE MIXTURES IN PERMEABLE REACTIVE BARRIERS

EPA Science Inventory

The generation and release of acidic drainage from mine wastes is an environmental problem of international scale. The use of zero-valent iron and/or iron mixtures in subsurface Permeable Reactive Barriers (PRB) presents a possible passive alternative for remediating acidic grou...

Functional advantages conferred by extracellular prokaryotic membrane vesicles.

PubMed

Manning, Andrew J; Kuehn, Meta J

2013-01-01

The absence of subcellular organelles is a characteristic typically used to distinguish prokaryotic from eukaryotic cells. But recent discoveries do not support this dogma. Over the past 50 years, researchers have begun to appreciate and characterize Gram-negative bacterial outer membrane-derived vesicles and Gram-positive and archaeal membrane vesicles. These extracellular, membrane-bound organelles can perform a variety of functions, including binding and delivery of DNA, transport of virulence factors, protection of the cell from outer membrane targeting antimicrobials and ridding the cell of toxic envelope proteins. Here, we review the contributions of these extracellular organelles to prokaryotic physiology and compare these with the contributions of the bacterial interior membrane-bound organelles responsible for harvesting light energy and for generating magnetic crystals of heavy metals. Understanding the roles of these multifunctional extracellular vesicle organelles as microbial tools will help us to better realize the diverse interactions that occur in our polymicrobial world. Copyright © 2013 S. Karger AG, Basel.

Effects of reactive oxygen species and interplay of antioxidants during physical exercise in skeletal muscles.

PubMed

Thirupathi, Anand; Pinho, Ricardo A

2018-05-01

A large number of researches have led to a substantial growth of knowledge about exercise and oxidative stress. Initial investigations reported that physical exercise generates free radical-mediated damages to cells; however, in recent years, studies have shown that regular exercise can upregulate endogenous antioxidants and reduce oxidative damage. Yet, strenuous exercise perturbs the antioxidant system by increasing the reactive oxygen species (ROS) content. These alterations in the cellular environment seem to occur in an exercise type-dependent manner. The source of ROS generation during exercise is debatable, but now it is well established that both contracting and relaxing skeletal muscles generate reactive oxygen species and reactive nitrogen species. In particular, exercises of higher intensity and longer duration can cause oxidative damage to lipids, proteins, and nucleotides in myocytes. In this review, we summarize the ROS effects and interplay of antioxidants in skeletal muscle during physical exercise. Additionally, we discuss how ROS-mediated signaling influences physical exercise in antioxidant system.

An improved AVC strategy applied in distributed wind power system

NASA Astrophysics Data System (ADS)

Zhao, Y. N.; Liu, Q. H.; Song, S. Y.; Mao, W.

2016-08-01

Traditional AVC strategy is mainly used in wind farm and only concerns about grid connection point, which is not suitable for distributed wind power system. Therefore, this paper comes up with an improved AVC strategy applied in distributed wind power system. The strategy takes all nodes of distribution network into consideration and chooses the node having the most serious voltage deviation as control point to calculate the reactive power reference. In addition, distribution principles can be divided into two conditions: when wind generators access to network on single node, the reactive power reference is distributed according to reactive power capacity; when wind generators access to network on multi-node, the reference is distributed according to sensitivity. Simulation results show the correctness and reliability of the strategy. Compared with traditional control strategy, the strategy described in this paper can make full use of generators reactive power output ability according to the distribution network voltage condition and improve the distribution network voltage level effectively.

Drug metabolism and hypersensitivity reactions to drugs.

PubMed

Agúndez, José A G; Mayorga, Cristobalina; García-Martin, Elena

2015-08-01

The aim of the present review was to discuss recent advances supporting a role of drug metabolism, and particularly of the generation of reactive metabolites, in hypersensitivity reactions to drugs. The development of novel mass-spectrometry procedures has allowed the identification of reactive metabolites from drugs known to be involved in hypersensitivity reactions, including amoxicillin and nonsteroidal antiinflammatory drugs such as aspirin, diclofenac or metamizole. Recent studies demonstrated that reactive metabolites may efficiently bind plasma proteins, thus suggesting that drug metabolites, rather than - or in addition to - parent drugs, may elicit an immune response. As drug metabolic profiles are often determined by variability in the genes coding for drug-metabolizing enzymes, it is conceivable that an altered drug metabolism may predispose to the generation of reactive drug metabolites and hence to hypersensitivity reactions. These findings support the potential for the use of pharmacogenomics tests in hypersensitivity (type B) adverse reactions, in addition to the well known utility of these tests in type A adverse reactions. Growing evidence supports a link between genetically determined drug metabolism, altered metabolic profiles, generation of highly reactive metabolites and haptenization. Additional research is required to developing robust biomarkers for drug-induced hypersensitivity reactions.

Effect of Morphologic Features of Neurons on the Extracellular Electric Potential: A Simulation Study Using Cable Theory and Electro-Quasi-Static Equations.

PubMed

Bestel, R; Appali, R; van Rienen, U; Thielemann, C

2017-11-01

Microelectrode arrays serve as an indispensable tool in electro-physiological research to study the electrical activity of neural cells, enabling measurements of single cell as well as network communication analysis. Recent experimental studies have reported that the neuronal geometry has an influence on electrical signaling and extracellular recordings. However, the corresponding mechanisms are not yet fully understood and require further investigation. Allowing systematic parameter studies, computational modeling provides the opportunity to examine the underlying effects that influence extracellular potentials. In this letter, we present an in silico single cell model to analyze the effect of geometrical variability on the extracellular electric potentials. We describe finite element models of a single neuron with varying geometric complexity in three-dimensional space. The electric potential generation of the neuron is modeled using Hodgkin-Huxley equations. The signal propagation is described with electro-quasi-static equations, and results are compared with corresponding cable equation descriptions. Our results show that both the geometric dimensions and the distribution of ion channels of a neuron are critical factors that significantly influence both the amplitude and shape of extracellular potentials.

Method for generating a highly reactive plasma for exhaust gas aftertreatment and enhanced catalyst reactivity

DOEpatents

Whealton, John H.; Hanson, Gregory R.; Storey, John M.; Raridon, Richard J.; Armfield, Jeffrey S.; Bigelow, Timothy S.; Graves, Ronald L.

2001-01-01

A method for non-thermal plasma aftertreatment of exhaust gases the method comprising the steps of providing short risetime (about 40 ps), high frequency (about 5G hz), high power bursts of low-duty factor microwaves sufficient to generate a dielectric barrier discharge and passing a gas to treated through the discharge so as to cause dissociative reduction of the exhaust gases. The invention also includes a reactor for generating the non-thermal plasma.

Participation of intracellular and extracellular pH changes in photosynthetic response development induced by variation potential in pumpkin seedlings.

PubMed

Sherstneva, O N; Vodeneev, V A; Katicheva, L A; Surova, L M; Sukhov, V S

2015-06-01

Electrical signals presented in plants by action potential and by variation potential (VP) can induce a reversible inactivation of photosynthesis. Changes in the intracellular and extracellular pH during VP generation are a potential mechanism of photosynthetic response induction; however, this hypothesis requires additional experimental investigation. The purpose of the present work was to analyze the influence of pH changes on induction of the photosynthetic response in pumpkin. It was shown that a burning of the cotyledon induced VP propagation into true leaves of pumpkin seedlings inducing a decrease in the photosynthetic CO2 assimilation and an increase in non-photochemical quenching of fluorescence, whereas respiration was activated insignificantly. The photosynthetic response magnitude depended linearly on the VP amplitude. The intracellular and extracellular concentrations of protons were analyzed using pH-sensitive fluorescent probes, and the VP generation was shown to be accompanied by apoplast alkalization (0.4 pH unit) and cytoplasm acidification (0.3 pH unit). The influence of changes in the incubation medium pH on the non-photochemical quenching of fluorescence of isolated chloroplasts was also investigated. It was found that acidification of the medium stimulated the non-photochemical quenching, and the magnitude of this increase depended on the decrease in pH. Our results confirm the contribution of changes in intracellular and extracellular pH to induction of the photosynthetic response caused by VP. Possible mechanisms of the influence of pH changes on photosynthesis are discussed.

mGluR5 stimulating Homer–PIKE formation initiates icariin induced cardiomyogenesis of mouse embryonic stem cells by activating reactive oxygen species

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou, Limin; Huang, Yujie; Zhang, Yingying

2013-06-10

Icariin (ICA) has been reported to facilitate cardiac differentiation of mouse embryonic stem (ES) cells; however, the mechanism by which ICA induced cardiomyogenesis has not been fully elucidated yet. Here, an underlying signaling network including metabotropic glutamate receptor 5 (mGluR5), Homer, phosphatidylinositol 3-Kinase Enhancer (PIKE), phosphatidylinositol 3-Kinase (PI3K), reactive oxygen species (ROS) and nuclear factor-kappaB (NF-κB) was investigated in ICA induced cardiomyogenesis. Our results showed that the co-expression of mGluR5 together with α-actinin or Troponin T in embryoid bodies (EBs) treated with ICA was elevated to 10.86% and 9.62%, compared with the case in the control (4.04% and 3.45%, respectively).more » Exposure of EBs to ICA for 2 h remarkably increased the dimeric form of mGluR5, which was inhibited by small interfering RNA targeting mGluR5 (si-mGluR5). Moreover, the extracellular glutamate concentration in ICA treatment medium was elevated to 28.9±3.5 μM. Furthermore, the activation of mGluR5 by ICA triggered the formation of Homer–PIKE complex and activated PI3K, stimulating ROS generation and NF-κB nuclear translocation. Knockdown of mGluR5 or inhibition of PI3K by LY294002 blocked ICA induced cardiomyogenesis via repressing mGluR5 pathway, reducing ROS and NF-κB activation. These results revealed that the inducible mechanisms of ICA were related to activate mGluR5 pathway. -- Highlights: • ICA increased mGluR5 expression in cardiac differentiation of ES cells. • ICA enhanced the glutamate level and the receptor mGluR5 dimerization, stimulating the formation of Homer–PIKE complex. • Knockdown of mGluR5 or inhibition of PI3K by LY294002 inhibited ICA induced ROS generation and NF-κB nuclear translocation.« less

The inhibitory effect of CIL-102 on the growth of human astrocytoma cells is mediated by the generation of reactive oxygen species and induction of ERK1/2 MAPK

DOE Office of Scientific and Technical Information (OSTI.GOV)

Teng, Chih-Chuan; Institute of Basic Medicine Science, National Cheng Kung University, Tainan, Taiwan; Kuo, Hsing-Chun

2012-08-15

CIL-102 (1-[4-(furo[2,3-b]quinolin-4-ylamino)phenyl]ethanone) is the major active agent of the alkaloid derivative of Camptotheca acuminata, with multiple pharmacological activities, including anticancer effects and promotion of apoptosis. The mechanism by which CIL-102 inhibits growth remains poorly understood in human astrocytoma cells. Herein, we investigated the molecular mechanisms by which CIL-102 affects the generation of reactive oxygen species (ROS) and cell cycle G2/M arrest in glioma cells. Treatment of U87 cells with 1.0 μM CIL-102 resulted in phosphorylation of extracellular signal-related kinase (ERK1/2), downregulation of cell cycle-related proteins (cyclin A, cyclin B, cyclin D1, and cdk1), and phosphorylation of cdk1Tyr{sup 15} and Cdc25cSer{supmore » 216}. Furthermore, treatment with the ERK1/2 inhibitor PD98059 abolished CIL-102-induced Cdc25cSer{sup 216} expression and reversed CIL-102-inhibited cdk1 activation. In addition, N-acetyl cysteine (NAC), an ROS scavenger, blocked cell cycle G2/M arrest and phosphorylation of ERK1/2 and Cdc25cSer{sup 216} in U87 cells. CIL-102-mediated ERK1/2 and ROS production, and cell cycle arrest were blocked by treatment with specific inhibitors. In conclusion, we have identified a novel CIL-102-inhibited proliferation in U87 cells by activating the ERK1/2 and Cdc25cSer{sup 216} cell cycle-related proteins and inducing ROS production; this might be a new mechanism in human astrocytoma cells. -- Highlights: ► We show the effects of CIL-102 on the G2/M arrest of human astrocytoma cells. ► ROS and the Ras/ERK1/2 triggering pathways are involved in the CIL-102 treatment. ► CIL-102 induces sustained activation of ERK1/2 and Cdc25c and ROS are required.« less

Intracellular and non-neuronal targets of voltage-gated potassium channel complex antibodies

PubMed Central

Lang, Bethan; Makuch, Mateusz; Moloney, Teresa; Dettmann, Inga; Mindorf, Swantje; Probst, Christian; Stoecker, Winfried; Buckley, Camilla; Newton, Charles R; Leite, M Isabel; Maddison, Paul; Komorowski, Lars; Adcock, Jane; Vincent, Angela; Waters, Patrick; Irani, Sarosh R

2017-01-01

Objectives Autoantibodies against the extracellular domains of the voltage-gated potassium channel (VGKC) complex proteins, leucine-rich glioma-inactivated 1 (LGI1) and contactin-associated protein-2 (CASPR2), are found in patients with limbic encephalitis, faciobrachial dystonic seizures, Morvan's syndrome and neuromyotonia. However, in routine testing, VGKC complex antibodies without LGI1 or CASPR2 reactivities (double-negative) are more common than LGI1 or CASPR2 specificities. Therefore, the target(s) and clinical associations of double-negative antibodies need to be determined. Methods Sera (n=1131) from several clinically defined cohorts were tested for IgG radioimmunoprecipitation of radioiodinated α-dendrotoxin (125I-αDTX)-labelled VGKC complexes from mammalian brain extracts. Positive samples were systematically tested for live hippocampal neuron reactivity, IgG precipitation of 125I-αDTX and 125I-αDTX-labelled Kv1 subunits, and by cell-based assays which expressed Kv1 subunits, LGI1 and CASPR2. Results VGKC complex antibodies were found in 162 of 1131 (14%) sera. 90 of these (56%) had antibodies targeting the extracellular domains of LGI1 or CASPR2. Of the remaining 72 double-negative sera, 10 (14%) immunoprecipitated 125I-αDTX itself, and 27 (38%) bound to solubilised co-expressed Kv1.1/1.2/1.6 subunits and/or Kv1.2 subunits alone, at levels proportionate to VGKC complex antibody levels (r=0.57, p=0.0017). The sera with LGI1 and CASPR2 antibodies immunoprecipitated neither preparation. None of the 27 Kv1-precipitating samples bound live hippocampal neurons or Kv1 extracellular domains, but 16 (59%) bound to permeabilised Kv1-expressing human embryonic kidney 293T cells. These intracellular Kv1 antibodies mainly associated with non-immune disease aetiologies, poor longitudinal clinical–serological correlations and a limited immunotherapy response. Conclusions Double-negative VGKC complex antibodies are often directed against cytosolic epitopes of Kv1 subunits and occasionally against non-mammalian αDTX. These antibodies should no longer be classified as neuronal-surface antibodies. They consequently lack pathogenic potential and do not in themselves support the use of immunotherapies. PMID:28115470

Sulforaphane Inhibits Lipopolysaccharide-Induced Inflammation, Cytotoxicity, Oxidative Stress, and miR-155 Expression and Switches to Mox Phenotype through Activating Extracellular Signal-Regulated Kinase 1/2-Nuclear Factor Erythroid 2-Related Factor 2/Antioxidant Response Element Pathway in Murine Microglial Cells.

PubMed

Eren, Erden; Tufekci, Kemal Ugur; Isci, Kamer Burak; Tastan, Bora; Genc, Kursad; Genc, Sermin

2018-01-01

Sulforaphane (SFN) is a natural product with cytoprotective, anti-inflammatory, and antioxidant effects. In this study, we evaluated the mechanisms of its effects on lipopolysaccharide (LPS)-induced cell death, inflammation, oxidative stress, and polarization in murine microglia. We found that SFN protects N9 microglial cells upon LPS-induced cell death and suppresses LPS-induced levels of secreted pro-inflammatory cytokines, tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-6. SFN is also a potent inducer of redox sensitive transcription factor, nuclear factor erythroid 2-related factor 2 (Nrf2), which is responsible for the transcription of antioxidant, cytoprotective, and anti-inflammatory genes. SFN induced translocation of Nrf2 to the nucleus via extracellular signal-regulated kinase 1/2 (ERK1/2) pathway activation. siRNA-mediated knockdown study showed that the effects of SFN on LPS-induced reactive oxygen species, reactive nitrogen species, and pro-inflammatory cytokine production and cell death are partly Nrf2 dependent. Mox phenotype is a novel microglial phenotype that has roles in oxidative stress responses. Our results suggested that SFN induced the Mox phenotype in murine microglia through Nrf2 pathway. SFN also alleviated LPS-induced expression of inflammatory microRNA, miR-155. Finally, SFN inhibits microglia-mediated neurotoxicity as demonstrated by conditioned medium and co-culture experiments. In conclusion, SFN exerts protective effects on microglia and modulates the microglial activation state.

Sulforaphane Inhibits Lipopolysaccharide-Induced Inflammation, Cytotoxicity, Oxidative Stress, and miR-155 Expression and Switches to Mox Phenotype through Activating Extracellular Signal-Regulated Kinase 1/2–Nuclear Factor Erythroid 2-Related Factor 2/Antioxidant Response Element Pathway in Murine Microglial Cells

PubMed Central

Eren, Erden; Tufekci, Kemal Ugur; Isci, Kamer Burak; Tastan, Bora; Genc, Kursad; Genc, Sermin

2018-01-01

Sulforaphane (SFN) is a natural product with cytoprotective, anti-inflammatory, and antioxidant effects. In this study, we evaluated the mechanisms of its effects on lipopolysaccharide (LPS)-induced cell death, inflammation, oxidative stress, and polarization in murine microglia. We found that SFN protects N9 microglial cells upon LPS-induced cell death and suppresses LPS-induced levels of secreted pro-inflammatory cytokines, tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-6. SFN is also a potent inducer of redox sensitive transcription factor, nuclear factor erythroid 2-related factor 2 (Nrf2), which is responsible for the transcription of antioxidant, cytoprotective, and anti-inflammatory genes. SFN induced translocation of Nrf2 to the nucleus via extracellular signal-regulated kinase 1/2 (ERK1/2) pathway activation. siRNA-mediated knockdown study showed that the effects of SFN on LPS-induced reactive oxygen species, reactive nitrogen species, and pro-inflammatory cytokine production and cell death are partly Nrf2 dependent. Mox phenotype is a novel microglial phenotype that has roles in oxidative stress responses. Our results suggested that SFN induced the Mox phenotype in murine microglia through Nrf2 pathway. SFN also alleviated LPS-induced expression of inflammatory microRNA, miR-155. Finally, SFN inhibits microglia-mediated neurotoxicity as demonstrated by conditioned medium and co-culture experiments. In conclusion, SFN exerts protective effects on microglia and modulates the microglial activation state. PMID:29410668

Modulation of Neutrophil Extracellular Trap and Reactive Oxygen Species Release by Periodontal Bacteria

PubMed Central

White, Phillipa C.; Milward, Michael R.; Cooper, Paul R.

2017-01-01

ABSTRACT Oral bacteria are the main trigger for the development of periodontitis, and some species are known to modulate neutrophil function. This study aimed to explore the release of neutrophil extracellular traps (NETs), associated antimicrobial proteins, and reactive oxygen species (ROS) in response to periodontal bacteria, as well as the underlying pathways. Isolated peripheral blood neutrophils were stimulated with 19 periodontal bacteria. NET and ROS release, as well as the expression of NET-bound antimicrobial proteins, elastase, myeloperoxidase, and cathepsin G, in response to these species was measured using fluorescence-based assays. NET and ROS release was monitored after the addition of NADP (NADPH) oxidase pathway modulators and inhibitors of Toll-like receptors (TLRs). Moreover, bacterial entrapment by NETs was visualized microscopically, and bacterial killing was assessed by bacterial culture. Certain microorganisms, e.g., Veillonella parvula and Streptococcus gordonii, stimulated higher levels of ROS and NET release than others. NETs were found to entrap, but not kill, all periodontal bacteria tested. NADPH oxidase pathway modulators decreased ROS production but not NET production in response to the bacteria. Interestingly, TLR inhibitors did not impact ROS and NET release. These data suggest that the variability in the neutrophil response toward different bacteria may contribute to the pathogenesis of periodontal diseases by mechanisms such as bacterial avoidance of host responses and activation of neutrophils. Moreover, our results indicate that bacterium-stimulated NET release may arise in part via NADPH oxidase-independent mechanisms. The role of TLR signaling in bacterium-induced ROS and NET release needs to be further elucidated. PMID:28947649

Modulation of Neutrophil Extracellular Trap and Reactive Oxygen Species Release by Periodontal Bacteria.

PubMed

Hirschfeld, Josefine; White, Phillipa C; Milward, Michael R; Cooper, Paul R; Chapple, Iain L C

2017-12-01

Oral bacteria are the main trigger for the development of periodontitis, and some species are known to modulate neutrophil function. This study aimed to explore the release of neutrophil extracellular traps (NETs), associated antimicrobial proteins, and reactive oxygen species (ROS) in response to periodontal bacteria, as well as the underlying pathways. Isolated peripheral blood neutrophils were stimulated with 19 periodontal bacteria. NET and ROS release, as well as the expression of NET-bound antimicrobial proteins, elastase, myeloperoxidase, and cathepsin G, in response to these species was measured using fluorescence-based assays. NET and ROS release was monitored after the addition of NADP (NADPH) oxidase pathway modulators and inhibitors of Toll-like receptors (TLRs). Moreover, bacterial entrapment by NETs was visualized microscopically, and bacterial killing was assessed by bacterial culture. Certain microorganisms, e.g., Veillonella parvula and Streptococcus gordonii , stimulated higher levels of ROS and NET release than others. NETs were found to entrap, but not kill, all periodontal bacteria tested. NADPH oxidase pathway modulators decreased ROS production but not NET production in response to the bacteria. Interestingly, TLR inhibitors did not impact ROS and NET release. These data suggest that the variability in the neutrophil response toward different bacteria may contribute to the pathogenesis of periodontal diseases by mechanisms such as bacterial avoidance of host responses and activation of neutrophils. Moreover, our results indicate that bacterium-stimulated NET release may arise in part via NADPH oxidase-independent mechanisms. The role of TLR signaling in bacterium-induced ROS and NET release needs to be further elucidated. Copyright © 2017 American Society for Microbiology.

Effect of cefodizime and ceftriaxone on phagocytic function in patients with severe infections.

PubMed Central

Wenisch, C; Parschalk, B; Hasenhündl, M; Wiesinger, E; Graninger, W

1995-01-01

Thirty patients with severe bacterial infections were treated with 50 mg of cefodizime per kg of body weight once daily or 50 mg of ceftriaxone per kg once daily for 10 +/- 3 days. The effect of cefodizime and ceftriaxone on the phagocytic capacity and generation of reactive oxygen intermediates after phagocytosis by granulocytes was assessed prior to, during, and after therapy. Flow cytometry was used to study phagocytic capacity by measuring the uptake of fluorescein-labeled bacteria. The generation of reactive oxygen intermediates after phagocytosis was estimated by the quantification of the intracellular conversion of dihydrorhodamine 123 to rhodamine 123. Prior to therapy, patients in both groups exhibited a decreased capacity to phagocytize Escherichia coli and subsequently to generate reactive oxygen intermediates. Granulocyte function increased after the initiation of therapy and normalized within 7 days for the ceftriaxone-treated patients and within 3 days for the cefodizime group (P < 0.05). In the cefodizime group, an enhancement of phagocytic capacity was observed 14 days after the initiation of therapy (P < 0.05). Prior to therapy, phagocytic capacity was significantly correlated with the generation of reactive oxygen products (r = 0.674 and P < 0.005). PMID:7793871

Microfluidic vascularized bone tissue model with hydroxyapatite-incorporated extracellular matrix.

PubMed

Jusoh, Norhana; Oh, Soojung; Kim, Sudong; Kim, Jangho; Jeon, Noo Li

2015-10-21

Current in vitro systems mimicking bone tissues fail to fully integrate the three-dimensional (3D) microvasculature and bone tissue microenvironments, decreasing their similarity to in vivo conditions. Here, we propose 3D microvascular networks in a hydroxyapatite (HA)-incorporated extracellular matrix (ECM) for designing and manipulating a vascularized bone tissue model in a microfluidic device. Incorporation of HA of various concentrations resulted in ECM with varying mechanical properties. Sprouting angiogenesis was affected by mechanically modulated HA-extracellular matrix interactions, generating a model of vascularized bone microenvironment. Using this platform, we observed that hydroxyapatite enhanced angiogenic properties such as sprout length, sprouting speed, sprout number, and lumen diameter. This new platform integrates fibrin ECM with the synthetic bone mineral HA to provide in vivo-like microenvironments for bone vessel sprouting.

[Establishment and evaluation of an in vitro method for neutrophil extracellular trap generation and degradation].

PubMed

Li, Jinlong; Zhang, Yidan; Zhou, Xin; Ji, Wenjie; Zhao, Jihong; Wei, Luqing; Li, Yuming

2014-09-01

To evaluate a novel method for in vitro generation and degradation of neutrophil extracellular traps (NETs), which are a newly recognized structure that is involved in the pathogenesis of autoimmune diseases and thrombosis. Neutrophils from peripheral blood of healthy donors were obtained by Ficoll-Histopaque gradient separation. NET release was initiated by phorbol myristate acetate (PMA) and validated by immunofluorescence staining and agarose gel electrophoresis. NETs degraded by DNase I and healthy human plasma were quantified by fluorescence spectrometry after staining with PicoGreen. HE staining showed that the purity of neutrophils was up to 95% after Ficoll-Histopaque gradient separation. NET immunofluorescent staining revealed that the network structure was mainly composed of DNA and histones, with molecular length more than 10 kb as demonstrated by agarose gel electrophoresis. Moreover, both DNase and healthy human plasma could induce the degradation of NETs, in varying degrees. This work established an efficient method for in vitro generation and degradation of human NETs.

Aging effects on reactivity of an aluminum-based drinking-water treatment residual as a soil amendment.

PubMed

Agyin-Birikorang, S; O'Connor, G A

2009-01-01

Several studies have shown that drinking-water treatment residuals (WTR) could be used to control mobility of excess phosphorus (P) and other oxyanions in poorly sorbing soils. Presently, only "aged" WTRs (those left, or manipulated, to dewater) are land applied. However, if demand for WTRs increase in the near future, freshly-generated WTRs could be considered for land application. To our knowledge, few studies have examined the reactivity and equilibration time of freshly-generated alum-based WTR (Al-WTR). A laboratory thermal incubation study was, therefore, conducted to determine various extractable Al forms in Al-WTR as a function of WTR "age", and the time required for freshly generated Al-WTR to stabilize. Freshly-generated Al-WTR samples were collected directly from the discharge pumps of a drinking-water treatment plant, and thermally incubated at 52 degrees C, either with or without moisture control, for < or = 24 wk. Additional dewatered Al-WTR samples of various ages (2 wk- to 2 y old) were also included in the study. Various methods of extracting Al [total-, oxalate (200 and 5 mM), and Mehlich 1 extractants] were utilized to assess Al extractability over time. Freshly-generated Al-WTR samples were potentially more reactive (as reflected in greater 5 mM oxalate extractable Al concentration) than dewatered Al-WTR samples stockpiled for > or = 6 mo. Aluminum reactivity of the freshly-generated Al-WTR decreased with time. At least 6 wk of thermal incubation (corresponding to > or = 6 mo of field drying) was required to stabilize the most reactive Al form (5mM oxalate extractable Al concentration) of the Al-WTR. Although no adverse Al-WTR effects have been reported on plants and grazing animals (apparently because of low availability of free Al(3+) in Al-WTR), land application of freshly-generated Al-WTRs (at least, those with similar physicochemical characteristics as the one utilized for the study) should be avoided.

Propagation of thrombosis by neutrophils and extracellular nucleosome networks

PubMed Central

Pfeiler, Susanne; Stark, Konstantin; Massberg, Steffen; Engelmann, Bernd

2017-01-01

Neutrophils, early mediators of the innate immune defense, are recruited to developing thrombi in different types of thrombosis. They amplify intravascular coagulation by stimulating the tissue factor-dependent extrinsic pathway via inactivation of endogenous anticoagulants, enhancing factor XII activation or decreasing plasmin generation. Neutrophil-dependent prothrombotic mechanisms are supported by the externalization of decondensed nucleosomes and granule proteins that together form neutrophil extracellular traps. These traps, either in intact or fragmented form, are causally involved in various forms of experimental thrombosis as first indicated by their role in the enhancement of both microvascular thrombosis during bacterial infection and carotid artery thrombosis. Neutrophil extracellular traps can be induced by interactions of neutrophils with activated platelets; vice versa, these traps enhance adhesion of platelets via von Willebrand factor. Neutrophil-induced microvascular thrombus formation can restrict the dissemination and survival of blood-borne bacteria and thereby sustain intravascular immunity. Dysregulation of this innate immune pathway may support sepsis-associated coagulopathies. Notably, neutrophils and extracellular nucleosomes, together with platelets, critically promote fibrin formation during flow restriction-induced deep vein thrombosis. Neutrophil extracellular traps/extracellular nucleosomes are increased in thrombi and in the blood of patients with different vaso-occlusive pathologies and could be therapeutically targeted for the prevention of thrombosis. Thus, during infections and in response to blood vessel damage, neutrophils and externalized nucleosomes are major promoters of intravascular blood coagulation and thrombosis. PMID:27927771

Investigation of pathways of advanced glycation end-products accumulation in macrophages.

PubMed

Nagai, Ryoji; Fujiwara, Yukio; Mera, Katsumi; Otagiri, Masaki

2007-04-01

Advanced glycation end-products (AGE) play a role in the pathogenesis of several diseases, including diabetic complications and atherosclerosis. In atherosclerotic lesions of human aortas, AGE are localized in the extracellular matrix and intracellularly in foam cells. Two interpretations are possible for AGE accumulation inside macrophages, one is endocytic uptake of extracellular AGE-proteins by scavenger receptors; the other is intracellular AGE formation inside the macrophages. In the present study, we determined the pathways involved in AGE accumulation inside macrophages. RAW 264.7 cells, a murine macrophage cell line, incubated with BSA and 1600 mM glucose for 40 weeks, recognized heavily modified AGE- BSA. In contrast, the cells showed no ligand activity for mildly modified AGE-BSA, prepared by incubating BSA with 50 mM glucose for 24 weeks. Nepsilon-(carboxymethyl)lysine (CML)-modified proteins of about 65 kDa were detected in human monocyte-derived macrophages incubated for 7 days with 30 mM glucose and phorbol myristate acetate. Furthermore, CML was generated when glycated protein was incubated with hypochloric acid. Taken together, our results indicate that AGE detected inside foam cells in atherosclerotic lesions are generated intracellularly rather than representing endocytic uptake of extracellular AGE-proteins by scavenger receptors.

Acute Responses of Microorganisms from Membrane Bioreactors in the Presence of NaOCl: Protective Mechanisms of Extracellular Polymeric Substances.

PubMed

Han, Xiaomeng; Wang, Zhiwei; Chen, Mei; Zhang, Xingran; Tang, Chuyang Y; Wu, Zhichao

2017-03-21

Extracellular polymeric substances (EPS) are key foulants in membrane bioreactors (MBRs). However, their positive functions of protecting microorganisms from environmental stresses, e.g., during in situ hypochlorite chemical cleaning of membranes, have not been adequately elucidated. In this work, we investigated the response of microorganisms in an MBR to various dosages of NaOCl, with a particular emphasis on the mechanistic roles of EPS. Results showed that functional groups in EPS such as the hydroxyl and amino groups were attacked by NaOCl, causing the oxidation of polysaccharides, denaturation of amino acids, damage to protein secondary structure, and transformation of tryptophan protein-like substances to condensed aromatic ring substances. The presence of EPS alleviated the negative impacts on catalase and superoxide dismutase, which in turn reduced the concentration of reactive oxygen species (ROS) in microbial cells. The direct extracellular reaction and the mitigated intracellular oxidative responses facilitated the maintenance of microbial metabolism, as indicated by the quantity of adenosine triphosphate and the activity of dehydrogenase. The reaction with NaOCl also led to the changes of cell integrity and adhesion properties of EPS, which promoted the release of organic matter into bulk solution. Our results systematically demonstrate the protective roles of EPS and the underlying mechanisms in resisting the environmental stress caused by NaOCl, which provides important implications for in situ chemical cleaning in MBRs.

Alteration of Extracellular Enzymes in Pinto Bean Leaves upon Exposure to Air Pollutants, Ozone and Sulfur Dioxide.

PubMed

Peters, J L; Castillo, F J; Heath, R L

1989-01-01

Diamine oxidase and peroxidase, associated with the wall in pinto bean (Phaseolus vulgaris L. var Pinto) leaves, can be washed out by vacuum infiltration and assayed without grinding the leaf. The diamine oxidase activity is inhibited in vivo by exposure of the plants to ozone (dose of 0.6 microliters per liter x hour), whereas the peroxidase activity associated with the wall space is stimulated. This dose does not cause obvious necrosis or chlorosis of the leaf. These alterations are greater when the dose of ozone exposure is given as a triangular pulse (a slow rise to a peak of 0.24 microliters per liter followed by a slow fall) compared to that given as a constant square wave pulse of 0.15 microliters per liter for the same 4 hour period. Exposure of the plants to sulfur dioxide (at a concentration of 0.4 microliters per liter for 4 hours) does not result in any change in the diamine oxidase or peroxidase activities, yet the total sulfhydryl content of the leaf is increased, demonstrating the entry of sulfur dioxide. These two pollutants, with different chemical reactivities, affect the activities of the extracellular enzymes in different manners. In the case of ozone exposure, the inhibition of extracellular diamine oxidase could profoundly alter the movements of polyamines from cell to cell.

Hypoxic preconditioning potentiates the trophic effects of mesenchymal stem cells on co-cultured human primary hepatocytes.

PubMed

Qin, Harry H; Filippi, Céline; Sun, Song; Lehec, Sharon; Dhawan, Anil; Hughes, Robin D

2015-12-01

Mesenchymal stem/stromal cells (MSCs) improve the metabolic function of co-cultured hepatocytes. The present study aimed to further enhance the trophic effects of co-culture with hepatocytes using hypoxic preconditioning (HPc) of the MSCs and also to investigate the underlying molecular mechanisms involved. Human adipose tissue-derived MSCs were subjected to hypoxia (2 % O2; HPc) or normoxia (20 % O2) for 24 h and then co-cultured with isolated human hepatocytes. Assays of metabolic function and apoptosis were performed to investigate the hepatotrophic and anti-apoptotic effects of co-culture. Indirect co-cultures and co-culture with MSC-conditioned medium investigated the role of paracrine factors in the hepatotrophic effects of co-culture. Reactive oxygen species (ROS) activity was antagonised with N-acetylcysteine to investigate whether HPc potentiated the effects of MSCs by intracellular ROS-dependent mechanisms. Tumour necrosis factor (TNF)-α, transforming growth factor (TGF)-β1, and extracellular collagen production was determined and CASP9 and BAX/BCL-2 signalling pathways analysed to investigate the role of soluble factors, extracellular matrix deposition, and apoptosis-associated gene signalling in the effects of co-culture. HPc potentiated the hepatotrophic and anti-apoptotic effects of co-culture by ROS-dependent mechanisms. There was increased MSC TGF-β1 production, and enhanced MSC deposition of extracellular collagen, with reduced synthesis of TNF-α, as well as a downregulation of the expression of pro-apoptotic CASP9, BAX, BID and BLK genes and upregulated expression of anti-apoptotic BCL-2 in hepatocytes. HPc potentiated the trophic and anti-apoptotic effects of MSCs on hepatocytes via mechanisms including intracellular ROS, autocrine TGF-β, extracellular collagen and caspase and BAX/BCL-2 signalling pathways.

Sensitization of interferon-γ induced apoptosis in human osteosarcoma cells by extracellular S100A4

PubMed Central

Pedersen, Kjetil Boye; Andersen, Kristin; Fodstad, Øystein; Mælandsmo, Gunhild Mari

2004-01-01

Background S100A4 is a small Ca2+-binding protein of the S100 family with metastasis-promoting properties. Recently, secreted S100A4 protein has been shown to possess a number of functions, including induction of angiogenesis, stimulation of cell motility and neurite extension. Methods Cell cultures from two human osteosarcoma cell lines, OHS and its anti-S100A4 ribozyme transfected counterpart II-11b, was treated with IFN-γ and recombinant S100A4 in order to study the sensitizing effects of extracellular S100A4 on IFN-γ mediated apoptosis. Induction of apoptosis was demonstrated by DNA fragmentation, cleavage of poly (ADP-ribose) polymerase and Lamin B. Results In the present work, we found that the S100A4-expressing human osteosarcoma cell line OHS was more sensitive to IFN-γ-mediated apoptosis than the II-11b cells. S100A4 protein was detected in conditioned medium from OHS cells, but not from II-11b cells, and addition of recombinant S100A4 to the cell medium sensitized II-11b cells to apoptosis induced by IFN-γ. The S100A4/IFN-γ-mediated induction of apoptosis was shown to be independent of caspase activation, but dependent on the formation of reactive oxygen species. Furthermore, addition of extracellular S100A4 was demonstrated to activate nuclear factor-κB (NF-κB). Conclusion In conclusion, we have shown that S100A4 sensitizes osteosarcoma cells to IFN-γ-mediated induction of apoptosis. Additionally, extracellular S100A4 activates NF-κB, but whether these events are causally related remains unknown. PMID:15318945

A study of the free radical scavenging effects of Piper betle leaf extract in patients with vitiligo.

PubMed

Mitra, Sneha; Pati, Ayan Kumar; Manna, Alak; Ghosh, Arghyaprasun; Sen, Sumit; Chatterjee, Suparna; Chatterjee, Mitali

2017-01-01

Vitiligo is an idiopathic skin disease manifested by depigmented macules. It is characterised by melanocyte destruction, and redox imbalance is proposed to play a contributory role. The aim of this study was to analyze the effects of an ethanolic extract of Piper betle leaves on the generation of reactive oxygen species in erythrocytes sourced from vitiligo patients. The effect of Piper betle on the generation of reactive oxygen species in erythrocytes was measured by flow cytometry in patients with active and stable vitiligo versus healthy controls, using 5-(and-6)-chloromethyl-2'-7'-dichlorodihydrofluorescein diacetate. The generation of reactive oxygen species in erythrocytes was higher in patients with vitiligo (n = 23) compared to healthy controls (n = 18). The geometrical mean fluorescence channel was 23.05 ± 2.11 in patients versus 17.77 ± 1.79 in controls, P = 0.039. The levels of reactive oxygen species were higher in patients with active vitiligo. Treatment of erythrocytes with Piper betle in concentrations of 0.5 and 1.0 μg/ml significantly decreased the baseline levels of reactive oxygen species by 31.7% in healthy controls, and 47.6% and 44.3% in patients with active vitiligo, respectively. Piper betle effectively scavenged hydrogen peroxide, which was evident by a decrease in the geometrical mean fluorescence channel by 52.4% and 62.9% in healthy controls, and 45.0% and 57.0% in patients with active vitiligo. The study had a small sample size. Future studies should focus on evaluation of the antioxidant role of Piper betle at the lesional site. This pilot study indicates that patients with active vitiligo demonstrate enhanced generation of reactive oxygen species in erythrocytes, which was significantly reduced following ex vivo treatment with Piper betle.

Reactive astrocytes as neural stem or progenitor cells: In vivo lineage, In vitro potential, and Genome‐wide expression analysis

PubMed Central

Sirko, Swetlana; Beckers, Johannes; Irmler, Martin

2015-01-01

Here, we review the stem cell hallmarks of endogenous neural stem cells (NSCs) during development and in some niches of the adult mammalian brain to then compare these with reactive astrocytes acquiring stem cell hallmarks after traumatic and ischemic brain injury. Notably, even endogenous NSCs including the earliest NSCs, the neuroepithelial cells, generate in most cases only a single type of progeny and self‐renew only for a rather short time in vivo. In vitro, however, especially cells cultured under neurosphere conditions reveal a larger potential and long‐term self‐renewal under the influence of growth factors. This is rather well comparable to reactive astrocytes in the traumatic or ischemic brain some of which acquire neurosphere‐forming capacity including multipotency and long‐term self‐renewal in vitro, while they remain within their astrocyte lineage in vivo. Both reactive astrocytes and endogenous NSCs exhibit stem cell hallmarks largely in vitro, but their lineage differs in vivo. Both populations generate largely a single cell type in vivo, but endogenous NSCs generate neurons and reactive astrocytes remain in the astrocyte lineage. However, at some early postnatal stages or in some brain regions reactive astrocytes can be released from this fate restriction, demonstrating that they can also enact neurogenesis. Thus, reactive astrocytes and NSCs share many characteristic hallmarks, but also exhibit key differences. This conclusion is further substantiated by genome‐wide expression analysis comparing NSCs at different stages with astrocytes from the intact and injured brain parenchyma. GLIA 2015;63:1452–1468 PMID:25965557

Analysis of Coolant Options for Advanced Metal Cooled Nuclear Reactors

DTIC Science & Technology

2006-12-01

24 Table 3.3 Hazards of Sodium Reaction Products, Hydride And Oxide...........................26 Table 3.4 Chemical Reactivity Of Selected...Liquid Metal Fast Breeder Reactor ORIGEN Oak Ridge Isotope Generator ORIGENARP Oak Ridge Isotope Generator Automated Rapid Processing PWR ...nuclear reactors, both because of the possibility of increased reactivity due to boiling and the potential loss of effectiveness of coolant heat transfer

Small unilamellar liposomes as a membrane model for cell inactivation by cold atmospheric plasma treatment

NASA Astrophysics Data System (ADS)

Maheux, S.; Frache, G.; Thomann, J. S.; Clément, F.; Penny, C.; Belmonte, T.; Duday, D.

2016-09-01

Cold atmospheric plasma is thought to be a promising tool for numerous biomedical applications due to its ability to generate a large diversity of reactive species in a controlled way. In some cases, it can also generate pulsed electric fields at the zone of treatment, which can induce processes such as electroporation in cell membranes. However, the interaction of these reactive species and the pulse electric field with cells in a physiological medium is very complex, and we still need a better understanding in order to be useful for future applications. A way to reach this goal is to work with model cell membranes such as liposomes, with the simplest physiological liquid and in a controlled atmosphere in order to limit the number of parallel reactions and processes. In this paper, where this approach has been chosen, 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) small unilamellar vesicles (SUV) have been synthesized in a phosphate buffered aqueous solution, and this solution has been treated by a nanosecond pulsed plasma jet under a pure nitrogen atmosphere. It is only the composition of the plasma gas that has been changed in order to generate different cocktails of reactive species. After the quantification of the main plasma reactive species in the phosphate buffered saline (PBS) solution, structural, surface charge state, and chemical modifications generated on the plasma treated liposomes, due to the interaction with the plasma reactive species, have been carefully characterized. These results allow us to further understand the effect of plasma reactive species on model cell membranes in physiological liquids. The permeation through the liposomal membrane and the reaction of plasma reactive species with molecules encapsulated inside the liposomes have also been evaluated. New processes of degradation are finally presented and discussed, which come from the specific conditions of plasma treatment under the pure nitrogen atmosphere.

Methods for the physical characterization and quantification of extracellular vesicles in biological samples.

PubMed

Rupert, Déborah L M; Claudio, Virginia; Lässer, Cecilia; Bally, Marta

2017-01-01

Our body fluids contain a multitude of cell-derived vesicles, secreted by most cell types, commonly referred to as extracellular vesicles. They have attracted considerable attention for their function as intercellular communication vehicles in a broad range of physiological processes and pathological conditions. Extracellular vesicles and especially the smallest type, exosomes, have also generated a lot of excitement in view of their potential as disease biomarkers or as carriers for drug delivery. In this context, state-of-the-art techniques capable of comprehensively characterizing vesicles in biological fluids are urgently needed. This review presents the arsenal of techniques available for quantification and characterization of physical properties of extracellular vesicles, summarizes their working principles, discusses their advantages and limitations and further illustrates their implementation in extracellular vesicle research. The small size and physicochemical heterogeneity of extracellular vesicles make their physical characterization and quantification an extremely challenging task. Currently, structure, size, buoyant density, optical properties and zeta potential have most commonly been studied. The concentration of vesicles in suspension can be expressed in terms of biomolecular or particle content depending on the method at hand. In addition, common quantification methods may either provide a direct quantitative measurement of vesicle concentration or solely allow for relative comparison between samples. The combination of complementary methods capable of detecting, characterizing and quantifying extracellular vesicles at a single particle level promises to provide new exciting insights into their modes of action and to reveal the existence of vesicle subpopulations fulfilling key biological tasks. Copyright © 2016 Elsevier B.V. All rights reserved.

Extracellular deoxyribonuclease made by group A Streptococcus assists pathogenesis by enhancing evasion of the innate immune response.

PubMed

Sumby, Paul; Barbian, Kent D; Gardner, Donald J; Whitney, Adeline R; Welty, Diane M; Long, R Daniel; Bailey, John R; Parnell, Michael J; Hoe, Nancy P; Adams, Gerald G; Deleo, Frank R; Musser, James M

2005-02-01

Many pathogenic bacteria produce extracellular DNase, but the benefit of this enzymatic activity is not understood. For example, all strains of the human bacterial pathogen group A Streptococcus (GAS) produce at least one extracellular DNase, and most strains make several distinct enzymes. Despite six decades of study, it is not known whether production of DNase by GAS enhances virulence. To test the hypothesis that extracellular DNase is required for normal progression of GAS infection, we generated seven isogenic mutant strains in which the three chromosomal- and prophage-encoded DNases made by a contemporary serotype M1 GAS strain were inactivated. Compared to the wild-type parental strain, the isogenic triple-mutant strain was significantly less virulent in two mouse models of invasive infection. The triple-mutant strain was cleared from the skin injection site significantly faster than the wild-type strain. Preferential clearance of the mutant strain was related to the differential extracellular killing of the mutant and wild-type strains, possibly through degradation of neutrophil extracellular traps, innate immune structures composed of chromatin and granule proteins. The triple-mutant strain was also significantly compromised in its ability to cause experimental pharyngeal disease in cynomolgus macaques. Comparative analysis of the seven DNase mutant strains strongly suggested that the prophage-encoded SdaD2 enzyme is the major DNase that contributes to virulence in this clone. We conclude that extracellular DNase activity made by GAS contributes to disease progression, thereby resolving a long-standing question in bacterial pathogenesis research.

Generation of reactive species in atmospheric pressure dielectric barrier discharge with liquid water

NASA Astrophysics Data System (ADS)

Zelong, ZHANG; Jie, SHEN; Cheng, CHENG; Zimu, XU; Weidong, XIA

2018-04-01

Atmospheric pressure helium/water dielectric barrier discharge (DBD) plasma is used to investigate the generation of reactive species in a gas-liquid interface and in a liquid. The emission intensity of the reactive species is measured by optical emission spectroscopy (OES) with different discharge powers at the gas-liquid interface. Spectrophotometry is used to analyze the reactive species induced by the plasma in the liquid. The concentration of OH radicals reaches 2.2 μm after 3 min of discharge treatment. In addition, the concentration of primary long-lived reactive species such as H2O2, {{{{NO}}}3}- and O3 are measured based on plasma treatment time. After 5 min of discharge treatment, the concentration of H2O2, {{{{NO}}}3}-, and O3 increased from 0 mg · L-1 to 96 mg · L-1, 19.5 mg · L-1, and 3.5 mg · L-1, respectively. The water treated by plasma still contained a considerable concentration of reactive species after 6 h of storage. The results will contribute to optimizing the DBD plasma system for biological decontamination.

Pulmonary Inflammatory Responses to Acute Meteorite Dust Exposures - to Acute Meteorite Dust Exposures - Exploration

NASA Technical Reports Server (NTRS)

Harrington, A. D.; McCubbin, F. M.; Kaur, J.; Smirnov, A.; Galdanes, K.; Schoonen, M. A. A.; Chen, L. C.; Tsirka, S. E.; Gordon, T.

2017-01-01

New initiatives to begin lunar and martian colonization within the next few decades are illustrative of the resurgence of interest in space travel. One of NASA's major concerns with extended human space exploration is the inadvertent and repeated exposure to unknown dust. This highly interdisciplinary study evaluates both the geochemical reactivity (e.g. iron solubility and acellular reactive oxygen species (ROS) generation) and the relative toxicity (e.g. in vitro and in vivo pulmonary inflammation) of six meteorite samples representing either basalt or regolith breccia on the surface of the Moon, Mars, and Asteroid 4Vesta. Terrestrial mid-ocean ridge basalt (MORB) is also used for comparison. The MORB demonstrated higher geochemical reactivity than most of the meteorite samples but caused the lowest acute pulmonary inflammation (API). Notably, the two martian meteorites generated some of the highest API but only the basaltic sample is significantly reactive geochemically. Furthermore, while there is a correlation between a meteorite's soluble iron content and its ability to generate acellular ROS, there is no direct correlation between a particle's ability to generate ROS acellularly and its ability to generate API. However, assorted in vivo API markers did demonstrate strong positive correlations with increasing bulk Fenton metal content. In summary, this comprehensive dataset allows for not only the toxicological evaluation of astromaterials but also clarifies important correlations between geochemistry and health.

Understanding Collagen Organization in Breast Tumors to Predict and Prevent Metastasis

DTIC Science & Technology

2014-09-01

Harmonic Generation to Image the Extracellular Matrix During Tumor Progression. Invited Perspective Intravital Manuscript Submitted. Sullivan K...harmonic generation (the SHG “F/B ratio”) in thick intact tissue, with a single image scan. This will be necessary for us to pursue our goal of...quantifying matrix changes dynamically, in intact tumor models. The first method determines F/B by generating a series of backscattered images using a series

On the origin of the extracellular field potential in the nucleus laminaris of the barn owl (Tyto alba).

PubMed

Kuokkanen, Paula T; Wagner, Hermann; Ashida, Go; Carr, Catherine E; Kempter, Richard

2010-10-01

The neurophonic is a sound-evoked, frequency-following potential that can be recorded extracellularly in nucleus laminaris of the barn owl. The origin of the neurophonic, and thus the mechanisms that give rise to its exceptional temporal precision, has not yet been identified. Putative generators of the neurophonic are the activity of afferent axons, synaptic activation of laminaris neurons, or action potentials in laminaris neurons. To identify the generators, we analyzed the neurophonic in the high-frequency (>2.5 kHz) region of nucleus laminaris in response to monaural pure-tone stimulation. The amplitude of the neurophonic is typically in the millivolt range. The signal-to-noise ratio reaches values beyond 30 dB. To assess which generators could give rise to these large, synchronous extracellular potentials, we developed a computational model. Spike trains were produced by an inhomogeneous Poisson process and convolved with a spike waveform. The model explained the dependence of the simulated neurophonic on parameters such as the mean rate, the vector strength of phase locking, the number of statistically independent sources, and why the signal-to-noise ratio is independent of the spike waveform and subsequent filtering of the signal. We found that several hundred sources are needed to reach the observed signal-to-noise ratio. The summed coherent signal from the densely packed afferent axons and activation of their synapses on laminaris neurons are alone sufficient to explain the measured properties of the neurophonic.

Collagen-IV supported embryoid bodies formation and differentiation from buffalo (Bubalus bubalis) embryonic stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Taru Sharma, G., E-mail: gts553@gmail.com; Dubey, Pawan K.; Verma, Om Prakash

Graphical abstract: EBs formation, characterization and expression of germinal layers marker genes of in vivo developed teratoma using four different types of extracellular matrices. Highlights: Black-Right-Pointing-Pointer Collagen-IV matrix is found cytocompatible for EBs formation and differentiation. Black-Right-Pointing-Pointer Established 3D microenvironment for ES cells development and differentiation into three germ layers. Black-Right-Pointing-Pointer Collagen-IV may be useful as promising candidate for ES cells based therapeutic applications. -- Abstract: Embryoid bodies (EBs) are used as in vitro model to study early extraembryonic tissue formation and differentiation. In this study, a novel method using three dimensional extracellular matrices for in vitro generation of EBsmore » from buffalo embryonic stem (ES) cells and its differentiation potential by teratoma formation was successfully established. In vitro derived inner cell masses (ICMs) of hatched buffalo blastocyst were cultured on buffalo fetal fibroblast feeder layer for primary cell colony formation. For generation of EBs, pluripotent ES cells were seeded onto four different types of extracellular matrices viz; collagen-IV, laminin, fibronectin and matrigel using undifferentiating ES cell culture medium. After 5 days of culture, ESCs gradually grew into aggregates and formed simple EBs having circular structures. Twenty-six days later, they formed cystic EBs over collagen matrix with higher EBs formation and greater proliferation rate as compared to other extracellular matrices. Studies involving histological observations, fluorescence microscopy and RT-PCR analysis of the in vivo developed teratoma revealed that presence of all the three germ layer derivatives viz. ectoderm (NCAM), mesoderm (Flk-1) and endoderm (AFP). In conclusion, the method described here demonstrates a simple and cost-effective way of generating EBs from buffalo ES cells. Collagen-IV matrix was found cytocompatible as it supported buffalo EBs formation, their subsequent differentiation could prove to be useful as promising candidate for ES cells based therapeutic applications.« less

Reactive species in non-equilibrium atmospheric-pressure plasmas: Generation, transport, and biological effects

NASA Astrophysics Data System (ADS)

Lu, X.; Naidis, G. V.; Laroussi, M.; Reuter, S.; Graves, D. B.; Ostrikov, K.

2016-05-01

Non-equilibrium atmospheric-pressure plasmas have recently become a topical area of research owing to their diverse applications in health care and medicine, environmental remediation and pollution control, materials processing, electrochemistry, nanotechnology and other fields. This review focuses on the reactive electrons and ionic, atomic, molecular, and radical species that are produced in these plasmas and then transported from the point of generation to the point of interaction with the material, medium, living cells or tissues being processed. The most important mechanisms of generation and transport of the key species in the plasmas of atmospheric-pressure plasma jets and other non-equilibrium atmospheric-pressure plasmas are introduced and examined from the viewpoint of their applications in plasma hygiene and medicine and other relevant fields. Sophisticated high-precision, time-resolved plasma diagnostics approaches and techniques are presented and their applications to monitor the reactive species and plasma dynamics in the plasma jets and other discharges, both in the gas phase and during the plasma interaction with liquid media, are critically reviewed. The large amount of experimental data is supported by the theoretical models of reactive species generation and transport in the plasmas, surrounding gaseous environments, and plasma interaction with liquid media. These models are presented and their limitations are discussed. Special attention is paid to biological effects of the plasma-generated reactive oxygen and nitrogen (and some other) species in basic biological processes such as cell metabolism, proliferation, survival, etc. as well as plasma applications in bacterial inactivation, wound healing, cancer treatment and some others. Challenges and opportunities for theoretical and experimental research are discussed and the authors' vision for the emerging convergence trends across several disciplines and application domains is presented to stimulate critical discussions and collaborations in the future.

Use of DFIWG for Improvement of Voltage Stability Condition of a Power System

NASA Astrophysics Data System (ADS)

Hazarika, Durlav; Das, Ranjay

2017-12-01

This paper describes a method for improvement of voltage stability condition of a multi-bus power system by regulating reactive power generation at a Doubly Fed Induction Wind Generator (DFIWG). For this purpose, sensitivity relation between changes in voltage stability index at a bus with respect to change in reactive power generation of the DFIWG is derived. This relation is used to determine the required amount of change in rotor current of the DFIWG to improve the voltage stability index of the bus.

Effect of CoQ homologues on reactive oxygen generation by mitochondria.

PubMed

Imada, Isuke; Sato, Eisuke F; Kira, Yukimi; Inoue, Masayasu

2008-01-01

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

Luteolin as reactive oxygen generator by X-ray and UV irradiation

NASA Astrophysics Data System (ADS)

Toyama, Michiru; Mori, Takashi; Takahashi, Junko; Iwahashi, Hitoshi

2018-05-01

Non-toxic X-ray-responsive substances can be used in the radiosensitization of cancer, like porphyrin mediated radiotherapy. However, most X-ray-responsive substances are toxic. To find novel non-toxic X-ray-responsive substances, we studied the X-ray and UV reactivity of 40 non-toxic compounds extracted from plants. Dihydroethidium was used as an indicator to detect reactive oxygen species (ROS) generated by the compounds under X-ray or UV irradiation. We found that 13 of the investigated compounds generated ROS under X-ray irradiation and 17 generated ROS under UV irradiation. Only 4 substances generated ROS under both X-ray and UV. In particular, luteolin exhibited the highest activity among the investigated compounds; therefore, the ROS generated by luteolin were thoroughly characterized. To identify the ROS, we employed a combination of ROS detection reagents and their quenchers. O2·- generation by luteolin was monitored using dihydroethidium and superoxide dismutase (as an O2·- quencher). OH· and 1O2 generation was determined using aminophenyl fluorescein with ethanol (OH· quencher) and Singlet Oxygen Sensor Green® with NaN3 (1O2 quencher), respectively. Generation of O2·- under X-ray and UV irradiation was observed; however, no OH· or 1O2 was detected. The production of ROS from luteolin is surprising, because luteolin is a well-known antioxidant.

Regulation of death induction and chemosensitizing action of 3-bromopyruvate in myeloid leukemia cells: energy depletion, oxidative stress, and protein kinase activity modulation.

PubMed

Calviño, Eva; Estañ, María Cristina; Sánchez-Martín, Carlos; Brea, Rocío; de Blas, Elena; Boyano-Adánez, María del Carmen; Rial, Eduardo; Aller, Patricio

2014-02-01

3-Bromopyruvate (3-BrP) is an alkylating, energy-depleting drug that is of interest in antitumor therapies, although the mechanisms underlying its cytotoxicity are ill-defined. We show here that 3-BrP causes concentration-dependent cell death of HL60 and other human myeloid leukemia cells, inducing both apoptosis and necrosis at 20-30 μM and a pure necrotic response at 60 μM. Low concentrations of 3-BrP (10-20 μM) brought about a rapid inhibition of glycolysis, which at higher concentrations was followed by the inhibition of mitochondrial respiration. The combination of these effects causes concentration-dependent ATP depletion, although this cannot explain the lethality at intermediate 3-BrP concentrations (20-30 μM). The oxidative stress caused by exposure to 3-BrP was evident as a moderate overproduction of reactive oxygen species and a concentration-dependent depletion of glutathione, which was an important determinant of 3-BrP toxicity. In addition, 3-BrP caused glutathione-dependent stimulation of p38 mitogen-activated protein kinase (MAPK), mitogen-induced extracellular kinase (MEK)/extracellular signal-regulated kinase (ERK), and protein kinase B (Akt)/mammalian target of rapamycin/p70S6K phosphorylation or activation, as well as rapid LKB-1/AMP kinase (AMPK) activation, which was later followed by Akt-mediated inactivation. Experiments with pharmacological inhibitors revealed that p38 MAPK activation enhances 3-BrP toxicity, which is conversely restrained by ERK and Akt activity. Finally, 3-BrP was seen to cooperate with antitumor agents like arsenic trioxide and curcumin in causing cell death, a response apparently mediated by both the generation of oxidative stress induced by 3-BrP and the attenuation of Akt and ERK activation by curcumin. In summary, 3-BrP cytotoxicity is the result of several combined regulatory mechanisms that might represent important targets to improve therapeutic efficacy.

Size-dependent cytotoxicity of yttrium oxide nanoparticles on primary osteoblasts in vitro

NASA Astrophysics Data System (ADS)

Zhou, Guoqiang; Li, Yunfei; Ma, Yanyan; Liu, Zhu; Cao, Lili; Wang, Da; Liu, Sudan; Xu, Wenshi; Wang, Wenying

2016-05-01

Yttrium oxide nanoparticles are an excellent host material for the rare earth metals and have high luminescence efficiency providing a potential application in photodynamic therapy and biological imaging. In this study, the effects of yttrium oxide nanoparticles with four different sizes were investigated using primary osteoblasts in vitro. The results demonstrated that the cytotoxicity generated by yttrium oxide nanoparticles depended on the particle size, and smaller particles possessed higher toxicological effects. For the purpose to elucidate the relationship between reactive oxygen species generation and cell damage, cytomembrane integrity, intracellular reactive oxygen species level, mitochondrial membrane potential, cell apoptosis rate, and activity of caspase-3 in cells were then measured. Increased reactive oxygen species level was also observed in a size-dependent way. Thus, our data demonstrated that exposure to yttrium oxide nanoparticles resulted in a size-dependent cytotoxicity in cultured primary osteoblasts, and reactive oxygen species generation should be one possible damage pathway for the toxicological effects produced by yttrium oxide particles. The results may provide useful information for more rational applications of yttrium oxide nanoparticles in the future.

Comparison of turnaround time and total cost of HIV testing before and after implementation of the 2014 CDC/APHL Laboratory Testing Algorithm for diagnosis of HIV infection.

PubMed

Chen, Derrick J; Yao, Joseph D

2017-06-01

Updated recommendations for HIV diagnostic laboratory testing published by the Centers for Disease Control and Prevention and the Association of Public Health Laboratories incorporate 4th generation HIV immunoassays, which are capable of identifying HIV infection prior to seroconversion. The purpose of this study was to compare turnaround time and cost between 3rd and 4th generation HIV immunoassay-based testing algorithms for initially reactive results. The clinical microbiology laboratory database at Mayo Clinic, Rochester, MN was queried for 3rd generation (from November 2012 to May 2014) and 4th generation (from May 2014 to November 2015) HIV immunoassay results. All results from downstream supplemental testing were recorded. Turnaround time (defined as the time of initial sample receipt in the laboratory to the time the final supplemental test in the algorithm was resulted) and cost (based on 2016 Medicare reimbursement rates) were assessed. A total of 76,454 and 78,998 initial tests were performed during the study period using the 3rd generation and 4th generation HIV immunoassays, respectively. There were 516 (0.7%) and 581 (0.7%) total initially reactive results, respectively. Of these, 304 (58.9%) and 457 (78.7%) were positive by supplemental testing. There were 10 (0.01%) cases of acute HIV infection identified with the 4th generation algorithm. The most frequent tests performed to confirm an HIV-positive case using the 3rd generation algorithm, which were reactive initial immunoassay and positive HIV-1 Western blot, took a median time of 1.1 days to complete at a cost of $45.00. In contrast, the most frequent tests performed to confirm an HIV-positive case using the 4th generation algorithm, which included a reactive initial immunoassay and positive HIV-1/-2 antibody differentiation immunoassay for HIV-1, took a median time of 0.4 days and cost $63.25. Overall median turnaround time was 2.2 and 1.5 days, and overall median cost was $63.90 and $72.50 for 3rd and 4th generation algorithms, respectively. Both 3rd and 4th generation HIV immunoassays had similar total numbers of tests performed and positivity rates during the study period. A greater proportion of reactive 4th generation immunoassays were confirmed to be positive, and the 4th generation algorithm identified several cases of acute HIV infection that would have been missed by the 3rd generation algorithm. The 4th generation algorithm had a more rapid turnaround time but higher cost for confirmed positive HIV infections and overall, compared to the 3rd generation algorithm. Copyright © 2017 Elsevier B.V. All rights reserved.

Requirement of extracellular Ca2+ binding to specific amino acids for heat‐evoked activation of TRPA1

PubMed Central

Kurganov, Erkin; Saito, Shigeru; Tanaka Saito, Claire

2017-01-01

Key points We found that extracellular Ca2+, but not other divalent cations (Mg2+ and Ba2+) or intracellular Ca2+, is involved in heat‐evoked activation of green anole (ga) TRPA1.Heat‐evoked activation of chicken (ch) and rat snake (rs) TRPA1 does not depend solely on extracellular Ca2+.Neutralization of acidic amino acids on the outer surface of TRPA1 by extracellular Ca2+ is important for heat‐evoked large activation of gaTRPA1, chTRPA1 and rsTRPA1. Abstract Transient receptor potential ankyrin 1 (TRPA1) is a homotetrameric non‐selective cation‐permeable channel that has six transmembrane domains and cytoplasmic N‐ and C‐termini. The N‐terminus is characterized by an unusually large number of ankyrin repeats. Although the 3‐dimensional structure of human TRPA1 has been determined, and TRPA1 channels from insects to birds are known to be activated by heat stimulus, the mechanism for temperature‐dependent TRPA1 activation is unclear. We previously reported that extracellular Ca2+, but not intracellular Ca2+, plays an important role in heat‐evoked TRPA1 activation in green anole lizards (gaTRPA1). Here we focus on extracellular Ca2+‐dependent heat sensitivity of gaTRPA1 by comparing gaTRPA1 with heat‐activated TRPA1 channels from rat snake (rsTRPA1) and chicken (chTRPA1). In the absence of extracellular Ca2+, rsTRPA1 and chTRPA1 are activated by heat and generate small inward currents. A comparison of extracellular amino acids in TRPA1 identified three negatively charged amino acid residues (glutamate and aspartate) near the outer pore vestibule that are involved in heat‐evoked TRPA1 activation in the presence of extracellular Ca2+. These results suggest that neutralization of acidic amino acids by extracellular Ca2+ is important for heat‐evoked activation of gaTRPA1, chTRPA1, and rsTRPA1, which could clarify mechanisms of heat‐evoked channel activation. PMID:28194754

A second-generation constrained reaction volume shock tube

NASA Astrophysics Data System (ADS)

Campbell, M. F.; Tulgestke, A. M.; Davidson, D. F.; Hanson, R. K.

2014-05-01

We have developed a shock tube that features a sliding gate valve in order to mechanically constrain the reactive test gas mixture to an area close to the shock tube endwall, separating it from a specially formulated non-reactive buffer gas mixture. This second-generation Constrained Reaction Volume (CRV) strategy enables near-constant-pressure shock tube test conditions for reactive experiments behind reflected shocks, thereby enabling improved modeling of the reactive flow field. Here we provide details of the design and operation of the new shock tube. In addition, we detail special buffer gas tailoring procedures, analyze the buffer/test gas interactions that occur on gate valve opening, and outline the size range of fuels that can be studied using the CRV technique in this facility. Finally, we present example low-temperature ignition delay time data to illustrate the CRV shock tube's performance.

Development of an electron paramagnetic resonance methodology for studying the photo-generation of reactive species in semiconductor nano-particle assembled films

NASA Astrophysics Data System (ADS)

Twardoch, Marek; Messai, Youcef; Vileno, Bertrand; Hoarau, Yannick; Mekki, Djamel E.; Felix, Olivier; Turek, Philippe; Weiss, Jean; Decher, Gero; Martel, David

2018-06-01

An experimental approach involving electron paramagnetic resonance is proposed for studying photo-generated reactive species in semiconductor nano-particle-based films deposited on the internal wall of glass capillaries. This methodology is applied here to nano-TiO2 and allows a semi-quantitative analysis of the kinetic evolutions of radical production using a spin scavenger probe.

Rapid Functional Decline of Activated and Memory Graft-vs-Host-Reactive T Cells Encountering Host Antigens in the Absence of Inflammation

PubMed Central

Li, Hao Wei; Andreola, Giovanna; Carlson, Alicia; Shao, Steven; Lin, Charles; Zhao, Guiling; Sykes, Megan

2015-01-01

Inflammation in the priming host environment has critical effects on the graft-vs-host (GVH) responses mediated by naïve donor T cells. However, it is unclear how a quiescent or inflammatory environment impacts the activity of GVH-reactive primed T and memory cells. We show here that GVH-reactive primed donor T cells generated in irradiated recipients had diminished ability compared to naïve T cells to increase donor chimerism when transferred to quiescent mixed allogeneic chimeras. GVH-reactive primed T cells showed marked loss of cytotoxic function and activation and delayed but not decreased proliferation or accumulation in lymphoid tissues when transferred to quiescent mixed chimeras compared to freshly irradiated secondary recipients. Primed CD4 and CD8 T cells provided mutual help to sustain these functions in both subsets. CD8 help for CD4 cells was largely IFN-γ-dependent. Toll-like receptor (TLR) stimulation following transfer of GVH-reactive primed T cells to mixed chimeras restored their cytotoxic effector function and permitted the generation of more effective T cell memory in association with reduced PD-1 expression on CD4 memory cells. Our data indicate that an inflammatory host environment is required for the maintenance of GVH-reactive primed T cell functions and the generation of memory T cells that can rapidly acquire effector functions. These findings have important implications for GVHD and T cell-mediated immunotherapies. PMID:26085679

Extracellular proteome analysis of Leptospira interrogans serovar Lai.

PubMed

Zeng, Lingbing; Zhang, Yunyi; Zhu, Yongzhang; Yin, Haidi; Zhuang, Xuran; Zhu, Weinan; Guo, Xiaokui; Qin, Jinhong

2013-10-01

Abstract Leptospirosis is one of the most important zoonoses. Leptospira interrogans serovar Lai is a pathogenic spirochete that is responsible for leptospirosis. Extracellular proteins play an important role in the pathogenicity of this bacterium. In this study, L. interrogans serovar Lai was grown in protein-free medium; the supernatant was collected and subsequently analyzed as the extracellular proteome. A total of 66 proteins with more than two unique peptides were detected by MS/MS, and 33 of these were predicted to be extracellular proteins by a combination of bioinformatics analyses, including Psortb, cello, SoSuiGramN and SignalP. Comparisons of the transcriptional levels of these 33 genes between in vivo and in vitro conditions revealed that 15 genes were upregulated and two genes were downregulated in vivo compared to in vitro. A BLAST search for the components of secretion system at the genomic and proteomic levels revealed the presence of the complete type I secretion system and type II secretion system in this strain. Moreover, this strain also exhibits complete Sec translocase and Tat translocase systems. The extracellular proteome analysis of L. interrogans will supplement the previously generated whole proteome data and provide more information for studying the functions of specific proteins in the infection process and for selecting candidate molecules for vaccines or diagnostic tools for leptospirosis.

Pulmonary immunity and extracellular matrix interactions.

PubMed

O'Dwyer, David N; Gurczynski, Stephen J; Moore, Bethany B

2018-04-09

The lung harbors a complex immune system composed of both innate and adaptive immune cells. Recognition of infection and injury by receptors on lung innate immune cells is crucial for generation of antigen-specific responses by adaptive immune cells. The extracellular matrix of the lung, comprising the interstitium and basement membrane, plays a key role in the regulation of these immune systems. The matrix consists of several hundred assembled proteins that interact to form a bioactive scaffold. This template, modified by enzymes, acts to facilitate cell function and differentiation and changes dynamically with age and lung disease. Herein, we explore relationships between innate and adaptive immunity and the lung extracellular matrix. We discuss the interactions between extracellular matrix proteins, including glycosaminoglycans, with prominent effects on innate immune signaling effectors such as toll-like receptors. We describe the relationship of extracellular matrix proteins with adaptive immunity and leukocyte migration to sites of injury within the lung. Further study of these interactions will lead to greater knowledge of the role of matrix biology in lung immunity. The development of novel therapies for acute and chronic lung disease is dependent on a comprehensive understanding of these complex matrix-immunity interactions. Copyright © 2017 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.

Procoagulant activity of extracellular vesicles as a potential biomarker for risk of thrombosis and DIC in patients with acute leukaemia.

PubMed

Gheldof, Damien; Haguet, Hélène; Dogné, Jean-Michel; Bouvy, Céline; Graux, Carlos; George, Fabienne; Sonet, Anne; Chatelain, Christian; Chatelain, Bernard; Mullier, François

2017-02-01

Haemostatic complication is common for patients with hematologic malignancies. Recent studies suggest that the procoagulant activity (PCA) of extracellular vesicles (EV) may play a major role in venous thromboembolism and disseminated intravascular coagulation (DIC) in acute leukaemia. To study the impact of EVs from leukaemic patients on thrombin generation and to assess EV-PCA as a potential biomarker for thrombotic complications in patients with acute leukaemia. Blood samples from a cohort of patients with newly diagnosed acute leukaemia were obtained before treatment (D-0), 3 and 7 days after treatment (D-3 and D-7). Extracellular vesicles were isolated and concentrated by ultracentrifugation. EV-PCA was assessed by thrombin generation assay, and EV-associated tissue factor activity was measured using a commercial bio-immunoassay (Zymuphen MP-TF®). Of the 53 patients, 6 had increased EV-PCA at D-0 and 4 had a thrombotic event. Patients without thrombotic events (n = 47) had no elevated EV-PCA. One patient had increased EVs with procoagulant activity at D-3 and developed a DIC at D-5. This patient had no increased EVs-related tissue factor activity from D-0 to D-7 (<2 pg/ml). Eight patients had increased EVs with tissue factor activity (>2 pg/ml), of these, four had a thrombosis and two had haemorrhages. Procoagulant activity of extracellular vesicles could have a predictive value in excluding the risk of thrombotic events. Our findings also suggest a possible association between thrombotic events and EV-PCA.

Mice selected for high versus low stress reactivity: a new animal model for affective disorders.

PubMed

Touma, Chadi; Bunck, Mirjam; Glasl, Lisa; Nussbaumer, Markus; Palme, Rupert; Stein, Hendrik; Wolferstätter, Michael; Zeh, Ramona; Zimbelmann, Marina; Holsboer, Florian; Landgraf, Rainer

2008-07-01

Affective disorders such as major depression are among the most prevalent and costly diseases of the central nervous system, but the underlying mechanisms are still poorly understood. In recent years, it has become evident that alterations of the stress hormone system, in particular dysfunctions (hyper- or hypo-activity) of the hypothalamic-pituitary-adrenal (HPA) axis, play a prominent role in the development of major depressive disorders. Therefore, we aimed to generate a new animal model comprising these neuroendocrine core symptoms in order to unravel parameters underlying increased or decreased stress reactivity. Starting from a population of outbred mice (parental generation: 100 males and 100 females of the CD-1 strain), two breeding lines were established according to the outcome of a 'stress reactivity test' (SRT), consisting of a 15-min restraint period and tail blood samplings immediately before and after exposure to the stressor. Mice showing a very high or a very low secretion of corticosterone in the SRT, i.e. animals expressing a hyper- or a hypo-reactivity of the HPA axis, were selected for the 'high reactivity' (HR) and the 'low reactivity' (LR) breeding line, respectively. Additionally, a third breeding line was established consisting of animals with an 'intermediate reactivity' (IR) in the SRT. Already in the first generation, i.e. animals derived from breeding pairs selected from the parental generation, significant differences in the reactivity of the HPA axis between HR, IR, and LR mice were observed. Moreover, these differences were found across all subsequent generations and could be increased by selective breeding, which indicates a genetic basis of the respective phenotype. Repeated testing of individuals in the SRT furthermore proved that the observed differences in stress responsiveness are present already early in life and can be regarded as a robust genetic predisposition. Tests investigating the animal's emotionality including anxiety-related behavior, exploratory drive, locomotor activity, and depression-like behavior point to phenotypic similarities with behavioral changes observed in depressive patients. In general, HR males and females were 'hyperactive' in some behavioral paradigms, resembling symptoms of restlessness and agitation often seen in melancholic depression. LR mice, on the other hand, showed more passive-aggressive coping styles, corresponding to signs of retardation and retreat observed in atypical depression. Several morphometric and neuroendocrine findings further support this view. For example, monitoring the circadian rhythm of glucocorticoid secretion revealed clearly increased trough levels in HR mice, resulting in a flattened diurnal rhythm, again adding to the neuroendocrine similarities to patients suffering from melancholic depression. Taken together, our results suggest that distinct mechanisms influencing the function and regulation of the HPA axis are involved in the respective behavioral and neurobiological endophenotypes. Thus, the generated HR/IR/LR mouse lines can be a valuable model to elucidate molecular genetic, neuroendocrine, and behavioral parameters associated with altered stress reactivity, thereby improving our understanding of affective disorders, presumably including the symptomatology and pathophysiology of specific subtypes of major depression.

Shikonin induces apoptosis of HaCaT cells via the mitochondrial, Erk and Akt pathways

PubMed Central

JING, HUILING; SUN, WENYAN; FAN, JINGHUA; ZHANG, YANMIN; YANG, JIAO; JIA, JINJING; LI, JICHANG; GUO, JIAQI; LUO, SUJU; ZHENG, YAN

2016-01-01

Shikonin, which is a major ingredient of the traditional Chinese herb Lithospermum erythrorhizon, possesses various biological functions, including antimicrobial, anti-inflammatory, and antitumor activities. The present study aimed to determine the molecular mechanisms underlying the effects of shikonin on HaCaT cell apoptosis. Treatment with shikonin significantly inhibited the viability of HaCaT cells in a dose- and time-dependent manner, and promoted cell cycle arrest at G0/G1 phase and apoptosis. In addition, shikonin treatment reduced the mitochondrial membrane potential and induced reactive oxygen species generation. The results of a western blot analysis demonstrated that shikonin significantly activated caspase 3 expression, downregulated B-cell lymphoma 2 (Bcl-2) expression, and upregulated Bcl-2-associated X protein and Bcl-2 homologous antagonist killer expression in a dose-dependent manner in HaCaT cells. Furthermore, shikonin decreased extracellular signal-regulated kinase (Erk) and Akt phosphorylation. These results indicated that shikonin may exert its anti-proliferative effects by inducing apoptosis via activation of the mitochondrial signaling pathway and inactivation of the Akt and Erk pathways in HaCaT cells. Therefore, the present study suggested that shikonin may have potential as a component of therapeutic strategies for the treatment of skin diseases. PMID:26935874

Dual-Component Gelatinous Peptide/Reactive Oligomer Formulations as Conduit Material and Luminal Filler for Peripheral Nerve Regeneration

PubMed Central

Kohn-Polster, Caroline; Bhatnagar, Divya; Woloszyn, Derek J.; Richtmyer, Matthew; Starke, Annett; Springwald, Alexandra H.; Franz, Sandra; Schulz-Siegmund, Michaela; Kaplan, Hilton M.; Kohn, Joachim; Hacker, Michael C.

2017-01-01

Toward the next generation of nerve guidance conduits (NGCs), novel biomaterials and functionalization concepts are required to address clinical demands in peripheral nerve regeneration (PNR). As a biological polymer with bioactive motifs, gelatinous peptides are promising building blocks. In combination with an anhydride-containing oligomer, a dual-component hydrogel system (cGEL) was established. First, hollow cGEL tubes were fabricated by a continuous dosing and templating process. Conduits were characterized concerning their mechanical strength, in vitro and in vivo degradation and biocompatibility. Second, cGEL was reformulated as injectable shear thinning filler for established NGCs, here tyrosine-derived polycarbonate-based braided conduits. Thereby, the formulation contained the small molecule LM11A-31. The biofunctionalized cGEL filler was assessed regarding building block integration, mechanical properties, in vitro cytotoxicity, and growth permissive effects on human adipose tissue-derived stem cells. A positive in vitro evaluation motivated further application of the filler material in a sciatic nerve defect. Compared to the empty conduit and pristine cGEL, the functionalization performed superior, though the autologous nerve graft remains the gold standard. In conclusion, LM11A-31 functionalized cGEL filler with extracellular matrix (ECM)-like characteristics and specific biochemical cues holds great potential to support PNR. PMID:28531139

The grafting of universal T-helper epitopes enhances immunogenicity of HIV-1 Tat concurrently improving its safety profile.

PubMed

Kashi, Venkatesh P; Jacob, Rajesh A; Shamanna, Raghavendra A; Menon, Malini; Balasiddaiah, Anangi; Varghese, Rebu K; Bachu, Mahesh; Ranga, Udaykumar

2014-01-01

Extracellular Tat (eTat) plays an important role in HIV-1 pathogenesis. The presence of anti-Tat antibodies is negatively correlated with disease progression, hence making Tat a potential vaccine candidate. The cytotoxicity and moderate immunogenicity of Tat however remain impediments for developing Tat-based vaccines. Here, we report a novel strategy to concurrently enhance the immunogenicity and safety profile of Tat. The grafting of universal helper T-lymphocyte (HTL) epitopes, Pan DR Epitope (PADRE) and Pol711 into the cysteine rich domain (CRD) and the basic domain (BD) abolished the transactivation potential of the Tat protein. The HTL-Tat proteins elicited a significantly higher titer of antibodies as compared to the wild-type Tat in BALB/c mice. While the N-terminal epitope remained immunodominant in HTL-Tat immunizations, an additional epitope in exon-2 was recognized with comparable magnitude suggesting a broader immune recognition. Additionally, the HTL-Tat proteins induced cross-reactive antibodies of high avidity that efficiently neutralized exogenous Tat, thus blocking the activation of a Tat-defective provirus. With advantages such as presentation of multiple B-cell epitopes, enhanced antibody response and importantly, transactivation-deficient Tat protein, this approach has potential application for the generation of Tat-based HIV/AIDS vaccines.

JNK and NADPH Oxidase Involved in Fluoride-Induced Oxidative Stress in BV-2 Microglia Cells

PubMed Central

Yan, Ling; Liu, Shengnan; Wang, Chen; Wang, Fei; Song, Yingli; Yan, Nan; Xi, Shuhua; Liu, Ziyou; Sun, Guifan

2013-01-01

Excessive fluoride may cause central nervous system (CNS) dysfunction, and oxidative stress is a recognized mode of action of fluoride toxicity. In CNS, activated microglial cells can release more reactive oxygen species (ROS), and NADPH oxidase (NOX) is the major enzyme for the production of extracellular superoxide in microglia. ROS have been characterized as an important secondary messenger and modulator for various mammalian intracellular signaling pathways, including the MAPK pathways. In this study we examined ROS production and TNF-α, IL-1β inflammatory cytokines releasing, and the expression of MAPKs in BV-2 microglia cells treated with fluoride. We found that fluoride increased JNK phosphorylation level of BV-2 cells and pretreatment with JNK inhibitor SP600125 markedly reduced the levels of intracellular O2 ·− and NO. NOX inhibitor apocynin and iNOS inhibitor SMT dramatically decreased NaF-induced ROS and NO generations, respectively. Antioxidant melatonin (MEL) resulted in a reduction in JNK phosphorylation in fluoride-stimulated BV-2 microglia. The results confirmed that NOX and iNOS played an important role in fluoride inducing oxidative stress and NO production and JNK took part in the oxidative stress induced by fluoride and meanwhile also could be activated by ROS in fluoride-treated BV-2 cells. PMID:24072958

The voltage dependence of NADPH oxidase reveals why phagocytes need proton channels

NASA Astrophysics Data System (ADS)

DeCoursey, Thomas E.; Morgan, Deri; Cherny, Vladimir V.

2003-04-01

The enzyme NADPH oxidase in phagocytes is important in the body's defence against microbes: it produces superoxide anions (O2-, precursors to bactericidal reactive oxygen species). Electrons move from intracellular NADPH, across a chain comprising FAD (flavin adenine dinucleotide) and two haems, to reduce extracellular O2 to O2-. NADPH oxidase is electrogenic, generating electron current (Ie) that is measurable under voltage-clamp conditions. Here we report the complete current-voltage relationship of NADPH oxidase, the first such measurement of a plasma membrane electron transporter. We find that Ie is voltage-independent from -100mV to >0mV, but is steeply inhibited by further depolarization, and is abolished at about +190mV. It was proposed that H+ efflux mediated by voltage-gated proton channels compensates Ie, because Zn2+ and Cd2+ inhibit both H+ currents and O2- production. Here we show that COS-7 cells transfected with four NADPH oxidase components, but lacking H+ channels, produce O2- in the presence of Zn2+ concentrations that inhibit O2- production in neutrophils and eosinophils. Zn2+ does not inhibit NADPH oxidase directly, but through effects on H+ channels. H+ channels optimize NADPH oxidase function by preventing membrane depolarization to inhibitory voltages.

CD73-derived adenosine and tenascin-C control cytokine production by epicardium-derived cells formed after myocardial infarction.

PubMed

Hesse, Julia; Leberling, Stella; Boden, Elisabeth; Friebe, Daniela; Schmidt, Timo; Ding, Zhaoping; Dieterich, Peter; Deussen, Andreas; Roderigo, Claudia; Rose, Christine R; Floss, Doreen M; Scheller, Jürgen; Schrader, Jürgen

2017-07-01

Epicardium-derived cells (EPDCs) play a fundamental role in embryonic cardiac development and are reactivated in the adult heart in response to myocardial infarction (MI). In this study, EPDCs from post-MI rat hearts highly expressed the ectoenzyme CD73 and secreted the profibrotic matricellular protein tenascin-C (TNC). CD73 on EPDCs extensively generated adenosine from both extracellular ATP and NAD. This in turn stimulated the release of additional nucleotides from a Brefeldin A-sensitive intracellular pool via adenosine-A 2B R signaling, forming a positive-feedback loop. A 2B R activation, in addition, strongly promoted the release of major regulatory cytokines, such as IL-6, IL-11, and VEGF. TNC was found to stimulate EPDC migration and, together with ATP-P2X 7 R signaling, to activate inflammasomes in EPDCs via TLR4. Our results demonstrate that EPDCs are an important source of various proinflammatory factors in the post-MI heart controlled by purinergic and TNC signaling.-Hesse, J., Leberling, S., Boden, E., Friebe, D., Schmidt, T., Ding, Z., Dieterich, P., Deussen, A., Roderigo, C., Rose, C. R., Floss, D. M., Scheller, J., Schrader, J. CD73-derived adenosine and tenascin-C control cytokine production by epicardium-derived cells formed after myocardial infarction. © FASEB.

An Early Tobacco Mosaic Virus-Induced Oxidative Burst in Tobacco Indicates Extracellular Perception of the Virus Coat Protein1

PubMed Central

Allan, Andrew C.; Lapidot, Moshe; Culver, James N.; Fluhr, Robert

2001-01-01

Induction of reactive oxygen species (ROS) was observed within seconds of the addition of exogenous tobacco mosaic virus (TMV) to the outside of tobacco (Nicotiana tabacum cv Samsun NN, EN, or nn) epidermal cells. Cell death was correlated with ROS production. Infectivity of the TMV virus was not a prerequisite for this elicitation and isolated coat protein (CP) subunits could also elicit the fast oxidative burst. The rapid induction of ROS was prevented by both inhibitors of plant signal transduction and inhibitors of NAD(P)H oxidases, suggesting activation of a multi-step signal transduction pathway. Induction of intracellular ROS by TMV was detected in TMV-resistant and -susceptible tobacco cultivars isogenic for the N allele. The burst was also detected with strains of virus that either elicit (ToMV) or fail to elicit (TMV U1) N′ gene-mediated responses. Hence, early ROS generation is independent or upstream of known genetic systems in tobacco that can mediate hypersensitive responses. Analysis of other viruses and TMV CP mutants showed marked differences in their ability to induce ROS showing specificity of the response. Thus, initial TMV-plant cell interactions that lead to early ROS induction occur outside the plasma membrane in an event requiring specific CP epitopes. PMID:11351074

Interactions of CuO nanoparticles with the algae Chlorella pyrenoidosa: adhesion, uptake, and toxicity.

PubMed

Zhao, Jian; Cao, Xuesong; Liu, Xiaoyu; Wang, Zhenyu; Zhang, Chenchen; White, Jason C; Xing, Baoshan

2016-11-01

The potential adverse effects of CuO nanoparticles (NPs) have increasingly attracted attention. Combining electron microscopic and toxicological investigations, we determined the adhesion, uptake, and toxicity of CuO NPs to eukaryotic alga Chlorella pyrenoidosa. CuO NPs were toxic to C. pyrenoidosa, with a 72 h EC50 of 45.7 mg/L. Scanning electron microscopy showed that CuO NPs were attached onto the surface of the algal cells and interacted with extracellular polymeric substances (EPS) excreted by the organisms. Transmission electron microscopy (TEM) showed that EPS layer of algae was thickened by nearly 4-fold after CuO NPs exposure, suggesting a possible protective mechanism. In spite of the thickening of EPS layer, CuO NPs were still internalized by endocytosis and were stored in algal vacuoles. TEM and electron diffraction analysis confirmed that the internalized CuO NPs were transformed to Cu2O NPs (d-spacing, ∼0.213 nm) with an average size approximately 5 nm. The toxicity investigation demonstrated that severe membrane damage was observed after attachment of CuO NPs with algae. Reactive oxygen species generation and mitochondrial depolarization were also noted upon exposure to CuO NPs. This work provides useful information on understanding the role of NPs-algae physical interactions in nanotoxicity.

Dual-Component Gelatinous Peptide/Reactive Oligomer Formulations as Conduit Material and Luminal Filler for Peripheral Nerve Regeneration.

PubMed

Kohn-Polster, Caroline; Bhatnagar, Divya; Woloszyn, Derek J; Richtmyer, Matthew; Starke, Annett; Springwald, Alexandra H; Franz, Sandra; Schulz-Siegmund, Michaela; Kaplan, Hilton M; Kohn, Joachim; Hacker, Michael C

2017-05-21

Toward the next generation of nerve guidance conduits (NGCs), novel biomaterials and functionalization concepts are required to address clinical demands in peripheral nerve regeneration (PNR). As a biological polymer with bioactive motifs, gelatinous peptides are promising building blocks. In combination with an anhydride-containing oligomer, a dual-component hydrogel system (cGEL) was established. First, hollow cGEL tubes were fabricated by a continuous dosing and templating process. Conduits were characterized concerning their mechanical strength, in vitro and in vivo degradation and biocompatibility. Second, cGEL was reformulated as injectable shear thinning filler for established NGCs, here tyrosine-derived polycarbonate-based braided conduits. Thereby, the formulation contained the small molecule LM11A-31. The biofunctionalized cGEL filler was assessed regarding building block integration, mechanical properties, in vitro cytotoxicity, and growth permissive effects on human adipose tissue-derived stem cells. A positive in vitro evaluation motivated further application of the filler material in a sciatic nerve defect. Compared to the empty conduit and pristine cGEL, the functionalization performed superior, though the autologous nerve graft remains the gold standard. In conclusion, LM11A-31 functionalized cGEL filler with extracellular matrix (ECM)-like characteristics and specific biochemical cues holds great potential to support PNR.

Extracellular cell wall β(1,3)glucan is required to couple septation to actomyosin ring contraction.

PubMed

Muñoz, Javier; Cortés, Juan Carlos G; Sipiczki, Matthias; Ramos, Mariona; Clemente-Ramos, José Angel; Moreno, M Belén; Martins, Ivone M; Pérez, Pilar; Ribas, Juan Carlos

2013-10-28

Cytokinesis has been extensively studied in different models, but the role of the extracellular cell wall is less understood. Here we studied this process in fission yeast. The essential protein Bgs4 synthesizes the main cell wall β(1,3)glucan. We show that Bgs4-derived β(1,3)glucan is required for correct and stable actomyosin ring positioning in the cell middle, before the start of septum formation and anchorage to the cell wall. Consequently, β(1,3)glucan loss generated ring sliding, oblique positioned rings and septa, misdirected septum synthesis indicative of relaxed rings, and uncoupling between a fast ring and membrane ingression and slow septum synthesis, suggesting that cytokinesis can progress with defective septum pushing and/or ring pulling forces. Moreover, Bgs4-derived β(1,3)glucan is essential for secondary septum formation and correct primary septum completion. Therefore, our results show that extracellular β(1,3)glucan is required for cytokinesis to connect the cell wall with the plasma membrane and for contractile ring function, as proposed for the equivalent extracellular matrix in animal cells.

Computational Modeling of Single Neuron Extracellular Electric Potentials and Network Local Field Potentials using LFPsim.

PubMed

Parasuram, Harilal; Nair, Bipin; D'Angelo, Egidio; Hines, Michael; Naldi, Giovanni; Diwakar, Shyam

2016-01-01

Local Field Potentials (LFPs) are population signals generated by complex spatiotemporal interaction of current sources and dipoles. Mathematical computations of LFPs allow the study of circuit functions and dysfunctions via simulations. This paper introduces LFPsim, a NEURON-based tool for computing population LFP activity and single neuron extracellular potentials. LFPsim was developed to be used on existing cable compartmental neuron and network models. Point source, line source, and RC based filter approximations can be used to compute extracellular activity. As a demonstration of efficient implementation, we showcase LFPs from mathematical models of electrotonically compact cerebellum granule neurons and morphologically complex neurons of the neocortical column. LFPsim reproduced neocortical LFP at 8, 32, and 56 Hz via current injection, in vitro post-synaptic N2a, N2b waves and in vivo T-C waves in cerebellum granular layer. LFPsim also includes a simulation of multi-electrode array of LFPs in network populations to aid computational inference between biophysical activity in neural networks and corresponding multi-unit activity resulting in extracellular and evoked LFP signals.

Extracellular cell wall β(1,3)glucan is required to couple septation to actomyosin ring contraction

PubMed Central

Muñoz, Javier; Cortés, Juan Carlos G.; Sipiczki, Matthias; Ramos, Mariona; Clemente-Ramos, José Angel; Moreno, M. Belén; Martins, Ivone M.; Pérez, Pilar

2013-01-01

Cytokinesis has been extensively studied in different models, but the role of the extracellular cell wall is less understood. Here we studied this process in fission yeast. The essential protein Bgs4 synthesizes the main cell wall β(1,3)glucan. We show that Bgs4-derived β(1,3)glucan is required for correct and stable actomyosin ring positioning in the cell middle, before the start of septum formation and anchorage to the cell wall. Consequently, β(1,3)glucan loss generated ring sliding, oblique positioned rings and septa, misdirected septum synthesis indicative of relaxed rings, and uncoupling between a fast ring and membrane ingression and slow septum synthesis, suggesting that cytokinesis can progress with defective septum pushing and/or ring pulling forces. Moreover, Bgs4-derived β(1,3)glucan is essential for secondary septum formation and correct primary septum completion. Therefore, our results show that extracellular β(1,3)glucan is required for cytokinesis to connect the cell wall with the plasma membrane and for contractile ring function, as proposed for the equivalent extracellular matrix in animal cells. PMID:24165938

Advanced Oxidative Protein Products Cause Pain Hypersensitivity in Rats by Inducing Dorsal Root Ganglion Neurons Apoptosis via NADPH Oxidase 4/c-Jun N-terminal Kinase Pathways

PubMed Central

Ding, Ruoting; Sun, Baihui; Liu, Zhongyuan; Yao, Xinqiang; Wang, Haiming; Shen, Xing; Jiang, Hui; Chen, Jianting

2017-01-01

Pain hypersensitivity is the most common category of chronic pain and is difficult to cure. Oxidative stress and certain cells apoptosis, such as dorsal root ganglion (DRG) neurons, play an essential role in the induction and development of pain hypersensitivity. The focus of this study is at a more specific molecular level. We investigated the role of advanced oxidative protein products (AOPPs) in inducing hypersensitivity and the cellular mechanism underlying the proapoptotic effect of AOPPs. Normal rats were injected by AOPPs-Rat serum albumin (AOPPs–RSA) to cause pain hypersensitivity. Primary cultured DRG neurons were treated with increasing concentrations of AOPPs–RSA or for increasing time durations. The MTT, flow cytometry and western blot analyses were performed in the DRG neurons. A loss of mitochondrial membrane potential (MMP) and an increase in intracellular reactive oxygen species (ROS) were observed. We found that AOPPs triggered DRG neurons apoptosis and MMP loss. After AOPPs treatment, intracellular ROS generation increased in a time- and dose-dependent manner, whereas, N-acetyl-L-cysteine (NAC), a specific ROS scavenger could inhibit the ROS generation. Proapoptotic proteins, such as Bax, caspase 9/caspase 3, and PARP-1 were activated, whereas anti-apoptotic Bcl-2 protein was down-regulated. AOPPs also increased Nox4 and JNK expression. Taken together, these findings suggest that AOPPs cause pain hypersensitivity in rats, and extracellular AOPPs accumulation triggered Nox4-dependent ROS production, which activated JNK, and induced DRG neurons apoptosis by activating caspase 3 and PARP-1. PMID:28674486

Redox Signaling and Bioenergetics Influence Lung Cancer Cell Line Sensitivity to the Isoflavone ME-344.

PubMed

Manevich, Yefim; Reyes, Leticia; Britten, Carolyn D; Townsend, Danyelle M; Tew, Kenneth D

2016-08-01

ME-344 [(3R,4S)-3,4-bis(4-hydroxyphenyl)-8-methyl-3,4-dihydro-2H-chromen-7-ol] is a second-generation derivative natural product isoflavone presently under clinical development. ME-344 effects were compared in lung cancer cell lines that are either intrinsically sensitive or resistant to the drug and in primary immortalized human lung embryonic fibroblasts (IHLEF). Cytotoxicity at low micromolar concentrations occurred only in sensitive cell lines, causing redox stress, decreased mitochondrial ATP production, and subsequent disruption of mitochondrial function. In a dose-dependent manner the drug caused instantaneous and pronounced inhibition of oxygen consumption rates (OCR) in drug-sensitive cells (quantitatively significantly less in drug-resistant cells). This was consistent with targeting of mitochondria by ME-344, with specific effects on the respiratory chain (resistance correlated with higher glycolytic indexes). OCR inhibition did not occur in primary IHLEF. ME-344 increased extracellular acidification rates in drug-resistant cells (significantly less in drug-sensitive cells), implying that ME-344 targets mitochondrial proton pumps. Only in drug-sensitive cells did ME-344 dose-dependently increase the intracellular generation of reactive oxygen species and cause oxidation of total (mainly glutathione) and protein thiols and the concomitant immediate increases in NADPH levels. We conclude that ME-344 causes complex, redox-specific, and mitochondria-targeted effects in lung cancer cells, which differ in extent from normal cells, correlate with drug sensitivity, and provide indications of a beneficial in vitro therapeutic index. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

Advanced Oxidative Protein Products Cause Pain Hypersensitivity in Rats by Inducing Dorsal Root Ganglion Neurons Apoptosis via NADPH Oxidase 4/c-Jun N-terminal Kinase Pathways.

PubMed

Ding, Ruoting; Sun, Baihui; Liu, Zhongyuan; Yao, Xinqiang; Wang, Haiming; Shen, Xing; Jiang, Hui; Chen, Jianting

2017-01-01

Pain hypersensitivity is the most common category of chronic pain and is difficult to cure. Oxidative stress and certain cells apoptosis, such as dorsal root ganglion (DRG) neurons, play an essential role in the induction and development of pain hypersensitivity. The focus of this study is at a more specific molecular level. We investigated the role of advanced oxidative protein products (AOPPs) in inducing hypersensitivity and the cellular mechanism underlying the proapoptotic effect of AOPPs. Normal rats were injected by AOPPs-Rat serum albumin (AOPPs-RSA) to cause pain hypersensitivity. Primary cultured DRG neurons were treated with increasing concentrations of AOPPs-RSA or for increasing time durations. The MTT, flow cytometry and western blot analyses were performed in the DRG neurons. A loss of mitochondrial membrane potential (MMP) and an increase in intracellular reactive oxygen species (ROS) were observed. We found that AOPPs triggered DRG neurons apoptosis and MMP loss. After AOPPs treatment, intracellular ROS generation increased in a time- and dose-dependent manner, whereas, N -acetyl-L-cysteine (NAC), a specific ROS scavenger could inhibit the ROS generation. Proapoptotic proteins, such as Bax, caspase 9/caspase 3, and PARP-1 were activated, whereas anti-apoptotic Bcl-2 protein was down-regulated. AOPPs also increased Nox4 and JNK expression. Taken together, these findings suggest that AOPPs cause pain hypersensitivity in rats, and extracellular AOPPs accumulation triggered Nox4-dependent ROS production, which activated JNK, and induced DRG neurons apoptosis by activating caspase 3 and PARP-1.

Microcalorimetric and potentiometric titration studies on the adsorption of copper by extracellular polymeric substances (EPS), minerals and their composites.

PubMed

Fang, Linchuan; Huang, Qiaoyun; Wei, Xing; Liang, Wei; Rong, Xinming; Chen, Wenli; Cai, Peng

2010-08-01

Equilibrium adsorption experiments, isothermal titration calorimetry and potentiometric titration techniques were employed to investigate the adsorption of Cu(II) by extracellular polymeric substances (EPS) extracted from Pseudomonas putida X4, minerals (montmorillonite and goethite) and their composites. Compared with predicted values of Cu(II) adsorption on composites, the measured values of Cu(II) on EPS-montmorillonite composite increased, however, those on EPS-goethite composite decreased. Potentiometric titration results also showed that more surface sites were observed on EPS-montmorillonite composite and less reactive sites were found on EPS-goethite composite. The adsorption of Cu(II) on EPS molecules and their composites with minerals was an endothermic reaction, while that on minerals was exothermic. The positive values of enthalpy change (Delta H) and entropy change (DeltaS) for Cu(II) adsorption on EPS and mineral-EPS composites indicated that Cu(II) mainly interacts with carboxyl and phosphoryl groups as inner-sphere complexes on EPS molecules and their composites with minerals. (c) 2010 Elsevier Ltd. All rights reserved.

Small extracellular vesicles secreted from senescent cells promote cancer cell proliferation through EphA2

PubMed Central

Takasugi, Masaki; Okada, Ryo; Takahashi, Akiko; Virya Chen, David; Watanabe, Sugiko; Hara, Eiji

2017-01-01

Cellular senescence prevents the proliferation of cells at risk for neoplastic transformation. However, the altered secretome of senescent cells can promote the growth of the surrounding cancer cells. Although extracellular vesicles (EVs) have emerged as new players in intercellular communication, their role in the function of senescent cell secretome has been largely unexplored. Here, we show that exosome-like small EVs (sEVs) are important mediators of the pro-tumorigenic function of senescent cells. sEV-associated EphA2 secreted from senescent cells binds to ephrin-A1, that is, highly expressed in several types of cancer cells and promotes cell proliferation through EphA2/ephrin-A1 reverse signalling. sEV sorting of EphA2 is increased in senescent cells because of its enhanced phosphorylation resulting from oxidative inactivation of PTP1B phosphatase. Our results demonstrate a novel mechanism of reactive oxygen species (ROS)-regulated cargo sorting into sEVs, which is critical for the potentially deleterious growth-promoting effect of the senescent cell secretome. PMID:28585531

Regulation of cell growth by redox-mediated extracellular proteolysis of platelet-derived growth factor receptor beta.

PubMed

Okuyama, H; Shimahara, Y; Kawada, N; Seki, S; Kristensen, D B; Yoshizato, K; Uyama, N; Yamaoka, Y

2001-07-27

Redox-regulated processes are important elements in various cellular functions. Reducing agents, such as N-acetyl-l-cysteine (NAC), are known to regulate signal transduction and cell growth through their radical scavenging action. However, recent studies have shown that reactive oxygen species are not always involved in ligand-stimulated intracellular signaling. Here, we report a novel mechanism by which NAC blocks platelet-derived growth factor (PDGF)-induced signaling pathways in hepatic stellate cells, a fibrogenic player in the liver. Unlike in vascular smooth muscle cells, we found that reducing agents, including NAC, triggered extracellular proteolysis of PDGF receptor-beta, leading to desensitization of hepatic stellate cells toward PDGF-BB. This effect was mediated by secreted mature cathepsin B. In addition, type II transforming growth factor-beta receptor was also down-regulated. Furthermore, these events seemed to cause a dramatic improvement of rat liver fibrosis. These results indicated that redox processes impact the cell's response to growth factors by regulating the turnover of growth factor receptors and that "redox therapy" is promising for fibrosis-related disease.

A Metabolic Shift toward Pentose Phosphate Pathway Is Necessary for Amyloid Fibril- and Phorbol 12-Myristate 13-Acetate-induced Neutrophil Extracellular Trap (NET) Formation*

PubMed Central

Azevedo, Estefania P.; Rochael, Natalia C.; Guimarães-Costa, Anderson B.; de Souza-Vieira, Thiago S.; Ganilho, Juliana; Saraiva, Elvira M.; Palhano, Fernando L.; Foguel, Debora

2015-01-01

Neutrophils are the main defense cells of the innate immune system. Upon stimulation, neutrophils release their chromosomal DNA to trap and kill microorganisms and inhibit their dissemination. These chromatin traps are termed neutrophil extracellular traps (NETs) and are decorated with granular and cytoplasm proteins. NET release can be induced by several microorganism membrane components, phorbol 12-myristate 13-acetate as well as by amyloid fibrils, insoluble proteinaceous molecules associated with more than 40 different pathologies among other stimuli. The intracellular signaling involved in NET formation is complex and remains unclear for most tested stimuli. Herein we demonstrate that a metabolic shift toward the pentose phosphate pathway (PPP) is necessary for NET release because glucose-6-phosphate dehydrogenase (G6PD), an important enzyme from PPP, fuels NADPH oxidase with NADPH to produce superoxide and thus induce NETs. In addition, we observed that mitochondrial reactive oxygen species, which are NADPH-independent, are not effective in producing NETs. These data shed new light on how the PPP and glucose metabolism contributes to NET formation. PMID:26198639

Bacteriophages and Biofilms

PubMed Central

Harper, David R.; Parracho, Helena M. R. T.; Walker, James; Sharp, Richard; Hughes, Gavin; Werthén, Maria; Lehman, Susan; Morales, Sandra

2014-01-01

Biofilms are an extremely common adaptation, allowing bacteria to colonize hostile environments. They present unique problems for antibiotics and biocides, both due to the nature of the extracellular matrix and to the presence within the biofilm of metabolically inactive persister cells. Such chemicals can be highly effective against planktonic bacterial cells, while being essentially ineffective against biofilms. By contrast, bacteriophages seem to have a greater ability to target this common form of bacterial growth. The high numbers of bacteria present within biofilms actually facilitate the action of bacteriophages by allowing rapid and efficient infection of the host and consequent amplification of the bacteriophage. Bacteriophages also have a number of properties that make biofilms susceptible to their action. They are known to produce (or to be able to induce) enzymes that degrade the extracellular matrix. They are also able to infect persister cells, remaining dormant within them, but re-activating when they become metabolically active. Some cultured biofilms also seem better able to support the replication of bacteriophages than comparable planktonic systems. It is perhaps unsurprising that bacteriophages, as the natural predators of bacteria, have the ability to target this common form of bacterial life.

Comparison in copper accumulation and physiological responses of Gracilaria lemaneiformis and G. lichenoides (Rhodophyceae)

NASA Astrophysics Data System (ADS)

Huang, Hezhong; Liang, Jiansheng; Wu, Xiaosong; Zhang, Hao; Li, Qianqian; Zhang, Qunying

2013-07-01

Heavy metal pollution has become a worldwide problem in aquaculture. We studied copper (Cu2+) accumulation and physiological responses of two red algae Gracilaria lemaneiformis and Gracilaria lichenoides from China under Cu2+ exposure of 0-500 μg/L in concentration. Compared with G. lemaneiformis, G. lichenoides was more capable in accumulating Cu2+, specifically, more Cu2+ on extracellular side (cell wall) than on intracellular side (cytoplasm) and in cell organelles (especially chloroplast, cell nucleus, mitochondria, and ribosome). In addition, G. lichenoides contained more insoluble polysaccharide in cell wall, which might promote the extracellular Cu2+-binding as an efficient barrier against metal toxicity. Conversely, G. lemaneiformis was more vulnerable than G. lichenoides to Cu2+ toxin for decreases in growth, pigment (chlorophyll a, chlorophyll b, phycobiliprotein, and β-carotene) content, and photosynthetic activity. Moreover, more serious oxidative damages in G. lemaneiformis than in G. lichenoides, in accumulation of reactive oxidative species and malondialdehyde, and in electrolyte leakage, because of lower antioxidant enzyme (superoxide dismutase and glutathione reductase) activities. Therefore, G. lichenoides was less susceptible to Cu2+ stress than G. lemaneiformis.

[Effect of smoking on neutrophil oxidative metabolism].

PubMed

Zasimauskas, Darius; Zekonis, Gediminas

2008-01-01

Alterations in neutrophil function by tobacco products may play a central role in the pathogenesis of periodontal diseases and several smoking-related systemic diseases. The aim of the study was to evaluate the effect of smoking on neutrophil oxidative metabolism. The study included 17 smoking men free of systemic diseases who were referred for treatment of various odontological diseases to outpatient department of Kaunas University of Medicine Hospital. The age of subjects varied from 22 to 43 years. All subjects answered the questions about smoking habits. Clinical examination included assessment of oral hygiene status according to the OHI-s index and periodontal status according to Russell and Ramfjord indices. To evaluate the oxidative metabolism of neutrophils, luminol- and liucigenin-dependent chemiluminescence and nitroblue tetrazolium test were used. After smoking, extracellular liucigenin-dependent chemiluminescence response was higher as compared to the response before smoking, but total (intra- and extracellular) luminol-dependent chemiluminescence response was the same both before and after smoking. Exposure of neutrophils to smoking caused a significant increase in nitroblue tetrazolium reduction. The release of reactive oxygen species in neutrophils exposed to smoking may alter the pathogenic processes in periodontal diseases.

Cell-mediated immunity to herpes simplex virus: recognition of type-specific and type-common surface antigens by cytotoxic T cell populations.

PubMed Central

Eberle, R; Russell, R G; Rouse, B T

1981-01-01

In this communication, we examine the specificity of anti-herpes simplex virus (HSV) cytotoxic T lymphocytes (CTL). Serological studies of the two related HSV serotypes (HSV-1 and HSV-2) have revealed both type-specific and cross-reactive antigenic determinants in the viral envelope and on the surface of infected cells. By analysis of cytotoxicity of CTL, generated in vitro by restimulation of splenocytes from mice primed with one or the other HSV serotype, the recognition of both type-specific and cross-reactive determinants on infected target cells by anti-HSV CTL was detectable. Thus, effector cells generated by priming and restimulating with the same virus recognized both type-specific and cross-reactive determinants on target cells infected with the homologous virus, but only cross-reactive determinants on target cells infected with the heterologous HSV serotype. CTL generated by restimulation with the heterologous virus were capable of recognizing only the cross-reactive determinants on either HSV-1- or HSV-2-infected target cells. These results indicate that two subpopulations of CTL exist in a population of anti-HSV immune spleen cells--those which recognize type-specific determinants and those specific for cross-reactive antigenic determinants present on the surface of HSV infected cells. The type-specific subset of anti-HSV CTL was shown to recognize the gC glycoprotein of HSV-1 infected target cells. In addition to the gC glycoprotein, at least one other type-specific surface antigen was also recognized by anti-HSV CTL in addition to the cross-reactive determinants recognized by anti-HSV CTL. PMID:6277790

Broad cross-reactive IgG responses elicited by adjuvanted vaccination with recombinant influenza hemagglutinin (rHA) in ferrets and mice

PubMed Central

Wang, Jiong; Hilchey, Shannon P.; DeDiego, Marta; Perry, Sheldon; Hyrien, Ollivier; Nogales, Aitor; Garigen, Jessica; Amanat, Fatima; Huertas, Nelson; Krammer, Florian; Martinez-Sobrido, Luis; Topham, David J.; Treanor, John J.; Sangster, Mark Y.

2018-01-01

Annual immunization against influenza virus is a large international public health effort. Accumulating evidence suggests that antibody mediated cross-reactive immunity against influenza hemagglutinin (HA) strongly correlates with long-lasting cross-protection against influenza virus strains that differ from the primary infection or vaccination strain. However, the optimal strategies for achieving highly cross-reactive antibodies to the influenza virus HA have not yet to be defined. In the current study, using Luminex-based mPlex-Flu assay, developed by our laboratory, to quantitatively measure influenza specific IgG antibody mediated cross-reactivity, we found that prime-boost-boost vaccination of ferrets with rHA proteins admixed with adjuvant elicited higher magnitude and broader cross-reactive antibody responses than that induced by actual influenza viral infection, and this cross-reactive response likely correlated with increased anti-stalk reactive antibodies. We observed a similar phenomenon in mice receiving three sequential vaccinations with rHA proteins from either A/California/07/2009 (H1N1) or A/Hong Kong/1/1968 (H3N2) viruses admixed with Addavax, an MF59-like adjuvant. Using this same mouse vaccination model, we determined that Addavax plays a more significant role in the initial priming event than in subsequent boosts. We also characterized the generation of cross-reactive antibody secreting cells (ASCs) and memory B cells (MBCs) when comparing vaccination to viral infection. We have also found that adjuvant plays a critical role in the generation of long-lived ASCs and MBCs cross-reactive to influenza viruses as a result of vaccination with rHA of influenza virus, and the observed increase in stalk-reactive antibodies likely contributes to this IgG mediated broad cross-reactivity. PMID:29641537

Broad cross-reactive IgG responses elicited by adjuvanted vaccination with recombinant influenza hemagglutinin (rHA) in ferrets and mice.

PubMed

Wang, Jiong; Hilchey, Shannon P; DeDiego, Marta; Perry, Sheldon; Hyrien, Ollivier; Nogales, Aitor; Garigen, Jessica; Amanat, Fatima; Huertas, Nelson; Krammer, Florian; Martinez-Sobrido, Luis; Topham, David J; Treanor, John J; Sangster, Mark Y; Zand, Martin S

2018-01-01

Annual immunization against influenza virus is a large international public health effort. Accumulating evidence suggests that antibody mediated cross-reactive immunity against influenza hemagglutinin (HA) strongly correlates with long-lasting cross-protection against influenza virus strains that differ from the primary infection or vaccination strain. However, the optimal strategies for achieving highly cross-reactive antibodies to the influenza virus HA have not yet to be defined. In the current study, using Luminex-based mPlex-Flu assay, developed by our laboratory, to quantitatively measure influenza specific IgG antibody mediated cross-reactivity, we found that prime-boost-boost vaccination of ferrets with rHA proteins admixed with adjuvant elicited higher magnitude and broader cross-reactive antibody responses than that induced by actual influenza viral infection, and this cross-reactive response likely correlated with increased anti-stalk reactive antibodies. We observed a similar phenomenon in mice receiving three sequential vaccinations with rHA proteins from either A/California/07/2009 (H1N1) or A/Hong Kong/1/1968 (H3N2) viruses admixed with Addavax, an MF59-like adjuvant. Using this same mouse vaccination model, we determined that Addavax plays a more significant role in the initial priming event than in subsequent boosts. We also characterized the generation of cross-reactive antibody secreting cells (ASCs) and memory B cells (MBCs) when comparing vaccination to viral infection. We have also found that adjuvant plays a critical role in the generation of long-lived ASCs and MBCs cross-reactive to influenza viruses as a result of vaccination with rHA of influenza virus, and the observed increase in stalk-reactive antibodies likely contributes to this IgG mediated broad cross-reactivity.

The impact of extracellular syntaxin4 on HaCaT keratinocyte behavior

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kadono, Nanako; Miyazaki, Takafumi; Okugawa, Yoji

2012-01-27

Highlights: Black-Right-Pointing-Pointer A subpopulation of syntaxin4 localizes extracellularly in the keratinocytes. Black-Right-Pointing-Pointer Epimorphin and syntaxin4 confer the resistance to the oxidative stress. Black-Right-Pointing-Pointer Epimorphin suppresses and syntaxin4 accelerates the CCE formation. Black-Right-Pointing-Pointer The antagonistic peptide to syntaxin4 blocks the syntaxin4-dependent CCE formation. -- Abstract: Syntaxin4 belongs to t-SNARE protein family and functions as a vesicular fusion mediator in the plasma membrane in a wide variety of cell types. This protein resembles another family member, epimorphin, a subpopulation of which has been shown to be secreted extracellularly in order to exert signaling functions. Here, we demonstrate the secretion of syntaxin4 viamore » a non-classical pathway and its extracellular functions by using the functionally normal keratinocyte HaCaT. Extracellularly presented syntaxin4 appeared to elicit many cell responses similar to epimorphin with an important exception: it clearly facilitated keratinocyte cornification. The circularized peptide ST4n1 was synthesized from the putative functional core of syntaxin4 (a.a. 103-108), which is equivalent to the previously generated antagonist of epimorphin, and neutralized this contradictory effect. Intriguingly, an epimorphin mutant (EP4M) in which the functional core was replaced by that of syntaxin4 behaved like epimorphin, which was again antagonized by ST4n1. Electrophoresis-based analyses demonstrated the distinct structure of syntaxin4 compared to epimorphin or EP4M. These results revealed, for the first time, the extracellular role of syntaxin4 and shed light on the division of the extracellular effects exerted by epimorphin and syntaxin4 on keratinocyte cornification.« less

Decreased dopamine brain reactivity in marijuana abusers is associated with negative emotionality and addiction severity

PubMed Central

Volkow, Nora D.; Wang, Gene-Jack; Telang, Frank; Fowler, Joanna S.; Alexoff, David; Logan, Jean; Jayne, Millard; Wong, Christopher; Tomasi, Dardo

2014-01-01

Moves to legalize marijuana highlight the urgency to investigate effects of chronic marijuana in the human brain. Here, we challenged 48 participants (24 controls and 24 marijuana abusers) with methylphenidate (MP), a drug that elevates extracellular dopamine (DA) as a surrogate for probing the reactivity of the brain to DA stimulation. We compared the subjective, cardiovascular, and brain DA responses (measured with PET and [11C]raclopride) to MP between controls and marijuana abusers. Although baseline (placebo) measures of striatal DA D2 receptor availability did not differ between groups, the marijuana abusers showed markedly blunted responses when challenged with MP. Specifically, compared with controls, marijuana abusers had significantly attenuated behavioral (“self-reports” for high, drug effects, anxiety, and restlessness), cardiovascular (pulse rate and diastolic blood pressure), and brain DA [reduced decreases in distribution volumes (DVs) of [11C]raclopride, although normal reductions in striatal nondisplaceable binding potential (BPND)] responses to MP. In ventral striatum (key brain reward region), MP-induced reductions in DVs and BPND (reflecting DA increases) were inversely correlated with scores of negative emotionality, which were significantly higher for marijuana abusers than controls. In marijuana abusers, DA responses in ventral striatum were also inversely correlated with addiction severity and craving. The attenuated responses to MP, including reduced decreases in striatal DVs, are consistent with decreased brain reactivity to the DA stimulation in marijuana abusers that might contribute to their negative emotionality (increased stress reactivity and irritability) and addictive behaviors. PMID:25024177

Dynamic PET and SPECT imaging with radioiodinated, amyloid-reactive peptide p5 in mice: A positive role for peptide dehalogenation

PubMed Central

Martin, Emily B.; Kennel, Stephen J.; Richey, Tina; Wooliver, Craig; Osborne, Dustin; Williams, Angela; Stuckey, Alan; Wall, Jonathan S.

2014-01-01

Dynamic molecular imaging provides bio-kinetic data that is used to characterize novel radiolabeled tracers for the detection of disease. Amyloidosis is a rare protein misfolding disease that can affect many organs. It is characterized by extracellular deposits composed principally of fibrillar proteins and hypersulfated proteoglycans. We have previously described a peptide, p5, which binds preferentially to amyloid deposits in a murine model of reactive (AA) amyloidosis. We have determined the whole body distribution of amyloid by molecular imaging techniques using radioiodinated p5. The loss of radioiodide from imaging probes due to enzymatic reaction has plagued the use of radioiodinated peptides and antibodies. Therefore, we studied iodine-124-labeled p5 by using dynamic PET imaging of both amyloid-laden and healthy mice to assess the rates of amyloid binding, the relevance of dehalogenation and the fate of the radiolabeled peptide. Rates of blood pool clearance, tissue accumulation and dehalogenation of the peptide were estimated from the images. Comparisons of these properties between the amyloid-laden and healthy mice provided kinetic profiles whose differences may prove to be indicative of the disease state. Additionally, we performed longitudinal SPECT/CT imaging with iodine-125-labeled p5 up to 72 hours post injection to determine the stability of the radioiodinated peptide when bound to the extracellular amyloid. Our data show that amyloid-associated peptide, in contrast to the unbound peptide, is resistant to dehalogenation resulting in enhanced amyloid-specific imaging. These data further support the utility of this peptide for detecting amyloidosis and monitoring potential therapeutic strategies in patients. PMID:25102446

Dynamic PET and SPECT imaging with radioiodinated, amyloid-reactive peptide p5 in mice: a positive role for peptide dehalogenation.

PubMed

Martin, Emily B; Kennel, Stephen J; Richey, Tina; Wooliver, Craig; Osborne, Dustin; Williams, Angela; Stuckey, Alan; Wall, Jonathan S

2014-10-01

Dynamic molecular imaging provides bio-kinetic data that is used to characterize novel radiolabeled tracers for the detection of disease. Amyloidosis is a rare protein misfolding disease that can affect many organs. It is characterized by extracellular deposits composed principally of fibrillar proteins and hypersulfated proteoglycans. We have previously described a peptide, p5, which binds preferentially to amyloid deposits in a murine model of reactive (AA) amyloidosis. We have determined the whole body distribution of amyloid by molecular imaging techniques using radioiodinated p5. The loss of radioiodide from imaging probes due to enzymatic reaction has plagued the use of radioiodinated peptides and antibodies. Therefore, we studied iodine-124-labeled p5 by using dynamic PET imaging of both amyloid-laden and healthy mice to assess the rates of amyloid binding, the relevance of dehalogenation and the fate of the radiolabeled peptide. Rates of blood pool clearance, tissue accumulation and dehalogenation of the peptide were estimated from the images. Comparisons of these properties between the amyloid-laden and healthy mice provided kinetic profiles whose differences may prove to be indicative of the disease state. Additionally, we performed longitudinal SPECT/CT imaging with iodine-125-labeled p5 up to 72h post injection to determine the stability of the radioiodinated peptide when bound to the extracellular amyloid. Our data show that amyloid-associated peptide, in contrast to the unbound peptide, is resistant to dehalogenation resulting in enhanced amyloid-specific imaging. These data further support the utility of this peptide for detecting amyloidosis and monitoring potential therapeutic strategies in patients. Copyright © 2014 Elsevier Inc. All rights reserved.

Competition strategies for the decolorization of a textile-reactive dye with the white-rot fungi Trametes versicolor under non-sterile conditions.

PubMed

Libra, Judy A; Borchert, Maren; Banit, Salima

2003-06-20

A variety of white-rot fungi can oxidize textile dyes under sterile conditions; however, an important consideration for their use in treating wastewater containing textile dyes is whether similar degrees of treatment can be achieved under non-sterile conditions. Four strategies were investigated for their potential in optimizing the use of the fungus Trametes versicolor in non-sterile culture for treating wastewater containing the diazo textile dye C.I. Reactive Black 5 (RB5). Three strategies with suspended culture were designed to increase the decolorization activity in suspended culture from a given amount of T. versicolor inoculum based on its tolerance of low pH (pH reduction in medium), production of extracellular enzymes (use of suspended enzymes alone), and its ability to produce enzymes independent of growth (nitrogen limitation in medium). The results showed that reduction of the medium pH to 3 did not suppress bacterial growth, while enzyme production by T. versicolor ceased. The use of the extracellular enzymes alone would allow the decoupling of the process of fungal growth from wastewater treatment; however, the enzyme activity of an enzyme suspension decreased rapidly under non-sterile conditions. The strategy of limiting nitrogen in the medium to suppress bacterial growth has potential together with the fourth strategy, the cultivation of fungi on organic solids to produce inocula for a decolorization process under non-sterile conditions. A high degree of decolorization of RB5 under non-sterile conditions was achieved with T. versicolor grown on grains as sole substrate. The rate of decolorization was dependent on the amount of fungal inoculum used. Copyright 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 82: 736-744, 2003.

Reactive Oxygen Species and Inhibitors of Inflammatory Enzymes, NADPH Oxidase, and iNOS in Experimental Models of Parkinson's Disease

PubMed Central

Koppula, Sushruta; Kumar, Hemant; Kim, In Su; Choi, Dong-Kug

2012-01-01

Reactive oxygen species (ROSs) are emerging as important players in the etiology of neurodegenerative disorders including Parkinson's disease (PD). Out of several ROS-generating systems, the inflammatory enzymes nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and inducible nitric oxide synthase (iNOS) were believed to play major roles. Mounting evidence suggests that activation of NADPH oxidase and the expression of iNOS are directly linked to the generation of highly reactive ROS which affects various cellular components and preferentially damage midbrain dopaminergic neurons in PD. Therefore, appropriate management or inhibition of ROS generated by these enzymes may represent a therapeutic target to reduce neuronal degeneration seen in PD. Here, we have summarized recently developed agents and patents claimed as inhibitors of NADPH oxidase and iNOS enzymes in experimental models of PD. PMID:22577256

Detergent-enzymatic decellularization of swine blood vessels: insight on mechanical properties for vascular tissue engineering.

PubMed

Pellegata, Alessandro F; Asnaghi, M Adelaide; Stefani, Ilaria; Maestroni, Anna; Maestroni, Silvia; Dominioni, Tommaso; Zonta, Sandro; Zerbini, Gianpaolo; Mantero, Sara

2013-01-01

Small caliber vessels substitutes still remain an unmet clinical need; few autologous substitutes are available, while synthetic grafts show insufficient patency in the long term. Decellularization is the complete removal of all cellular and nuclear matters from a tissue while leaving a preserved extracellular matrix representing a promising tool for the generation of acellular scaffolds for tissue engineering, already used for various tissues with positive outcomes. The aim of this work is to investigate the effect of a detergent-enzymatic decellularization protocol on swine arteries in terms of cell removal, extracellular matrix preservation, and mechanical properties. Furthermore, the effect of storage at -80°C on the mechanical properties of the tissue is evaluated. Swine arteries were harvested, frozen, and decellularized; histological analysis revealed complete cell removal and preserved extracellular matrix. Furthermore, the residual DNA content in decellularized tissues was far low compared to native one. Mechanical testings were performed on native, defrozen, and decellularized tissues; no statistically significant differences were reported for Young's modulus, ultimate stress, compliance, burst pressure, and suture retention strength, while ultimate strain and stress relaxation of decellularized vessels were significantly different from the native ones. Considering the overall results, the process was confirmed to be suitable for the generation of acellular scaffolds for vascular tissue engineering.

Oxidant-induced DNA damage of target cells.

PubMed Central

Schraufstätter, I; Hyslop, P A; Jackson, J H; Cochrane, C G

1988-01-01

In this study we examined the leukocytic oxidant species that induce oxidant damage of DNA in whole cells. H2O2 added extracellularly in micromolar concentrations (10-100 microM) induced DNA strand breaks in various target cells. The sensitivity of a specific target cell was inversely correlated to its catalase content and the rate of removal of H2O2 by the target cell. Oxidant species produced by xanthine oxidase/purine or phorbol myristate acetate-stimulated monocytes induced DNA breakage of target cells in proportion to the amount of H2O2 generated. These DNA strand breaks were prevented by extracellular catalase, but not by superoxide dismutase. Cytotoxic doses of HOCl, added to target cells, did not induce DNA strand breakage, and myeloperoxidase added extracellularly in the presence of an H2O2-generating system, prevented the formation of DNA strand breaks in proportion to its H2O2 degrading capacity. The studies also indicated that H2O2 formed hydroxyl radical (.OH) intracellularly, which appeared to be the most likely free radical responsible for DNA damage: .OH was detected in cells exposed to H2O2; the DNA base, deoxyguanosine, was hydroxylated in cells exposed to H2O2; and intracellular iron was essential for induction of DNA strand breaks. PMID:2843565

Cell Adhesions: Actin-Based Modules that Mediate Cell-Extracellular Matrix and Cell-Cell Interactions

PubMed Central

Bachir, Alexia; Horwitz, Alan Rick; Nelson, W. James; Bianchini, Julie M.

2018-01-01

Cell adhesions link cells to the extracellular matrix (ECM) and to each other, and depend on interactions with the actin cytoskeleton. Both cell-ECM and cell-cell adhesion sites contain discrete, yet overlapping functional modules. These modules establish physical association with the actin cytoskeleton, locally modulate actin organization and dynamics, and trigger intracellular signaling pathways. Interplay between these modules generates distinct actin architectures that underlie different stages, types, and functions of cell-ECM and cell-cell adhesions. Actomyosin contractility is required to generate mature, stable adhesions, as well as sense and translate the mechanical properties of the cellular environment to changes in cell organization and behavior. In this chapter we discuss the organization and function of different adhesion modules and how they interact with the actin cytoskeleton. We highlight the molecular mechanisms of mechanotransduction in adhesions, and how adhesion molecules mediate crosstalk between cell-ECM and cell-cell adhesion sites. PMID:28679638

Extracellular electron transfer in yeast-based biofuel cells: A review.

PubMed

Hubenova, Yolina; Mitov, Mario

2015-12-01

This paper reviews the state-of-the art of the yeast-based biofuel cell research and development. The established extracellular electron transfer (EET) mechanisms in the presence and absence of exogenous mediators are summarized and discussed. The approaches applied for improvement of mediator-less yeast-based biofuel cells performance are also presented. The overview of the literature shows that biofuel cells utilizing yeasts as biocatalysts generate power density in the range of 20 to 2440 mW/m(2), which values are comparable with the power achieved when bacteria are used instead. The electrons' origin and the contribution of the glycolysis, fermentation, aerobic respiration, and phosphorylation to the EET are commented. The reported enhanced current generation in aerobic conditions presumes reconsideration of some basic MFC principles. The challenges towards the practical application of the yeast-based biofuel cells are outlined. Copyright © 2015 Elsevier B.V. All rights reserved.

Regulation of the basement membrane by epithelia generated forces

NASA Astrophysics Data System (ADS)

Tanner, Kandice

2012-12-01

Tumor metastasis involves a progressive loss of tissue architecture and dissolution of structural boundaries between the epithelium and connective tissue. The basement membrane (BM), a specialized network of extracellular matrix proteins forms a barrier that physically restricts pre-invasive lesions such that they remain as local insults. The BM is not a static structure, but one that is constantly regenerated and remodeled in the adult organism. Matrix organization also regulates cell function. Thus alterations in the balance of synthesis, remodeling and proteolytic degradation of the extracellular matrix proteins may contribute to a loss of structural integrity. However, the de novo assembly and maintenance of the complex structural properties of in vivo basement membranes remain elusive. Here, this paper highlights the current understanding on the structural properties and the establishment of the BM, and discusses the potential role of self-generated forces in adult tissue remodeling and the maintenance of the BM as a malignancy suppressor.

Multi-electrode array technologies for neuroscience and cardiology

NASA Astrophysics Data System (ADS)

Spira, Micha E.; Hai, Aviad

2013-02-01

At present, the prime methodology for studying neuronal circuit-connectivity, physiology and pathology under in vitro or in vivo conditions is by using substrate-integrated microelectrode arrays. Although this methodology permits simultaneous, cell-non-invasive, long-term recordings of extracellular field potentials generated by action potentials, it is 'blind' to subthreshold synaptic potentials generated by single cells. On the other hand, intracellular recordings of the full electrophysiological repertoire (subthreshold synaptic potentials, membrane oscillations and action potentials) are, at present, obtained only by sharp or patch microelectrodes. These, however, are limited to single cells at a time and for short durations. Recently a number of laboratories began to merge the advantages of extracellular microelectrode arrays and intracellular microelectrodes. This Review describes the novel approaches, identifying their strengths and limitations from the point of view of the end users -- with the intention to help steer the bioengineering efforts towards the needs of brain-circuit research.

Multi-electrode array technologies for neuroscience and cardiology.

PubMed

Spira, Micha E; Hai, Aviad

2013-02-01

At present, the prime methodology for studying neuronal circuit-connectivity, physiology and pathology under in vitro or in vivo conditions is by using substrate-integrated microelectrode arrays. Although this methodology permits simultaneous, cell-non-invasive, long-term recordings of extracellular field potentials generated by action potentials, it is 'blind' to subthreshold synaptic potentials generated by single cells. On the other hand, intracellular recordings of the full electrophysiological repertoire (subthreshold synaptic potentials, membrane oscillations and action potentials) are, at present, obtained only by sharp or patch microelectrodes. These, however, are limited to single cells at a time and for short durations. Recently a number of laboratories began to merge the advantages of extracellular microelectrode arrays and intracellular microelectrodes. This Review describes the novel approaches, identifying their strengths and limitations from the point of view of the end users--with the intention to help steer the bioengineering efforts towards the needs of brain-circuit research.

Mitochondria and Reactive Oxygen Species: Physiology and Pathophysiology

PubMed Central

Bolisetty, Subhashini; Jaimes, Edgar A.

2013-01-01

The air that we breathe contains nearly 21% oxygen, most of which is utilized by mitochondria during respiration. While we cannot live without it, it was perceived as a bane to aerobic organisms due to the generation of reactive oxygen and nitrogen metabolites by mitochondria and other cellular compartments. However, this dogma was challenged when these species were demonstrated to modulate cellular responses through altering signaling pathways. In fact, since this discovery of a dichotomous role of reactive species in immune function and signal transduction, research in this field grew at an exponential pace and the pursuit for mechanisms involved began. Due to a significant number of review articles present on the reactive species mediated cell death, we have focused on emerging novel pathways such as autophagy, signaling and maintenance of the mitochondrial network. Despite its role in several processes, increased reactive species generation has been associated with the origin and pathogenesis of a plethora of diseases. While it is tempting to speculate that anti-oxidant therapy would protect against these disorders, growing evidence suggests that this may not be true. This further supports our belief that these reactive species play a fundamental role in maintenance of cellular and tissue homeostasis. PMID:23528859

Method for generating a highly reactive plasma for exhaust gas aftertreatment and enhanced catalyst reactivity

DOEpatents

Whealton, John H.; Hanson, Gregory R.; Storey, John M.; Raridon, Richard J.; Armfield, Jeffrey S.; Bigelow, Timothy S.; Graves, Ronald L.

2002-01-01

A method for non-thermal plasma aftertreatment of exhaust gases the method comprising the steps of providing short risetime, high frequency, high power bursts of low-duty factor microwaves sufficient to generate a plasma discharge and passing a gas to be treated through the discharge so as to cause dissociative reduction of the exhaust gases and enhanced catalyst reactivity through application of the pulsed microwave fields directly to the catalyst material sufficient to cause a polarizability catastrophe and enhanced heating of the metal crystallite particles of the catalyst, and in the presence or absence of the plasma. The invention also includes a reactor for aftertreatment of exhaust gases.

GABA-independent GABAA Receptor Openings Maintain Tonic Currents

PubMed Central

Wlodarczyk, Agnieszka I.; Sylantyev, Sergiy; Herd, Murray B.; Kersanté, Flavie; Lambert, Jeremy J.; Rusakov, Dmitri A.; Linthorst, Astrid C.E.; Semyanov, Alexey; Belelli, Delia; Pavlov, Ivan; Walker, Matthew C.

2013-01-01

Activation of GABAA receptors (GABAARs) produces two forms of inhibition: ‘phasic’ inhibition generated by the rapid, transient activation of synaptic GABAARs by presynaptic GABA release, and tonic inhibition generated by the persistent activation of peri- or extrasynaptic GABAARs which can detect extracellular GABA. Such tonic GABAAR-mediated currents are particularly evident in dentate granule cells in which they play a major role in regulating cell excitability. Here we show that in rat dentate granule cells in ex-vivo hippocampal slices, tonic currents are predominantly generated by GABA-independent GABAA receptor openings. This tonic GABAAR conductance is resistant to the competitive GABAAR antagonist SR95531, which at high concentrations acts as a partial agonist, but can be blocked by an open channel blocker picrotoxin. When slices are perfused with 200 nM GABA, a concentration that is comparable to cerebrospinal fluid concentrations but is twice that measured by us in the hippocampus in vivo using zero-net-flux microdialysis, negligible GABA is detected by dentate granule cells. Spontaneously opening GABAARs, therefore, maintain dentate granule cell tonic currents in the face of low extracellular GABA concentrations. PMID:23447601

Inhibitory effect of self-generated extracellular dissolved organic carbon on carbon dioxide fixation in sulfur-oxidizing bacteria during a chemoautotrophic cultivation process and its elimination.

PubMed

Wang, Ya-Nan; Tsang, Yiu Fai; Wang, Lei; Fu, Xiaohua; Hu, Jiajun; Li, Huan; Le, Yiquan

2018-03-01

The features of extracellular dissolved organic carbon (EDOC) generation in two typical aerobic sulfur-oxidizing bacteria (Thiobacillus thioparus DSM 505 and Halothiobacillus neapolitanus DSM 15147) and its impact on CO 2 fixation during chemoautotrophic cultivation process were investigated. The results showed that EDOC accumulated in both strains during CO 2 fixation process. Large molecular weight (MW) EDOC derived from cell lysis and decay was dominant during the entire process in DSM 505, whereas small MW EDOC accounted for a large proportion during initial and middle stages of DSM 15147 as its cytoskeleton synthesis rate did not keep up with CO 2 assimilation rate. The self-generated EDOC feedback repressed cbb gene transcription and thus decreased total bacterial cell number and CO 2 fixation yield in both strains, but DSM 505 was more sensitive to this inhibition effect. Moreover, the membrane bioreactor effectively decreased the EDOC/TOC ratio and improved carbon fixation yield of DSM 505. Copyright © 2017 Elsevier Ltd. All rights reserved.

The Neutrophil’s Choice: Phagocytose vs Make Neutrophil Extracellular Traps

PubMed Central

Manfredi, Angelo A.; Ramirez, Giuseppe A.; Rovere-Querini, Patrizia; Maugeri, Norma

2018-01-01

Neutrophils recognize particulate substrates of microbial or endogenous origin and react by sequestering the cargo via phagocytosis or by releasing neutrophil extracellular traps (NETs) outside the cell, thus modifying and alerting the environment and bystander leukocytes. The signals that determine the choice between phagocytosis and the generation of NETs are still poorly characterized. Neutrophils that had phagocytosed bulky particulate substrates, such as apoptotic cells and activated platelets, appear to be “poised” in an unresponsive state. Environmental conditions, the metabolic, adhesive and activation state of the phagocyte, and the size of and signals associated with the tethered phagocytic cargo influence the choice of the neutrophils, prompting either phagocytic clearance or the generation of NETs. The choice is dichotomic and apparently irreversible. Defects in phagocytosis may foster the intravascular generation of NETs, thus promoting vascular inflammation and morbidities associated with diseases characterized by defective phagocytic clearance, such as systemic lupus erythematosus. There is a strong potential for novel treatments based on new knowledge of the events determining the inflammatory and pro-thrombotic function of inflammatory leukocytes. PMID:29515586

Uric acid is released in the brain during seizure activity and increases severity of seizures in a mouse model for acute limbic seizures.

PubMed

Thyrion, Lisa; Raedt, Robrecht; Portelli, Jeanelle; Van Loo, Pieter; Wadman, Wytse J; Glorieux, Griet; Lambrecht, Bart N; Janssens, Sophie; Vonck, Kristl; Boon, Paul

2016-03-01

Recent evidence points at an important role of endogenous cell-damage induced pro-inflammatory molecules in the generation of epileptic seizures. Uric acid, under the form of monosodium urate crystals, has shown to have pro-inflammatory properties in the body, but less is known about its role in seizure generation. This study aimed to unravel the contribution of uric acid to seizure generation in a mouse model for acute limbic seizures. We measured extracellular levels of uric acid in the brain and modulated them using complementary pharmacological and genetic tools. Local extracellular uric acid levels increased three to four times during acute limbic seizures and peaked between 50 and 100 min after kainic acid infusion. Manipulating uric acid levels through administration of allopurinol or knock-out of urate oxidase significantly altered the number of generalized seizures, decreasing and increasing them by a twofold respectively. Taken together, our results consistently show that uric acid is released during limbic seizures and suggest that uric acid facilitates seizure generalization. Copyright © 2016 Elsevier Inc. All rights reserved.

Electron density reactivity indexes of the tautomeric/ionization forms of thiamin diphosphate.

PubMed

Jaña, Gonzalo A; Delgado, Eduardo J

2013-09-01

The generation of the highly reactive ylide in thiamin diphosphate catalysis is analyzed in terms of the nucleophilicity of key atoms, by means of density functional calculations at X3LYP/6-31++G(d,p) level of theory. The Fukui functions of all tautomeric/ionization forms are calculated in order to assess their reactivity. The results allow to conclude that the highly conserved glutamic residue does not protonate the N1' atom of the pyrimidyl ring, but it participates in a strong hydrogen bonding, stabilizing the eventual negative charge on the nitrogen, in all forms involved in the ylide generation. This condition provides the necessary reactivity on key atoms, N4' and C2, to carry out the formation of the ylide required to initiate the catalytic cycle of ThDP-dependent enzymes. This study represents a new approach for the ylide formation in ThDP catalysis.

Antibodies Reactive to Commensal Streptococcus mitis Show Cross-Reactivity With Virulent Streptococcus pneumoniae Serotypes.

PubMed

Shekhar, Sudhanshu; Khan, Rabia; Ferreira, Daniela M; Mitsi, Elena; German, Esther; Rørvik, Gro Herredsvela; Berild, Dag; Schenck, Karl; Kwon, Keehwan; Petersen, Fernanda

2018-01-01

Current vaccines against Streptococcus pneumoniae , a bacterial species that afflicts people by causing a wide spectrum of diseases, do not protect against all pneumococcal serotypes. Thus, alternative vaccines to fight pneumococcal infections that target common proteins are under investigation. One promising strategy is to take advantage of immune cross-reactivity between commensal and pathogenic microbes for cross-protection. In this study, we examined the antibody-mediated cross-reactivity between S. pneumoniae and Streptococcus mitis , a commensal species closely related to S. pneumoniae . Western blot analysis showed that rabbit antisera raised against S. mitis reacted with multiple proteins of virulent S. pneumoniae strains (6B, TIGR4, and D39). Rabbit anti- S. pneumoniae IgG antibodies also showed binding to S. mitis antigens. Incubation of rabbit antisera raised against S. mitis with heterologous or homologous bacterial lysates resulted in marked inhibition of the developments of bands in the Western blots. Furthermore, plasma IgG antibodies from adult human volunteers intranasally inoculated with S. pneumoniae 6B revealed enhanced S. mitis -specific IgG titers compared with the pre-inoculation samples. Using an on-chip protein microarray representing a number of selected membrane and extracellular S. pneumoniae proteins, we identified choline-binding protein D (CbpD), cell division protein (FtsH), and manganese ABC transporter or manganese-binding adhesion lipoprotein (PsaA) as common targets of the rabbit IgG antibodies raised against S. mitis or S. pneumoniae . Cumulatively, these findings provide evidence on the antibody-mediated cross-reactivity of proteins from S. mitis and S. pneumoniae , which may have implications for development of effective and wide-range pneumococcal vaccines.

Superoxide and Peroxynitrite in Atherosclerosis

NASA Astrophysics Data System (ADS)

White, C. Roger; Brock, Tommy A.; Chang, Ling-Yi; Crapo, James; Briscoe, Page; Ku, David; Bradley, William A.; Gianturco, Sandra H.; Gore, Jeri; Freeman, Bruce A.; Tarpey, Margaret M.

1994-02-01

The role of reactive oxygen species in the vascular pathology associated with atherosclerosis was examined by testing the hypothesis that impaired vascular reactivity results from the reaction of nitric oxide (^.NO) with superoxide (O^-_2), yielding the oxidant peroxynitrite (ONOO^-). Contractility studies were performed on femoral arteries from rabbits fed a cholesterol-supplemented diet. Cholesterol feeding shifted the EC50 for acetylcholine (ACh)-induced relaxation and impaired the maximal response to ACh. We used pH-sensitive liposomes to deliver CuZn superoxide dismutase (SOD; superoxide:superoxide oxidoreductase, EC 1.15.1.1) to critical sites of ^.NO reaction with O^-_2. Intravenously injected liposomes (3000 units of SOD per ml) augmented ACh-induced relaxation in the cholesterol-fed group to a greater extent than in controls. Quantitative immunocytochemistry demonstrated enhanced distribution of SOD in both endothelial and vascular smooth muscle cells as well as in the extracellular matrix. SOD activity in vessel homogenates of liposome-treated rabbits was also increased. Incubation of β very low density lipoprotein with ONOO^- resulted in the rapid formation of conjugated dienes and thiobarbituric acid-reactive substances. Our results suggest that the reaction of O^-_2 with ^.NO is involved in the development of atherosclerotic disease by yielding a potent mediator of lipoprotein oxidation, as well as by limiting ^.NO stimulation of vascular smooth muscle guanylate cyclase activity.

Expression of the plasminogen activator system and the inhibitors PAI-1 and PAI-2 in posttraumatic lesions of the CNS and brain injuries following dramatic circulatory arrests: an immunohistochemical study.

PubMed

Dietzmann, K; von Bossanyi, P; Krause, D; Wittig, H; Mawrin, C; Kirches, E

2000-01-01

Plasminogen activators as inducible extracellular serine proteases are involved in a variety of processes, such as the degradation of brain structures. In regions of brain degradation, an increase in the expression of genes encoding cytokines and proteinases has recently been demonstrated. We tested the hypothesis, whether the plasminogen activator system as well as the plasminogen activator inhibitors are expressed and possibly involved in a proteolytic cascade that breaks down the extracellular matrix as a result of ischemic or posttraumatic brain destructions. To study this supposition, we investigated immunohistochemically the expression of tPA, uPA and its receptor, the plasminogen activator inhibitors PAI-1 and PAI-2, tetranectin as well as the laminin breakdown as an event of secondary brain injury. Brain tissue from 21 autopsy cases with severe brain injuries, material from 14 ischemic infarcts and 11 controls with acute hypoxia were used. All components of the plasminogen activator system studied were over-expressed immunohistochemically in reactive astrocytes, microglia and endothelial cells around the lesion zone. Tetranectin showed an analogous distribution to the plasminogen activator system. A reduced immunoreactivity of laminin within the identical region of destruction was detected concomitant with laminin remnants in perivascular macrophages, so that a remarkable role of the plasmin cascade in the degradation of extracellular matrix proteins in the brain is taken into consideration.

Cell wall-associated ROOT HAIR SPECIFIC 10, a proline-rich receptor-like kinase, is a negative modulator of Arabidopsis root hair growth

PubMed Central

Hwang, Youra; Lee, Hyodong; Lee, Young-Sook; Cho, Hyung-Taeg

2016-01-01

Plant cell growth is restricted by the cell wall, and cell wall dynamics act as signals for the cytoplasmic and nuclear events of cell growth. Among various receptor kinases, ROOT HAIR SPECIFIC 10 (RHS10) belongs to a poorly known receptor kinase subfamily with a proline-rich extracellular domain. Here, we report that RHS10 defines the root hair length of Arabidopsis thaliana by negatively regulating hair growth. RHS10 modulates the duration of root hair growth rather than the growth rate. As poplar and rice RHS10 orthologs also showed a root hair-inhibitory function, this receptor kinase-mediated function appears to be conserved in angiosperms. RHS10 showed a strong association with the cell wall, most probably through its extracellular proline-rich domain (ECD). Deletion analysis of the ECD demonstrated that a minimal extracellular part, which includes a few proline residues, is required for RHS10-mediated root hair inhibition. RHS10 suppressed the accumulation of reactive oxygen species (ROS) in the root, which are necessary for root hair growth. A yeast two-hybrid screening identified an RNase (RNS2) as a putative downstream target of RHS10. Accordingly, RHS10 overexpression decreased and RHS10 loss increased RNA levels in the hair-growing root region. Our results suggest that RHS10 mediates cell wall-associated signals to maintain proper root hair length, at least in part by regulating RNA catabolism and ROS accumulation. PMID:26884603

Neuroprotective effects of angiotensin II type 1 receptor (AT1-R) blocker via modulating AT1-R signaling and decreased extracellular glutamate levels.

PubMed

Fujita, Tomoyoshi; Hirooka, Kazuyuki; Nakamura, Takehiro; Itano, Toshifumi; Nishiyama, Akira; Nagai, Yukiko; Shiraga, Fumio

2012-06-26

To investigate the mechanism of the neuroprotective effects of the angiotensin II type 1 receptor (AT1-R) blocker against retinal ischemia-reperfusion injury in the rat. Retinal ischemia was induced by increasing intraocular pressure. Glutamate release from the rat retina and intravitreal PO(2) (partial pressure of oxygen) profiles were monitored during and after ischemia using a microdialysis biosensor and oxygen-sensitive microelectrodes. ELISA was used to measure changes in the expression of AT1-R. Retinal mRNA expressions of p47phox and p67phox were measured by real-time polymerase chain reaction. Reactive oxygen species (ROS) were measured using dihydroethidium. Administration of candesartan, which is an AT1-R blocker (ARB), suppressed ischemia-induced increases in the extracellular glutamate. Candesartan also attenuated the increase in intravitreal PO(2) during reperfusion. AT1-R expression peaked at 12 hours after reperfusion. Although there was an increase in the retinal mRNA expression of p47phox and p64phox at 12 hours after the reperfusion, administration of candesartan suppressed these expressions. The production of ROS that was detected at 12 hours after reperfusion was also suppressed by the administration of candesartan or apocynin. NADPH oxidase-mediated ROS production increased at 12 hours after reperfusion. Candesartan may protect neurons by decreasing extracellular glutamate immediately after reperfusion and by attenuating oxidative stress via a modulation of the AT1-R signaling that occurs during ischemic insult.

Gelatinized Copper–Capillary Alginate Gel Functions as an Injectable Tissue Scaffolding System for Stem Cell Transplants

PubMed Central

Willenberg, Bradley Jay; Zheng, Tong; Meng, Fan-Wei; Meneses, Juan Carlos; Rossignol, Candace; Batich, Christopher D.; Terada, Naohiro; Steindler, Dennis A.; Weiss, Michael D.

2013-01-01

In severe hypoxic–ischemic brain injury, cellular components such as neurons and astrocytes are injured or destroyed along with the supporting extracellular matrix. This presents a challenge to the field of regenerative medicine since the lack of extracellular matrix and supporting structures makes the transplant milieu inhospitable to the transplanted cells. A potential solution to this problem is the use of a biomaterial to provide the extracellular components needed to keep cells localized in cystic brain regions, allowing the cells to form connections and repair lost brain tissue. Ideally, this biomaterial would be combined with stem cells, which have been proven to have therapeutic potentials, and could be delivered via an injection. To study this approach, we derived a hydrogel biomaterial tissue scaffold from oligomeric gelatin and copper–capillary alginate gel (GCCAG). We then demonstrated that our multipotent astrocytic stem cells (MASCs) could be maintained in GCCAG scaffolds for up to 2 weeks in vitro and that the cells retained their multipotency. We next performed a pilot transplant study in which GCCAG was mixed with MASCs and injected into the brain of a neonatal rat pup. After a week in vivo, our results showed that: the GCCAG biomaterial did not cause a significant reactive gliosis; viable cells were retained within the injected scaffolds; and some delivered cells migrated into the surrounding brain tissue. Therefore, GCCAG tissue scaffolds are a promising, novel injectable system for transplantation of stem cells to the brain. PMID:20699061

Extracellular Superoxide Dismutase Inhibits Innate Immune Responses and Clearance of an Intracellular Bacterial Infection

PubMed Central

Break, Timothy J.; Jun, Sujung; Indramohan, Mohanalaxmi; Carr, Karen D.; Sieve, Amy N.; Dory, Ladislav; Berg, Rance E.

2012-01-01

Reactive oxygen and nitrogen species (ROS/RNS) play important roles during immune responses to bacterial pathogens. Extracellular superoxide dismutase (ecSOD) regulates extracellular concentrations of ROS/RNS and contributes to tissue protection during inflammatory insults. The participation of ecSOD in immune responses seems therefore intuitive, yet is poorly understood. In the present study, we utilized mice with varying levels of ecSOD activity to investigate the involvement of this enzyme in immune responses against Listeria monocytogenes. Surprisingly, our data demonstrate that, despite enhanced neutrophil recruitment to the liver, ecSOD activity negatively impacted host survival and bacterial clearance. Increased ecSOD activity was accompanied by decreased co-localization of neutrophils with bacteria, as well as increased neutrophil apoptosis, which reduced overall and neutrophil-specific TNF-α production. Liver leukocytes from mice lacking ecSOD produced equivalent nitric oxide (NO·) when compared to mice expressing ecSOD. However, during infection, there were higher levels of peroxynitrite (NO3·−) in livers from mice lacking ecSOD compared to mice expressing ecSOD. Neutrophil depletion studies revealed that high levels of ecSOD activity resulted in neutrophils with limited protective capacity, whereas neutrophils from mice lacking ecSOD provided superior protection compared to neutrophils from wild-type mice. Taken together, our data demonstrate that ecSOD activity reduces innate immune responses during bacterial infection and provides a potential target for therapeutic intervention. PMID:22393157

Printing three-dimensional tissue analogues with decellularized extracellular matrix bioink

PubMed Central

Pati, Falguni; Jang, Jinah; Ha, Dong-Heon; Won Kim, Sung; Rhie, Jong-Won; Shim, Jin-Hyung; Kim, Deok-Ho; Cho, Dong-Woo

2014-01-01

The ability to print and pattern all the components that make up a tissue (cells and matrix materials) in three dimensions to generate structures similar to tissues is an exciting prospect of bioprinting. However, the majority of the matrix materials used so far for bioprinting cannot represent the complexity of natural extracellular matrix (ECM) and thus are unable to reconstitute the intrinsic cellular morphologies and functions. Here, we develop a method for the bioprinting of cell-laden constructs with novel decellularized extracellular matrix (dECM) bioink capable of providing an optimized microenvironment conducive to the growth of three-dimensional structured tissue. We show the versatility and flexibility of the developed bioprinting process using tissue-specific dECM bioinks, including adipose, cartilage and heart tissues, capable of providing crucial cues for cells engraftment, survival and long-term function. We achieve high cell viability and functionality of the printed dECM structures using our bioprinting method. PMID:24887553

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bose, A; Gardel, EJ; Vidoudez, C

Oxidation-reduction reactions underlie energy generation in nearly all life forms. Although most organisms use soluble oxidants and reductants, some microbes can access solid-phase materials as electron-acceptors or -donors via extracellular electron transfer. Many studies have focused on the reduction of solid-phase oxidants. Far less is known about electron uptake via microbial extracellular electron transfer, and almost nothing is known about the associated mechanisms. Here we show that the iron-oxidizing photoautotroph Rhodopseudomonas palustris TIE-1 accepts electrons from a poised electrode, with carbon dioxide as the sole carbon source/electron acceptor. Both electron uptake and ruBisCo form I expression are stimulated by light.more » Electron uptake also occurs in the dark, uncoupled from photosynthesis. Notably, the pioABC operon, which encodes a protein system essential for photoautotrophic growth by ferrous iron oxidation, influences electron uptake. These data reveal a previously unknown metabolic versatility of photoferrotrophs to use extracellular electron transfer for electron uptake.« less

Printing three-dimensional tissue analogues with decellularized extracellular matrix bioink

NASA Astrophysics Data System (ADS)

Pati, Falguni; Jang, Jinah; Ha, Dong-Heon; Won Kim, Sung; Rhie, Jong-Won; Shim, Jin-Hyung; Kim, Deok-Ho; Cho, Dong-Woo

2014-06-01

The ability to print and pattern all the components that make up a tissue (cells and matrix materials) in three dimensions to generate structures similar to tissues is an exciting prospect of bioprinting. However, the majority of the matrix materials used so far for bioprinting cannot represent the complexity of natural extracellular matrix (ECM) and thus are unable to reconstitute the intrinsic cellular morphologies and functions. Here, we develop a method for the bioprinting of cell-laden constructs with novel decellularized extracellular matrix (dECM) bioink capable of providing an optimized microenvironment conducive to the growth of three-dimensional structured tissue. We show the versatility and flexibility of the developed bioprinting process using tissue-specific dECM bioinks, including adipose, cartilage and heart tissues, capable of providing crucial cues for cells engraftment, survival and long-term function. We achieve high cell viability and functionality of the printed dECM structures using our bioprinting method.

Bioprinting of 3D Tissue Models Using Decellularized Extracellular Matrix Bioink.

PubMed

Pati, Falguni; Cho, Dong-Woo

2017-01-01

Bioprinting provides an exciting opportunity to print and pattern all the components that make up a tissue-cells and extracellular matrix (ECM) material-in three dimensions (3D) to generate tissue analogues. A large number of materials have been used for making bioinks; however, majority of them cannot represent the complexity of natural ECM and thus are unable to reconstitute the intrinsic cellular morphologies and functions. We present here a method for making of bioink from decellularized extracellular matrices (dECMs) and a protocol for bioprinting of cell-laden constructs with this novel bioink. The dECM bioink is capable of providing an optimized microenvironment that is conducive to the growth of 3D structured tissue. We have prepared bioinks from different tissues, including adipose, cartilage and heart tissues and achieved high cell viability and functionality of the bioprinted tissue structures using our novel bioink.

Reactive documentation system

NASA Astrophysics Data System (ADS)

Boehnlein, Thomas R.; Kramb, Victoria

2018-04-01

Proper formal documentation of computer acquired NDE experimental data generated during research is critical to the longevity and usefulness of the data. Without documentation describing how and why the data was acquired, NDE research teams lose capability such as their ability to generate new information from previously collected data or provide adequate information so that their work can be replicated by others seeking to validate their research. Despite the critical nature of this issue, NDE data is still being generated in research labs without appropriate documentation. By generating documentation in series with data, equal priority is given to both activities during the research process. One way to achieve this is to use a reactive documentation system (RDS). RDS prompts an operator to document the data as it is generated rather than relying on the operator to decide when and what to document. This paper discusses how such a system can be implemented in a dynamic environment made up of in-house and third party NDE data acquisition systems without creating additional burden on the operator. The reactive documentation approach presented here is agnostic enough that the principles can be applied to any operator controlled, computer based, data acquisition system.