scholarly journals A cytochemical stain for glutathione in rat hepatocytes cultured on plastic.

1987 ◽  
Vol 35 (2) ◽  
pp. 271-274 ◽  
Author(s):  
A Larrauri ◽  
P López ◽  
M J Gómez-Lechón ◽  
J V Castell
Keyword(s):  
Azo Dye ◽  
Room Temperature ◽  
Cultured Cells ◽  
Rat Hepatocytes ◽  
Thiol Groups ◽  
Cytochemical Localization ◽  
Sh Groups ◽  
Acetone Water ◽  
Mercury Orange ◽  
Orange Fluorescence

Thiol groups of glutathione react with the organomercurial azo dye mercury orange at a faster rate than with -SH groups of proteins. This property makes possible visualization of glutathione in cells without appreciable interference from other -SH groups. To render this method useful for cytochemical localization of glutathione in plastic cultured cells, it was necessary to adapt this reaction to the specific characteristics of the biological samples to be assayed. First, the choice of a solvent that would allow a convenient solubility of the dye and at the same time be compatible with the plastic culture plate was crucial. Second, to avoid diffusion of glutathione out of the cell the procedure for staining cells was also important. Satisfactory results were obtained after 30-40 sec reaction with 50 microM mercury orange in acetone/water 9:1, v/v, at room temperature. Glutathione-mercury orange complexes exhibited orange fluorescence on excitation with blue light. No diffusion of glutathione out of the cells was observed, and the hepatocytes stained with the dye showed orange fluorescence which paralleled their glutathione content.

A Novel Alkali-Metal Hydrido-tris(pyrazolyl)borate (Tp*) Complex. Isolation and Crystal Structure of [(Me2CO)3(NaTp*)2]

2014 ◽  
Vol 69 (7) ◽  
pp. 793-798
Author(s):  
Laurent Plasseraud ◽  
Hélène Cattey
Keyword(s):  
Crystal Structure ◽  
Alkali Metal ◽  
Sodium Hydroxide ◽  
Title Compound ◽  
Room Temperature ◽  
Crystallographic Analysis ◽  
Monoclinic Space Group ◽  
Large Excess ◽  
X Ray ◽  
Acetone Water

The title compound was isolated from the treatment of Tp*Sn(Cl)2Bu (1) with a large excess of sodium hydroxide in a mixture of acetone-water at room temperature. [(Me2CO)3(NaTp*)2] (2) crystallizes at 4 °C as prismatic colorless crystals, in the monoclinic space group P21/c with Z = 4, a = 12.2837(6), b = 24.3197(12), c = 16.9547(8) Å, β = 110.017(1)°, and V = 4759.0(4) Å3. The X-ray crystallographic analysis revealed a dinuclear unit in which two Tp*Na moieties are held together by three bridging acetone molecules acting as oxygen-based donors.

Synthesis of an Fe Rich Amorphous Structure with a Catalytic Effect To Rapidly Decolorize Azo Dye at Room Temperature

2014 ◽  
Vol 6 (8) ◽  
pp. 5500-5505 ◽  
Author(s):  
Peng Liu ◽  
Ji Liang Zhang ◽  
Mei Qin Zha ◽  
Chan Hung Shek
Keyword(s):  
Azo Dye ◽  
Room Temperature ◽  
Catalytic Effect ◽  
Amorphous Structure

scholarly journals Insulin-responsive cultured foetal-rat hepatocytes. Their preparation and characterization

1984 ◽  
Vol 223 (1) ◽  
pp. 39-46 ◽  
Author(s):  
D C DeSante ◽  
L Little ◽  
D E Peavy ◽  
F Vinicor
Keyword(s):  
Monolayer Culture ◽  
Cultured Cells ◽  
Rat Hepatocytes ◽  
Dependent Manner ◽  
Deoxyribonuclease I ◽  
Haematopoietic Cells ◽  
Foetal Rat ◽  
The Relationship ◽  
Dose Dependent ◽  
Scatchard Plots

An improved non-perfusion method for the preparation of cultured foetal-rat hepatocytes is described. Digestion of the liver with collagenase and deoxyribonuclease I gave yields of 40 × 10(6) hepatocytes/g of liver. The plating efficiency of hepatocytes in medium with 10 microM-cortisol was 50%. Cell morphology and metabolism were maintained through 3 days of monolayer culture, with minimal contamination by haematopoietic cells or fibroblasts. The cultured cells bound and degraded 125I-insulin in a time- and dose-dependent manner. The estimated ED50 for competitive binding at 37 degrees C was 1.1 nM. Curvilinear Scatchard plots were observed, with estimates of 16 500 high-affinity sites (Kd = 813 pM) and 53 000 low-affinity sites (Kd = 23 nM) per cell. The cultured cells demonstrated a glycogenic response to insulin, with an estimated ED50 of 120 pM. The degree of glycogenic response to insulin varied with time in culture: 500% above basal on day 1, 200% on day 2, and only 150% on day 3. Cultured foetal cells also exhibited a time-dependent uptake of 2-aminoisobutyric acid, which, in contrast with previous reports with adult cells, was not stimulated by the presence of 10 nM-insulin. Cultured foetal hepatocytes may provide an interesting model with which to study the relationship between insulin-receptor binding and insulin action.

INTERACTION BETWEEN CULTURED ENDOTHELIAL CELLS AND ABNORMAL ANTITHROMBIN III "TOYAMA"

1987 ◽  
Author(s):  
N Sakuragawa ◽  
S Saitoh ◽  
K Takahashi
Keyword(s):  
Endothelial Cells ◽  
Room Temperature ◽  
Antithrombin Iii ◽  
Cultured Cells ◽  
Binding Activity ◽  
Endothelial Cell Culture ◽  
Antithrombin Activity ◽  
Thrombin Activity ◽  
Cultured Endothelial Cells ◽  
At Iii

Purpose: Abnormal antithrombin III(AT-III)Toyama showed non-affinity to heparin and heparinoid to show loss of immediate antithrombin activity. On the endothelial cells, there are heparinoids including heparan sulfate. We investigated on the interaction between cultured endothelial cells and abnormal AT-III"Toyama" from the viewpoint of antithrombin activity.Materials and methods: (1) Endothelial cell culture:^125I-labelled normal and abnormal AT-III were placed on the washed endothelial cultured cells in 0.2 ml of RPMI-1640 medium for 15 min at 37°C. The medium was suctioned off and the cell layer was washed with Hank's balanced salt solution. The cells were incubated with 1 ml of heparin(3 ug/ml) for 15 min at 4°C. The radioactivity in the supernatant was counted, and represented AT-III which bound to the cells surface. (2) Antithrombin activity: 0.23 ml of thrombin solution^ U/ml) and 0.03 ml of normal or abnormal AT-III plasma were mixed, and incubated on the cultured cell surface for 5 min at room temperature. The residual thrombin activity was assayed by 0.3 ml of the substrate (S-2238) solution(0.8mM)for 5 min. After these procedures,2 ml of 2% citric acid solution was added to stop the reaction, and 0D(405 nm) was recorded.Results: Abnormal AT-III showed reduced binding-activity to cultured cells to one fifth compared with normal AT-III, and the residual thrombin activity in the abnormal was higher compared with that in normal plasma.Conclusion: Abnormal AT-III showed less binding activity to the cultured endothelial cells, and less thrombin neutralizing activity to show thrombogenic tendency.

scholarly journals Measurement of the metabolism of cytochrome P-450 in cultured hepatocytes by a quantitative and specific immunochemical method

1982 ◽  
Vol 204 (1) ◽  
pp. 281-290 ◽  
Author(s):  
Sammye L. Newman ◽  
Joyce L. Barwick ◽  
Nabil A. Elshourbagy ◽  
Philip S. Guzelian
Keyword(s):  
Cultured Cells ◽  
De Novo ◽  
Internal Standard ◽  
Rat Hepatocytes ◽  
Cellular Protein ◽  
Small Samples ◽  
Cultured Hepatocytes ◽  
Immunochemical Method ◽  
Adult Rat ◽  
Cytochrome P 450

We have defined conditions that permit quantitative and specific measurement of the metabolism of the major phenobarbital-inducible form of cytochrome P-450 protein in primary non-proliferating monolayer cultures of adult rat hepatocytes. Isolated antibodies specifically directed against phenobarbital cytochrome P-450 are used to immunoprecipitate the cytochrome from lysates of cultured hepatocytes pulse-labelled with [3H]leucine. Phenobarbital cytochrome P-450 protein is then isolated from the immunoprecipitate by electrophoresis on polyacrylamide gradient slab gels. Specificity of the assay for phenobarbital cytochrome P-450 was established by competition experiments involving other forms of purified cytochrome P-450 as well as by testing antibodies directed against these other forms of the cytochrome. Using purified phenobarbital cytochrome P-450, radiolabelled in both its haem and apoprotein portions, as an internal standard, we demonstrated that, with this immunoassay, recovery of cytochrome P-450 from microsomal samples is nearly complete. Basal rates of synthesis of phenobarbital cytochrome P-450 representing as little as 0.02–0.05% of total cellular protein synthesis were reliably and reproducibly detected in hepatocyte culture maintained in serum-free medium for 72h. Moreover, inclusion of phenobarbital in the culture medium for 96h stimulated not only synthesis de novo of phenobarbital cytochrome P-450 protein, but also accumulation of spectrally and catalytically active cytochrome P-450. Advantages of this immunoassay are that metabolism (synthesis or degradation) of the haem or protein of this important form of the cytochrome can be measured conveniently in the small samples available from cultured cells without the necessity of preparing subcellular fractions.

scholarly journals Evidence that binding of CTP:phosphocholine cytidylyltransferase to membranes in rat hepatocytes is modulated by the ratio of bilayer- to non-bilayer-forming lipids

1993 ◽  
Vol 291 (2) ◽  
pp. 419-427 ◽  
Author(s):  
H Jamil ◽  
G M Hatch ◽  
D E Vance
Keyword(s):  
Phospholipase A2 ◽  
Phospholipase C ◽  
Cultured Cells ◽  
Normal Values ◽  
Common Factor ◽  
Rat Hepatocytes ◽  
Cellular Membranes ◽  
Ctp:Phosphocholine Cytidylyltransferase ◽  
Cellular Concentration

The mechanism by which phospholipase C (PLC) digestion of cultured cells mediates binding of CTP:phosphocholine cytidylyltransferase to cellular membranes was investigated. Incubation of choline-depleted rat hepatocytes with PLC caused a translocation of enzyme from cytosol to membranes concomitant with a decrease in the concentration of phosphatidylcholine with no effect on the concentration of other phospholipids. Removal of PLC and supplementation with choline restored the amount of phosphatidylcholine in the cells and translocated cytidylyltransferase to the cytosol. However, when phosphatidylcholine levels were decreased by incubation with phospholipase A2 (PLA2), there was no significant redistribution of cytidylyltransferase activity. With PLA2 the concentration of phosphatidylethanolamine, as well as of phosphatidylcholine, was significantly decreased. Since PLC, but not phospholipase A2, raised the cellular concentration of diacylglycerol, possibly diacylglycerol mediated the binding of cytidylyltransferase to membranes. This possibility was examined, but is unlikely, since addition of lysophosphatidylcholine to PLC-treated cells restored the concentration of phosphatidylcholine and released cytidylyltransferase into the cytosol, but did not lower diacylglycerol levels to normal values. Studies in vitro, incubations of cells with choline analogues and a survey of the literature suggested that the over-riding common factor in regulation of cytidylyltransferase binding to membranes may be the ratio of bilayer to non-bilayer lipids in that membrane.

Cytochemical localization of glutaraldehyde-resistant NAD(P)H-oxidase in rat hepatocytes

1983 ◽  
Vol 79 (2) ◽  
pp. 259-267 ◽  
Author(s):  
Y. Mizukami ◽  
F. Matsubara ◽  
S. Matsukawa ◽  
R. Izumi
Keyword(s):  
Rat Hepatocytes ◽  
Cytochemical Localization
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