scholarly journals Cell type-dependent release of nitric oxide and/or reactive nitrogenoxide species from intracellular SIN-1: effects on cellular NAD(P)H

2004 ◽  
Vol 385 (7) ◽  
Author(s):  
A.U. Swintek ◽  
S. Christoph ◽  
F. Petrat ◽  
H. de Groot ◽  
M. Kirsch
Keyword(s):  
Nitric Oxide ◽  
Cell Culture ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Culture Studies ◽  
Aortic Endothelial Cells ◽  
No Formation ◽  
Cell Culture Systems ◽  
Zone Electrophoresis ◽  
Capillary Zone

AbstractSIN-1 is frequently used in cell culture studies as an extracellularly operating generator of peroxynitrite. However, little is known about the nature of the reactive species produced intracellulary from SIN-1. SIN-1 can easily penetrate cells as exemplified for both L-929 mouse fibroblasts and bovine aortic endothelial cells (BAECs) by utilizing capillary zone electrophoresis. In L-929 cells, SIN-1 produced nitric oxide (NO) as monitored by the fluorescent NO scavenger FNOCT-1 and by means of a NO electrode, as well as reactive nitrogenoxide species (RNOS, e.g. peroxynitrite, nitrogen dioxide, dinitrogen trioxide), as detected with the fluorescent indicator DAF-2. Laser scanning microscopy revealed that in L-929 cells SIN-1-derived species initially oxidized the major fraction of the NAD(P)H within the cytosol and the nuclei, whereas the mitochondrial NAD(P)H level was somewhat increased. In marked contrast to this, in BAECs no evidence for NO formation was found although the intracellular amount of SIN-1 was fourfold higher than in L-929 cells. In BAECs, the level of NAD(P)H was slightly decreased within the first 10 min after administration of SIN-1 in both the cytosol/nuclei and mitochondria. These observations reflect the capability of SIN-1 to generate intracellularly either almost exclusively RNOS as in BAECs, or RNOS and freely diffusing NO as in L-929 cells. Nitric oxide as well as RNOS may decisively affect cellular metabolism as indicated by the alterations in the NAD(P)H level. Hence, care should be taken when applying SIN-1 as an exclusively peroxynitrite-generating compound in cell culture systems.

scholarly journals Cephalosporin nitric oxide-donor prodrug DEA-C3D disperses biofilms formed by clinical cystic fibrosis isolates of Pseudomonas aeruginosa

2019 ◽  
Vol 75 (1) ◽  
pp. 117-125 ◽  
Author(s):  
Odel Soren ◽  
Ardeshir Rineh ◽  
Diogo G Silva ◽  
Yuming Cai ◽  
Robert P Howlin ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Clinical Isolates ◽  
Nitric Oxide Donor ◽  
Confocal Laser ◽  
Broth Microdilution Method

Abstract Objectives The cephalosporin nitric oxide (NO)-donor prodrug DEA-C3D (‘DiEthylAmin-Cephalosporin-3′-Diazeniumdiolate’) has been shown to initiate the dispersal of biofilms formed by the Pseudomonas aeruginosa laboratory strain PAO1. In this study, we investigated whether DEA-C3D disperses biofilms formed by clinical cystic fibrosis (CF) isolates of P. aeruginosa and its effect in combination with two antipseudomonal antibiotics, tobramycin and colistin, in vitro. Methods β-Lactamase-triggered release of NO from DEA-C3D was confirmed using a gas-phase chemiluminescence detector. MICs for P. aeruginosa clinical isolates were determined using the broth microdilution method. A crystal violet staining technique and confocal laser scanning microscopy were used to evaluate the effects of DEA-C3D on P. aeruginosa biofilms alone and in combination with tobramycin and colistin. Results DEA-C3D was confirmed to selectively release NO in response to contact with bacterial β-lactamase. Despite lacking direct, cephalosporin/β-lactam-based antibacterial activity, DEA-C3D was able to disperse biofilms formed by three P. aeruginosa clinical isolates. Confocal microscopy revealed that DEA-C3D in combination with tobramycin produces similar reductions in biofilm to DEA-C3D alone, whereas the combination with colistin causes near complete eradication of P. aeruginosa biofilms in vitro. Conclusions DEA-C3D is effective in dispersing biofilms formed by multiple clinical isolates of P. aeruginosa and could hold promise as a new adjunctive therapy to patients with CF.

LTCC Based Multi-Electrode Arrays for In-Vitro Cell Culture

2015 ◽  
Vol 2015 (CICMT) ◽  
pp. 000269-000274
Author(s):  
Heike Bartsch ◽  
Dirk Stöpel ◽  
Marcel Himmerlich ◽  
Martin Baca ◽  
Philipp Stadie ◽  
...  
Keyword(s):  
Cell Culture ◽  
Laser Scanning ◽  
Neuronal Cell ◽  
Laser Scanning Microscopy ◽  
Gold Electrodes ◽  
Neural Cells ◽  
Electrode Arrays ◽  
Green Tape ◽  
High Level

Neurobiological concepts based on state-of-the art technology have so far lacked the complexity of actual high-level neurobiological systems. Two key advances are needed to improve our understanding of such systems: in vitro 3D-neuronal cell culture and 3D MEA systems for measuring such 3D-cultures. These requirements call for smart multilayer and packaging technology. The material Green Tape TM from DuPont Nemours is chosen for the presented works, because its compatibility and those of available metallisation with cell cultures is already proven. An LTCC multilayer circuit with gold electrodes is the base of the 3D MEA. The layout of the 3D MEA is designed to fit the MEA2100-System for in vitro recording from Multi Channel Systems and enable thus a comparable data processing to established 2D MEAs Slots. The surface topography of the thick film electrodes and the surface state is investigated with laser scanning microscopy, SEM, XPS and measurements of the wetting angle of contact. The impedance of the screen printed electrodes is discussed taking these data into account. Their impedance amounts to 24 kΩ and are falls thus below the impedance of commercially available electroplated gold electrodes of 30 kΩ. First promising results have been achieved using 3D MEAs for 2D culture of human pluripotent stem cell derived neural cells.

scholarly journals A Stepwise Protocol for Induction and Selection of Prominent Coniferous Cell Cultures for the Production of β-Thujaplicin

2015 ◽  
Vol 10 (5) ◽  
pp. 1934578X1501000
Author(s):  
Shinjiro Ogita ◽  
Masahito Shichiken ◽  
Chizuru Ito ◽  
Toshiyuki Yamashita ◽  
Taiji Nomura ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cell Cultures ◽  
High Performance ◽  
Laser Scanning ◽  
Bioactive Compound ◽  
Laser Scanning Microscopy ◽  
Induction Phase ◽  
Conifer Species ◽  
Biosynthetic Activity ◽  
Habituation Phase

In order to demonstrate the potential of plant cell culture systems to produce a target natural bioactive compound, we proposed a stepwise protocol for β-thujaplicin production as follows. 1. Induction phase: Characteristics of callus cultures originating from newly flushed shoots of 10 conifer species were evaluated on different basal media such as Murashige and Skoog (MS), Schenk and Hildebrandt (SH), and Lloyd and McCown's Woody Plant medium (WP) containing 10 μM 2,4-dichlorophenoxyacetic acid (2,4-D) either alone or in combination with 1 μM of N6-benzyladenine (BA). The conifer species used were as follows: Chamaecyparis ( C. obtusa Sieb. et Zucc. and C. pisifera Sieb. et Zucc), Juniperus (J. chinensis L. ‘Kaizuka', J. chinensis L. var. sargentii, and J. conferta Parlatore), Thuja (T. occidentals L. and T. standishii (Gord.) Carr.), Thujopsis (T. dolabrata Sieb. et Zucc. and T. dolabrata Sieb. et Zucc. var. hondae), and Cryptomeria (C. japonica D. Don). We observed the phenotypes of each callus to determine the optimal conditions for callus induction and to infer biosynthetic activity of the calli over 4–8 weeks. 2. Habituation phase: Each of the cell cultures obtained was transferred to a modified MS medium containing 680 mg L−1 KH2PO4 and 10 μM Picloram to select the habituated cells with synchronous growth pattern. The growth of each cell culture was highly improved in the habituation medium, except that of J. chinensis ‘Kaizuka'. 3. Metabolite-production phase: The concentration of β-thujaplicin (known as hinokitiol in Japan) in the shoots of donor trees and the habituated cell cultures was analyzed via high-performance liquid chromatography (HPLC). Histochemical characteristics of the cells were also observed using laser scanning microscopy (LSM) imaging. After the third step, we tested the biosynthetic activity of two habituated calli ( C. obtusa and J. conferta) on a 0.3%, w/v, yeast extract (YE)-containing medium. We found significant improvement in β-thujaplicin production in J. conferta callus (4600 μg g DW-1), which was up to 20-fold higher than in the habituation phase.

Metabolism of S-nitrosoglutathione by endothelial cells

2001 ◽  
Vol 281 (1) ◽  
pp. H432-H439 ◽  
Author(s):  
Hong Zeng ◽  
Netanya Y. Spencer ◽  
Neil Hogg
Keyword(s):  
Nitric Oxide ◽  
Cell Culture ◽  
Endothelial Cells ◽  
Buthionine Sulfoximine ◽  
Synthesis Inhibitor ◽  
Aortic Endothelial Cells ◽  
Absolute Dependence ◽  
Major Factors ◽  
Nitrite Nitrate ◽  
A Minor

S-nitrosoglutathione (GSNO) is an inhibitor of platelet aggregation and has also been shown to protect the ischemic heart from reperfusion-mediated injury. Although GSNO is often used in cell culture as a source of nitric oxide, the mechanisms of GSNO metabolism are not well established. We show here that GSNO decomposition by bovine aortic endothelial cells has an absolute dependence on the presence of cystine in the cell culture medium. In addition, GSNO decay is inhibited by diethyl maleate, an intracellular glutathione scavenger, but not by buthionine sulfoximine, a glutathione synthesis inhibitor. This indicates that thiols in general, rather than specifically glutathione, are the major factors that influence GSNO decay. Only 40% of the nitroso group of GSNO could be recovered as nitrite/nitrate, suggesting that the primary route of GSNO decay is reductive and that nitric oxide is only a minor product of GSNO decay. We conclude that the intracellular thiol pool causes the reduction of extracellular disulfides to thiols, which then directly reduce GSNO.

scholarly journals Development of an advanced diagnostic concept for intestinal inflammation: molecular visualisation of nitric oxide in macrophages by functional poly(lactic-co-glycolic acid) microspheres

2017 ◽  
Vol 8 ◽  
pp. 1637-1641
Author(s):  
Kathleen Lange ◽  
Christian Lautenschläger ◽  
Maria Wallert ◽  
Stefan Lorkowski ◽  
Andreas Stallmach ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Laser Scanning ◽  
Glycolic Acid ◽  
Intestinal Inflammation ◽  
Laser Scanning Microscopy ◽  
Hydrodynamic Diameter ◽  
New Approach ◽  
Early Evaluation ◽  
Griess Reaction ◽  
No Release

We here describe a new approach to visualise nitric oxide (NO) in living macrophages by fluorescent NO-sensitive microspheres based on poly(lactic-co-glycolic acid) (PLGA). PLGA microspheres loaded with NO550 dye were prepared through a modified solvent-evaporation method. Microparticles were characterized by a mean hydrodynamic diameter of 3000 nm, zeta potential of −26.000 ± 0.351 mV and a PDI of 0.828 ± 0.298. Under abiotic conditions, NO release was triggered through UV radiation (254 nm) of 10 mM sodium nitroprusside dehydrate (SNP). After incubation, AZO550 microspheres exhibited an about 8-fold increased emission at 550 nm compared to NO550 particles. For biotic NO release, RAW 264.7 murine macrophages were activated with lipopolysaccharide (LPS) of Salmonella typhimurium. After treatment with NO550 microparticles, only activated cells caused a green particle fluorescence and could be detected by laser scanning microscopy. NO release was confirmed indirectly with Griess reaction. Our functional NO550 particles enable a simple and early evaluation of inflammatory and immunological processes. Furthermore, our results on particle-based NO sensing and previous studies in targeting intestinal inflammation via (PLGA)-based microspheres demonstrate that an advanced concept for visualizing intestinal inflammation is tangible.

Synchronous detection of vascular tension and nitric oxide release in pulmonary artery: A combined application of confocal wire myograph with confocal laser scanning microscopy

Vascular ◽  
2020 ◽  
Vol 28 (5) ◽  
pp. 619-628
Author(s):  
Xiao-Ling Zhuang ◽  
Zhuang-Li Zhu ◽  
Jie-Ling Zhu ◽  
Su-Mei Lai ◽  
Long-Xin Gui ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Pulmonary Artery ◽  
Confocal Laser Scanning Microscopy ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Confocal Laser ◽  
Combined Application ◽  
M 10 ◽  
No Release

Objectives To detect the vascular tension and nitric oxide (NO) release synchronously in mice pulmonary artery, we perform two experiments and present a novel application of confocal wire myograph coupled with the confocal laser scanning microscopy. Methods In the first experiment, viable endothelium-intact mouse pulmonary artery (outer diameter 100–300 μM) rings underwent a one-hour preincubation with a NO-specific fluorescent dye, 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate Calbiochem (2.5 μM), and then precontracted with phenylephrine (Phen, 10−6 M), and subsequently dilated in acetylcholine (ACh, 10−6 M – 10−4 M). The endothelium-dependent vasorelaxation and NO generation in pulmonary artery rings were simultaneously recorded. In the second experiment, after 30-min incubation with the former NO fluorescent dye, the qualified pulmonary artery rings were co-incubated for another 30 min with a nitric oxide synthase inhibitor, 10−4 M Nω-nitro-L-arginine-methyl-ester (L-NAME), and then pretreated with Phen (10−6 M) followed by ACh (10−5 M). The Ach-induced vasodilation and NO release were recorded simultaneously. Results ACh (10−6 M – 10−4 M) promoted pulmonary artery relaxation and intracellular NO release in a dose-dependent manner. Additionally, L-NAME (10−4 M) significantly attenuated the vasodilatation and the intracellular NO release. Conclusions This combined application visually confirms that the synchronous changes in Ach induced vasodilation and NO release, which provides a new method for cardiovascular research.

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