scholarly journals Direct Determination of Glutathione Reductase in Cells Cultured in Microtitre Plates as a Biomarker for Oxidative Stress

1995 ◽  
Vol 23 (4) ◽  
pp. 531-538
Author(s):  
Concepción García-Alfonso ◽  
Pilar Sanz ◽  
Guillermo Repetto ◽  
Manuel Repetto ◽  
Juan López-Barea
Keyword(s):  
Oxidative Stress ◽  
Glutathione Reductase ◽  
Potassium Phosphate ◽  
Direct Determination ◽  
Vero Cells ◽  
Control Activity ◽  
Wide Range ◽  
Cells Cultured ◽  
In Cells

— A new method was developed for the direct determination of glutathione reductase (GOR) activity in Vero cells cultured in microtitre plates, avoiding cell-free extract preparation. The cells in each well were washed twice with phosphate-buffered saline, lysed with Triton X-100, and assayed in 0.1M potassium phosphate, pH 7.0. After subtracting oxidase activity, which increased with NADPH concentration, the net GOR activity was similar at different oxidised glutathione (GSSG) and NADPH concentrations, thus confirming enzyme saturation. The optimised GOR assay used 2.5mM GSSG and 0.12mM NADPH; 5mM EDTA was also added to prevent the enzyme from redox inactivation. The GOR activity was directly proportional to the number of cells per well for a wide range of cell densities, thus supporting the assay's validity for use with cultured cells. The effects on GOR activity of three chemicals which induce oxidative stress, namely, paraquat, iron (II) chloride and iron (III) chloride, were examined to validate the assay under experimental conditions. The specific enzymatic activity increased to 357% of untreated control activity in 5mM paraquat-treated cells, and to 407% of control activity in cells exposed to 7.5mM iron (II) chloride. By contrast, activity decreased to 56% of control activity in cells exposed to 5mM iron (III) chloride. In conclusion, the changes in GOR activity detected in Vero cells confirm that the new assay is suitable for routine in vitro screening of toxicants capable of inducing oxidative stress.

scholarly journals Direct Determination of Glutathione S-transferase and Glucose-6-phosphate Dehydrogenase Activities in Cells Cultured in Microtitre Plates as Biomarkers for Oxidative Stress

1998 ◽  
Vol 26 (3) ◽  
pp. 321-330 ◽  
Author(s):  
Concepción García-Alfonso ◽  
Guillermo Repetto ◽  
Pilar Sanz ◽  
Manuel Repetto ◽  
Juan López-Barea
Keyword(s):  
Oxidative Stress ◽  
Direct Determination ◽  
Cell Number ◽  
Glutathione S Transferase ◽  
Enzymatic Assays ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Gst Activity ◽  
In Cells

The enzymes glutathione S-transferase (GST) and glucose-6-phosphate dehydrogenase (G-6PDH) are implicated in the defence against oxidative stress. GST is mainly involved in the conjugation of electrophilic compounds with glutathione (GSH), although some of its isoenzymes display peroxidase activity. G-6PDH and glutathione reductase regenerate NADPH and GSH, respectively, to restore the reduced intracellular redox status following oxidative stress. Enzymatic assays for GST and G-6PDH were adapted and optimised to permit the direct in vitro determination of the effects of toxicants which induce oxidative stress in cells on microtitre plates, thereby avoiding the need to prepare cell-free extracts. To optimise the conditions of the enzymatic assays, GST activity’ was measured at substrate concentrations of 1–3mM GSH and 1–3mM 1-chloro-2,4-dinitrobenzene, while G-6PDH activity was measured at 7.5–37.5mM glucose-6-phosphate and 55–275mM NADP. Both enzymatic activities were directly proportional to cell number up to a density of 1 x 105 cells/well. The effects on GST and G-6PDH activities of three toxicants which induce oxidative stress — paraquat, iron (II) chloride and iron (III) chloride — were compared in cultured Vero cells to validate the new assays. Specific GST activity increased to 145% and 171% compared to the controls in cells treated with 5mM paraquat and 5mM iron (II) chloride, respectively, but was inhibited after exposure to 25mM iron (III) chloride. Specific G-6PDH activity increased to 136% compared to the control after exposure to 5mM paraquat, but was inhibited in cells exposed to 5mM iron (II) chloride and 25mM iron (III) chloride.

scholarly journals A new method for effective dose calculation based on the ambient dose height distribution

2017 ◽  
Vol 3 (2) ◽  
pp. 147-149
Author(s):  
Mario Liebmann ◽  
Niroojiny Sangarapillai ◽  
Björn Poppe ◽  
Heiner von Boetticher
Keyword(s):  
Effective Dose ◽  
Direct Determination ◽  
Dose Calculation ◽  
Gender Effects ◽  
Height Distribution ◽  
Wide Range ◽  
Personal Dosimetry ◽  
And Gender ◽  
Ambient Dose

AbstractThe realistic determination of effective dose of the staff in diagnostic radiology has been a challenge both for personal dosimetry and ambient dose measurement. A model for dosimetry of occupational exposure is presented that allows direct determination of effective dose from measured or even manufacturer given ambient dose distribution in front of the personnel. This model considers a wide range of radiation energies, different radiation protection situations, and gender effects.

scholarly journals Direct Determination of Phosphatase Activity from Physiological Substrates in Cells

PLoS ONE ◽  
2015 ◽  
Vol 10 (3) ◽  
pp. e0120087 ◽  
Author(s):  
Zhongyuan Ren ◽  
Le Duy Do ◽  
Géraldine Bechkoff ◽  
Saida Mebarek ◽  
Nermin Keloglu ◽  
...  
Keyword(s):  
Phosphatase Activity ◽  
Direct Determination ◽  
Physiological Substrates ◽  
In Cells

Elemental Speciation Measurements with Electrospray Mass Spectrometry: An Assessment

1994 ◽  
Vol 48 (11) ◽  
pp. 1347-1359 ◽  
Author(s):  
George R. Agnes ◽  
Ian I. Stewart ◽  
Gary Horlick
Keyword(s):  
Mass Spectrometry ◽  
Electrospray Mass Spectrometry ◽  
Molecular Form ◽  
Direct Determination ◽  
Organometallic Compounds ◽  
Ionic Species ◽  
Elemental Speciation ◽  
Wide Range ◽  
Inorganic Complexes

Electrospray mass spectrometry (ES-MS) is shown to be suited for the direct determination of ionic species in solution. Examples used in this investigation of ES-MS for elemental speciation determinations included several inorganic ionic species of chlorine, iodine, sulfur, and iron. These species were differentiated on the basis of differences in the molecular form and/or the oxidation state of the analyte ion. The inorganic complexes of silver-cyanide and iron-fluoride were also measured in this assessment. Finally it is shown that ES-MS is applicable to the determination of organometallic compounds. While ES-MS is not an ideal speciation technique, there is considerable evidence to indicate that ES-MS can become an invaluable tool with which species distributions in a wide range of sample types may be probed.

scholarly journals Evaluation of the P-solubilizing activity of soil microorganisms and its sensitivity to soluble phosphate

2011 ◽  
Vol 48 (No. 9) ◽  
pp. 397-400 ◽  
Author(s):  
O. Mikanová ◽  
J. Nováková
Keyword(s):  
Tricalcium Phosphate ◽  
Agricultural Soils ◽  
Potassium Phosphate ◽  
Direct Determination ◽  
Soluble Phosphate ◽  
Natural Ecosystems ◽  
High Concentrations ◽  
Filter Papers ◽  
Solubilizing Activity

Microbial solubilization of hardly soluble mineral phosphates in soil is an important process in natural ecosystems and in agricultural soils. Regulation of the P-solubilizing activity by the presence of soluble phosphates in medium was determined. For this reason we decided to test a&nbsp;number of soil bacteria showing a&nbsp;high P-solubilizing activity for its sensitivity to the presence of soluble dihydrogen potassium phosphate in medium. At these studies, the direct determination of the solubilized phosphate in medium was masked by the presence of relatively high concentrations of soluble phosphate added. Therefore, we have modified the method, determining the residual tricalcium phosphate. The effect of soluble phosphate in medium on the P-solubilizing activity of rhizosphere isolates and strains of Rhizobium were tested in liquid cultures with the addition of various concentrations of soluble KH<sub>2</sub>PO<sub>4</sub>. The medium was filtered after incubation and the remaining tricalcium phosphate was separated by filtration. Filter papers with the remaining tricalcium phosphate were hydrolysed with 2N H2SO4. Phosphorus was determined spectrophotometrically. The P-solubilizing activity was expressed as a&nbsp;difference between the tricalcium phosphate added and its remainder after the incubation. These results fully confirmed that there exist the strains, whose P-solubilizing activity is inhibited and other strains, whose P-solubilizing activity is not inhibited or is inhibited very little in the presence of soluble phosphate. The use of our adapted method was much more suitable for this type of experiments.

Apical and basolateral uptake and intracellular fate of dopamine precursor l-dopa in LLC-PK1 cells

1998 ◽  
Vol 274 (2) ◽  
pp. F243-F251 ◽  
Author(s):  
P. Soares-Da-Silva ◽  
M. P. Serrão ◽  
M. A. Vieira-Coelho
Keyword(s):  
Nonlinear Analysis ◽  
Basal Cell ◽  
Acid Decarboxylase ◽  
Maximal Velocity ◽  
Gbr 12909 ◽  
Apical Side ◽  
Wide Range ◽  
Cells Cultured ◽  
In Cells

The present study was aimed at the uptake ofl-3,4-dihydroxyphenylalanine (l-dopa) and its intracellular decarboxylation to dopamine. The accumulation ofl-dopa from the apical side in cells cultured in collagen-treated plastic was found to be a saturable process with a Michaelis constant ( K m) of 123 ± 17 μM and a maximal velocity ( V max) of 6.0 ± 0.2 nmol ⋅ mg protein−1 ⋅ 6 min−1. The uptake ofl-dopa applied from either the apical or basal cell borders in cells cultured in polycarbonate filters was also found to be saturable; nonlinear analysis of saturation curves for apical and basal application revealed K m values of 63.8 ± 17.0 and 42.5 ± 9.6 μM and V maxvalues of 32.0 ± 5.8 and 26.2 ± 3.4 nmol ⋅ mg protein−1 ⋅ 6 min−1, respectively. Cell monolayers incubated withl-dopa, applied from either the apical or the basal side, in the absence of benserazide, led to the accumulation of newly formed dopamine. The intracellular accumulation of newly formed dopamine was a saturable process with apparent K m values of 20.5 ± 8.2 and 247.3 ± 76.8 μM when the substrate was applied from the apical and basal side, respectively. Some of the newly formed dopamine escaped to the extracellular milieu. The basal outward transfer of dopamine was five- to sevenfold of that occurring at the apical side and was uniform over a wide range of concentrations of intracellular dopamine; the apical outward transfer of the amine depended on the intracellular concentration of dopamine and was a nonsaturable process. The apical and basal outward transfers of dopamine were insensitive to cocaine (10 and 30 μM) and GBR-12909 (1 and 3 μM). The accumulation of exogenous dopamine in LLC-PK1 cells was found to be saturable; nonlinear analysis of the saturation curves revealed for the apical and basal application of dopamine a K m of 17.7 ± 4.3 and 96.0 ± 28.1 μM and a V max of 2.0 ± 0.1 and 2.2 ± 0.3 nmol ⋅ mg protein−1 ⋅ 6 min−1, respectively. However, both cocaine (10, 30, or 100 μM) and GBR-12909 (1 or 3 μM) were found not to affect the uptake of 100 μM dopamine applied from either the apical or the basal cell border. In conclusion, the data presented here show that LLC-PK1cells are endowed with considerable aromaticl-amino acid decarboxylase (AADC) activity and transportl-dopa quite efficiently through both the apical and basal cell borders. On the other hand, our observations support the possibility of a basal-to-apical gradient of AADC activity and the possibility that LLC-PK1 cells might constitute an interesting in vitro model for the study of the renal dopaminergic physiology.

Improved Methods for the Prediction of Chatter in Turning, Part 2: Determination of Cutting Process Parameters

1990 ◽  
Vol 112 (1) ◽  
pp. 21-27 ◽  
Author(s):  
I. E. Minis ◽  
E. B. Magrab ◽  
I. O. Pandelidis
Keyword(s):  
Cutting Force ◽  
Metal Cutting ◽  
Transfer Functions ◽  
Direct Determination ◽  
Base Plate ◽  
Cutting Process ◽  
Input Output ◽  
Wide Range ◽  
Improved Methods

The dynamics of the metal cutting process are identified by a new and simple experimental method that permits the direct determination of the cutting transfer functions from input-output measurements. A specially designed stiff force dynamometer rigidly mounted to the machine tool’s turret is used for the measurement of the cutting force. An external force is applied to the base plate of the dynamometer in such a way that the meaurements of both the tool’s displacement and the corresponding cutting force are uncoupled from the influence of the machine tool structure’s dynamics. The cutting transfer functions are obtained in the vicinity of the structure’s first resonance, under a wide range of cutting conditions.

Virtual Compound Screening and Molecular Dynamics to Identify New Inhibitors for Human Glutathione Reductase

2020 ◽  
Vol 17 (12) ◽  
pp. 1465-1474
Author(s):  
Mohsen Sargolzaei
Keyword(s):  
Oxidative Stress ◽  
Molecular Dynamics ◽  
Free Energy ◽  
Glutathione Reductase ◽  
Binding Free Energy ◽  
Rational Drug Design ◽  
Dynamics Simulation ◽  
Reductase Inhibitors ◽  
Free Energy Decomposition

Background: Oxidative stress is a defense mechanism against malarial intracellular parasite infection. On the other hand, the Human glutathione reductase enzyme reduces oxidative stress in the cells, making the inhibitors of this enzyme a promising candidate for malaria treatment. Objective: Rational drug design was used in this work to plan new human glutathione reductase inhibitors. Methods: Virtual screening was performed using the ZINC database and molecular docking was used to detect appropriate human glutathione reductase inhibitors. Based on the docking scores obtained, the top three highest-ranked ligands were selected for the molecular dynamics simulation study. The MD simulation was performed for each complex in a length of 100 ns. Results: RMSD, RMSF and hydrogen bond analyzes were performed on the derived trajectories. Molecular mechanics generalized born surface area (MM-GBSA) and pairwise per-residue free energy decomposition analyzes were performed for the determination of binding free energy and the determination of dominant residues involved in the binding process, respectively. The binding free energy analysis showed that the molecule of 3-((7-(furan-2-ylmethyl)-5,6-diphenyl-7H-pyrrolo[2,3- d] pyrimidin-4-yl) amino) propan-1-ol is the most potent inhibitor among the molecules considered against human glutathione reductase enzyme. Conclusion: This molecule can be considered a novel candidate for antimalarial treatments.

Extraction concentrating of the acid-soluble forms of rare earth elements from oils of the Volga-Ural oil and gas province using rotating coiled columns

2021 ◽  
Vol 87 (12) ◽  
pp. 5-10
Author(s):  
E. Yu. Savonina ◽  
O. N. Katasonova ◽  
T. A. Maryutina
Keyword(s):  
Trace Elements ◽  
Rare Earth ◽  
Rare Earth Elements ◽  
Inductively Coupled Plasma ◽  
Oil And Gas ◽  
Direct Determination ◽  
Soluble Form ◽  
Rotating Coiled Columns ◽  
Wide Range

Trace elements in oils are the natural markers of the oil origin of and the mechanism of oil genesis. Direct determination of rare earth elements (REE) in crude oils is rather difficult due to their low concentrations (10–6 wt. %). Information about the forms of trace elements in oils is of particular interest. The goal of the study is determination of the content of acid-soluble forms of REE in oils from various fields of the Volga-Ural oil and gas province. Six samples of oils of heavy and medium density and viscosity were analyzed. Extraction of acid-soluble forms of REE was carried out using rotating coiled columns in 0.5 M aqueous solution of nitric acid. Toluene was used to dilute the oils up to the necessary values of the density and viscosity providing the possibility of extraction in the system aqueous acid solution – oil. The content of REE was determined by inductively coupled plasma mass spectrometry on an Agilent 7900 spectrometer. Their content in an acid-soluble form in oil samples under study ranged within 172 – 2173 ng/kg. Such a wide range of values is attributed to the difference in the geology and age of deposit formation. The highest content of acid-soluble forms of REE (2173 ng/kg) was observed in the oil sample from the Novo-Elkhovsky field, whereas the lowest value (172 ng/kg) was measured in the oil sample of the Pioneer field. It is shown that the content of acid-soluble forms of REE depends on tectonic zoning, productive horizons and layers, as well as on the physical and chemical properties of oils. The distribution of acid-soluble forms of light and heavy REE was evaluated. Most of the REE (75 – 99%) determined in the analyzed oil samples are light REE. It is noted that systematization of the data on the properties of oils, geological and geochemical characteristics of the deposits will provide reliable forecasting of the various forms of REE present in oils.

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