scholarly journals A Simple Method for Evaluation of Superoxide Radical Production in Neural Cells under Various Culture Conditions: Application to Hypoxia

1995 ◽  
Vol 15 (1) ◽  
pp. 71-77 ◽  
Author(s):  
Jean-Luc Daval ◽  
Jean-François Ghersi-Egea ◽  
Jean Oillet ◽  
Violette Koziel
Keyword(s):  
Cytochrome C ◽  
Superoxide Radical ◽  
Cell Injury ◽  
Acute Hypoxia ◽  
Culture Conditions ◽  
Superoxide Radicals ◽  
Extracellular Medium ◽  
Simple Method ◽  
In Cells

To evaluate the potential deleterious influence of oxygen-derived free radicals following hypoxia in a model of primary culture of neurons obtained from the fetal rat brain, superoxide radicals were measured as a function of time in the extracellular medium. Neuronal cells were grown for 8 days in the presence or absence of serum, then incubated in a buffered Krebs–Ringer solution containing 60 μ M acetyl-cytochrome c. The rate of superoxide radical formation was quantified spectrophotometrically by measuring the specific reduction of acetyl-cytochrome c. Under normoxic conditions (95% air-5% CO2), basal production of superoxide that increased with time was recorded. It was significantly more pronounced in cells grown in serum-free medium. Under both culture conditions, acute hypoxia (95% N2–5% CO2) for 6 h increased superoxide radical amounts in the extracellular medium, and they were still enhanced 3 h after reoxygenation. The addition of superoxide dismutase to the incubating medium abolished the detection of superoxide radicals. The present study describes a new reliable method for superoxide radical measurement in cells in vitro and demonstrates hypoxia/reoxygenation-induced overproduction of superoxide in cultured neurons that may account for cell injury.

scholarly journals EXPERIMENTAL STUDIES UPON LYMPHOCYTES

1917 ◽  
Vol 25 (5) ◽  
pp. 633-650 ◽  
Author(s):  
Alwin M. Pappenheimer
Keyword(s):  
Cell Injury ◽  
Experimental Studies ◽  
Gum Arabic ◽  
Sudden Increase ◽  
Human Tonsil ◽  
Colloidal Iron ◽  
Simple Method ◽  
Thymus Cells ◽  
Phosphate Solutions

A simple method is presented by which, with the diffusion of trypan blue into the nucleus as a criterion of cell injury, it is possible to study quantitatively the effect of various agencies upon the small thymus cells and upon the tissue lymphocytes. Preliminary studies with this method have led us to the following conclusions, which, however, unless otherwise stated, may be taken as applying only to the lymphocytes of the rat thymus. 1. The small thymus cells, when suspended in balanced phosphate solutions, show no distinct reaction to variations in hydrogen ion concentrations ranging between Ph 7.0 and PH 7.8. Beyond PH 7.0 there is a sudden increase in the permeability of the cells to the dye; plasmolysis of the cells occurs when the alkalinity exceeds PH 8.0. 2. Heating to 49° or 50°C. is accompanied by a critical increase in the permeability of the cells to the dye. 3. The injury caused by lack of oxygen can be demonstrated by the increase in the number of stained cells. 4. The addition of serum to suspensions of thymus cells or tonsil lymphocytes greatly inhibits the diffusion of the trypan into the cells. The protection afforded is roughly proportionate to the amount of serum added. Gelatin also exerts a marked protective influence; egg albumin affords a partial protection; starch and gum arabic are inert. Hemoglobin and cholesterol do not modify the stainability of the cells. Arsenious sulfide in weak concentrations partially inhibits the diffusion of the dye. Colloidal iron is without effect, and is precipitated about the cells. 5. The toxicity of the photodynamic substance, hematoporphyrin, and of an impure chlorophyll solution in the presence of sunlight could be strikingly demonstrated by the greatly increased permeability of the cells to the stain. 6. Acute and chronic inanition produces an increased fragility of the cells. The protective power of the serum in acute starvation appears to be increased. 7. The small thymus cells of old animals are more readily injured than are those of young ones, as indicated by the increased proportion of stained cells. 8. The method has been applied to the demonstration of the action of cytotoxic immune sera for rat thymus cells and for human tonsil lymphocytes in vitro. Further experiments dealing with the question of specificity are in progress. The cytotoxins are inactivated by the addition of complement. Thermostabile cytagglutinins have also been produced.

Alteration of activation energy of reaction in the catalase of human erythrocyte by cimetidine, an in vitro thermokinetics study

2020 ◽  
Vol 09 ◽  
Author(s):  
Dariush Minai-Tehrani
Keyword(s):  
Activation Energy ◽  
Human Erythrocyte ◽  
Cell Injury ◽  
Gastrointestinal Diseases ◽  
Maximum Activity ◽  
The Body ◽  
Effect Of Temperature ◽  
Histamine H2 Receptor ◽  
In Cells

Background: Hydrogen peroxide is normally formed during the metabolic pathway of the body. It is a toxic compound for vital cells, which can oxidize many macromolecules and cause damage in cells. Catalase can degrade H2O2 in cells and prevent cell injury. Cimetidine is a histamine H2 receptor blocker which decreases the release of stomach acid and is used for gastrointestinal diseases. Cimetidine inhibited catalase by mixed inhibition. Objective: In this study, effect of temperature on the binding of cimetidine to human erythrocyte catalase was investigated and kinetic factors of the binding were determined. Results: Dixon plot confirmed the mixed type of inhibition and determined the Ki of the drug. Maximum activity of the enzyme was observed at 30oC. Arrhenius plot demonstrated that the activation energy of the enzyme reaction in the absence and presence of cimetidine was about 4.7 and 8.13 kJ/mol, respectively. Temperature coefficient (Q30-40) was determined as about 1.11 and 1.09 in the absence and presence of cimetidine. Conclusion: Cimetidine was able to increase the activation energy of the reaction of catalase, which confirmed the inhibition of the enzyme based on the kinetic results.

No effect of superoxide dismutase on spontaneous development of diabetes in db/db mice

1988 ◽  
Vol 117 (1) ◽  
pp. 99-102
Author(s):  
Ove Berglund ◽  
Kjell Grankvist ◽  
Carina Albiin ◽  
Stefan L. Marklund
Keyword(s):  
Superoxide Dismutase ◽  
B Cells ◽  
Superoxide Radical ◽  
Superoxide Radicals ◽  
Cuzn Superoxide Dismutase

Abstract. B-cells have previously been shown to be very susceptible to damage induced by superoxide radicals, and protection against such damage has been achieved both in vitro and in vivo with superoxide dismutase. During maturation, db/db mice develop diabetes and accumulation of potentially superoxide radical-producing leucocytes can be demonstrated in the islets during the process. To test for the possibility that superoxide radical-induced damage contributes to the development of diabetes, db/db mice were given daily ip injections of 200 mg/kg polyethylene glycolsubstituted CuZn superoxide dismutase. No effect of the treatment could be demonstrated.

Aldosterone release from adrenal cells is inhibited by reduced oxygen levels in vitro during maturation in rabbits

1996 ◽  
Vol 8 (8) ◽  
pp. 1131 ◽  
Author(s):  
PE Papanek ◽  
BM Jankowski ◽  
H Raff
Keyword(s):  
Acute Hypoxia ◽  
Specific Effect ◽  
Adrenocorticotrophic Hormone ◽  
Adrenal Cells ◽  
New Zealand White Rabbits ◽  
Aldosterone Production ◽  
Different Levels ◽  
In Cells

Hypoxia in vivo leads to a decrease in aldosterone not completely explained by extrinsic controllers of adrenal function including adrenocorticotrophic hormone, renin-angiotensin II, and K+. The dissociation of renin and aldosterone during acute hypoxia in vivo may be explained by the finding that aldosterone synthesis in adrenal cells is reversibly and specifically inhibited by decreases in O2 levels within the physiological range. The present study investigated whether the direct effect of acute decreases in O2 levels on aldosteronogenic pathway is altered during maturation. Adrenal cells (whole adrenals) were prepared from fetal (27 days gestation), neonatal (1 day), and infant (10 days) New Zealand White rabbits, and capsular cells were prepared from young (21 days) and adult (3 months) rabbits. All cells were dispersed with collagenase. Basal and cAMP-stimulated aldosterone production were assessed under two different levels of O2 (pO2 = 20.0 kPa or pO2 = 8.7 kPa). Decreased O2 levels significantly inhibited cAMP-stimulated aldosterone production in cells obtained from rabbits of all ages by 60 +/- 5% cAMP-stimulated aldosterone production was significantly lower in cells obtained from neonates and premature animals under both normoxic and reduced O2 conditions as compared with animals > or = 10 days old. Corticosterone production by cells obtained from adults and 21-day-old rabbits was unaffected by reduced O2 conditions suggesting a specific effect on the aldosterone pathway. The data demonstrate that the O2 sensitivity of the aldosterone pathway is present throughout development.

A novel method to determine specificity and sensitivity of the TUNEL reaction in the quantitation of apoptosis

2003 ◽  
Vol 284 (5) ◽  
pp. C1309-C1318 ◽  
Author(s):  
K. J. Kelly ◽  
Ruben M. Sandoval ◽  
Kenneth W. Dunn ◽  
Bruce A. Molitoris ◽  
Pierre C. Dagher
Keyword(s):  
Cell Injury ◽  
Tunel Assay ◽  
Live Cells ◽  
Tunel Staining ◽  
Experimental Conditions ◽  
Simple Method ◽  
Specificity And Sensitivity ◽  
Tunel Reaction

Apoptosis is an important mode of cell death under both physiological and pathophysiological conditions. Numerous techniques are available for the study and quantitation of apoptosis in cell culture, but only few are useful when applied to complex tissues. Among these, the terminal transferase-mediated dUTP nick end-labeling (TUNEL) assay remains the most widely used technique. However, its specificity and sensitivity for the detection of apoptosis remain controversial. We developed a technique consisting of staining live cells and tissues with Hoechst 33342 and the vital dye propidium iodide (PI), followed by fixation and the TUNEL reaction. We demonstrate excellent retention of PI in necrotic cells after fixation. We also examined the distribution of TUNEL staining among necrotic and apoptotic cells in various models of cell injury in vitro and in vivo. We show that the sensitivity of the TUNEL varied between 61 and 90% in the models examined. The specificity exceeded 87% in all models but fell to 70% when a predominantly necrotic injury was induced. This novel and simple method will permit the determination of indices of sensitivity and specificity for the TUNEL assay in other tissues and experimental conditions.

Effect of Some Psychotropic Drugs on Luminol - Dependent Chemiluminescence Induced by O2–, •OH, HOCl

2002 ◽  
Vol 57 (11-12) ◽  
pp. 1066-1071 ◽  
Author(s):  
Vera Hadjimitova ◽  
Trayko Traykov ◽  
Milka Mileva ◽  
Stefan Ribarov
Keyword(s):  
Antioxidant Activity ◽  
Hydroxyl Radical ◽  
Psychotropic Drugs ◽  
Superoxide Radical ◽  
Hypochlorous Acid ◽  
Superoxide Radicals ◽  
Scavenge Activity ◽  
Biologically Relevant

We studied antioxidant activity of six neuroleptics (chlorpromazine, levomepromazine, promethazine, trifluoperazine and thioridazine) and two antidepressants (imipramine and amitriptyline) in the range of concentration of 10−7−10−4 м. We applied luminol-dependent chemiluminescence to test the ability of these drugs to scavenge the biologically relevant oxygen-derived species: hydroxyl radical, superoxide radical, hypochlorous acid in vitro. We found that the phenothiazines were powerful scavengers of hydroxyl and superoxide radicals. Chlorprothixene, amitriptyline and imipramine had no scavenge activity to the superoxide radical. All drugs showed a moderate scavenger effect on hypochloric anion.

scholarly journals Micromolecular inhibitors of superoxide radicals

2021 ◽  
Vol 64 (6) ◽  
pp. 5-9
Author(s):  
Lilia Andronache ◽  
◽  
Valeriana Pantea ◽  
Aurelian Gulea ◽  
Inna Svet ◽  
...  
Keyword(s):  
Biological Activity ◽  
Coordination Compounds ◽  
Superoxide Radical ◽  
Treatment Strategies ◽  
Superoxide Radicals ◽  
In Vitro Experiments ◽  
High Biological Activity ◽  
Acid Ligand ◽  
New Treatment

Background: Currently, there is a growing interest in new copper (Cu2+) heterocyclic coordination compounds (CC), isothiosemicarbazide derivates, which demonstrated multiple beneficial properties, but their effect on reactions with free radicals such as the superoxide radical has not been investigated. Material and methods: The action of new micromolecular complexes of copper (Cu2+) chloride and bromide with methyl n- (prop-2-en-1-yl) -2- (pyridin2-ylmethylidene) hydrazine carbimidothioate on capturing activity of the superoxide radical was determined by the spectrophotometric method in vitro experiments. Results: It was established that the micromolecular complexes of copper (II) chloride and bromide with methyl n-(prop-2-en-1-yl)-2-(pyridin-2- ylmethylidene) hydrazine carbimidothioate have been found to possess strong superoxide radical inhibitor properties when interacting with a superoxide radical. In addition to this, the IC50 of the studied compounds depends on the nature of the acid-ligand in the internal sphere of the complex and increases in the following sequence: Cl- –Br- . Conclusions: The established property of mentioned compounds is new, because their use as micromolecular inhibitors of superoxide radicals has not been described so far. The synthesized CC expand the arsenal of superoxide radical inhibitors with high biological activity. Their possible significance for the development of new treatment strategies for diseases associated with the overproduction of superoxide radicals is discussed.

scholarly journals Fe(III) heme sets an activation threshold for processing distinct groups of pri-miRNAs in mammalian cells

2020 ◽  
Author(s):  
Sara H. Weitz ◽  
Jen Quick-Cleveland ◽  
Jose P. Jacob ◽  
Ian Barr ◽  
Rachel Senturia ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Rna Binding ◽  
Biological Significance ◽  
Culture Conditions ◽  
Sequencing Data ◽  
Mirna Sequencing ◽  
Typical Cell ◽  
Genomic Scale ◽  
In Cells

ABSTRACTThe essential biological cofactor heme is synthesized in cells in the Fe(II) form. Oxidized Fe(III) heme is specifically required for processing primary transcripts of microRNAs (pri-miRNAs) by the RNA-binding protein DGCR8, a core component of the Microprocessor complex. It is unknown how readily available Fe(III) heme is in the largely reducing environment in human cells and how changes in cellular Fe(III) heme availability alter microRNA (miRNA) expression. Here we address the first question by characterizing DGCR8 mutants with various degrees of deficiency in heme-binding. We observed a strikingly simple correlation between Fe(III) heme affinity in vitro and the Microprocessor activity in HeLa cells, with the heme affinity threshold for activation estimated to be between 0.6-5 pM under typical cell culture conditions. The threshold is strongly influenced by cellular heme synthesis and uptake. We suggest that the threshold reflects a labile Fe(III) heme pool in cells. Based on our understanding of DGCR8 mutants, we reanalyzed recently reported miRNA sequencing data and conclude that heme is generally required for processing canonical pri-miRNAs, that heme modulates the specificity of Microprocessor, and that cellular heme level and differential DGCR8 heme occupancy alter the expression of distinct groups of miRNAs in a hierarchical fashion. Overall, our study provides the first glimpse of a labile Fe(III) heme pool important for a fundamental physiological function and reveal principles governing how Fe(III) heme modulates miRNA maturation at a genomic scale. We also discuss potential states and biological significance of the labile Fe(III) heme pool.

Neutrophil migration through in vitro interstitial matrix

1992 ◽  
Vol 50 (1) ◽  
pp. 894-895
Author(s):  
J. Roemer ◽  
S.R. Simon
Keyword(s):  
Extracellular Matrix ◽  
Smooth Muscle ◽  
Cell Migration ◽  
Smooth Muscle Cells ◽  
Inflammatory Cell ◽  
Neutrophil Migration ◽  
Culture Conditions ◽  
Aortic Smooth Muscle ◽  
Specific Culture

We are developing an in vitro interstitial extracellular matrix (ECM) system for study of inflammatory cell migration. Falcon brand Cyclopore membrane inserts of various pore sizes are used as a support substrate for production of ECM by R22 rat aortic smooth muscle cells. Under specific culture conditions these cells produce a highly insoluble matrix consisting of typical interstitial ECM components, i.e.: types I and III collagen, elastin, proteoglycans and fibronectin.

Superoxide, Xanthine Oxidase and Platelet Reactions: Further Studies on Mechanisms by which Oxidants Influence Platelets

1981 ◽  
Vol 45 (03) ◽  
pp. 290-293 ◽  
Author(s):  
Peter H Levine ◽  
Danielle G Sladdin ◽  
Norman I Krinsky
Keyword(s):  
Superoxide Dismutase ◽  
Cytochrome C ◽  
Xanthine Oxidase ◽  
Direct Effect ◽  
Platelet Function ◽  
Experimental Conditions ◽  
Release Reaction

SummaryIn the course of studying the effects on platelets of the oxidant species superoxide (O- 2), Of was generated by the interaction of xanthine oxidase plus xanthine. Surprisingly, gel-filtered platelets, when exposed to xanthine oxidase in the absence of xanthine substrate, were found to generate superoxide (O- 2), as determined by the reduction of added cytochrome c and by the inhibition of this reduction in the presence of superoxide dismutase.In addition to generating Of, the xanthine oxidase-treated platelets display both aggregation and evidence of the release reaction. This xanthine oxidase induced aggreagtion is not inhibited by the addition of either superoxide dismutase or cytochrome c, suggesting that it is due to either a further metabolite of O- 2, or that O- 2 itself exerts no important direct effect on platelet function under these experimental conditions. The ability of Of to modulate platelet reactions in vivo or in vitro remains in doubt, and xanthine oxidase is an unsuitable source of O- 2 in platelet studies because of its own effects on platelets.

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