SLCO/OATP-like transport of glutathione in FasL-induced apoptosis: glutathione efflux is coupled to an organic anion exchange and is necessary for the progression of the execution phase of apoptosis.
AuthorsFranco R,Cidlowski JA
JournalThe Journal of biological chemistry
PubMed ID16857677
A general strategy for site-specific double labeling of globular proteins for kinetic FRET studies.
AuthorsRatner V,Kahana E,Eichler M,Haas E
JournalBioconjugate chemistry
PubMed ID12236801
Site-directed mutagenesis provides a straightforward means of creating specific targets for chemical modifications of proteins. This capability enhanced the applications of spectroscopic methods adapted for addressing specific structural questions such as the characterization of partially folded and transient intermediate structures of globular proteins. Some applications such as the steady state ... More
Membrane translocation of charged residues at the tips of hydrophobic helices in the T domain of diphtheria toxin.
AuthorsRen J, Sharpe JC, Collier RJ, London E
JournalBiochemistry
PubMed ID9893993
'The low pH triggered membrane insertion of the T domain of diphtheria toxin is a critical step in the translocation of the C domain of the toxin across membranes in vivo. We previously established that the T domain can interact with membranes in two distinct conformations, one in which the ... More
Topography of diphtheria toxin A chain inserted into lipid vesicles.
AuthorsHayashibara M, London E
JournalBiochemistry
PubMed ID15697244
'The membrane-inserting T domain of diphtheria toxin aids the low-pH-triggered translocation of the catalytic A chain of the toxin across endosomal membranes. To evaluate the role of the isolated A chain in translocation, the topography of isolated A chain inserted into model membrane vesicles was investigated using a mixture either ... More
Topography of the hydrophilic helices of membrane-inserted diphtheria toxin T domain: TH1-TH3 as a hydrophilic tether.
AuthorsWang J, Rosconi MP, London E
JournalBiochemistry
PubMed ID16800637
'After low pH-triggered membrane insertion, the T domain of diphtheria toxin helps translocate the catalytic domain of the toxin across membranes. In this study, the hydrophilic N-terminal helices of the T domain (TH1-TH3) were studied. The conformation triggered by exposure to low pH and changes in topography upon membrane insertion ... More
Evaluation of methods for measuring cellular glutathione content using flow cytometry.
AuthorsHedley DW, Chow S
JournalCytometry
PubMed ID8026225
'The currently available flow cytometric stains for cellular glutathione were evaluated, examining the labelling of both human and rodent cell lines under various conditions of concentration, time, and temperature. Procedures were used that depleted glutathione (GSH) while having a minimal effect on other cellular sulphydryls in order to estimate linearity ... More
Formation, intracellular distribution and efflux of glutathione-bimane conjugates in drug-sensitive and -resistant MCF-7 cells.
AuthorsMillis KK, Lesko SA, Gamcsik MP
JournalCancer Chemother Pharmacol
PubMed ID9182830
'The rate of reaction of monochlorobimane with glutathione (GSH) was measured in native human mammary MCF-7 adenocarcinoma cells (MCF-7wt) and sublines displaying resistance to 4-hydroperoxycyclophosphamide (MCF-7hc) and adriamycin (MCF-7adr) prior to examination by epifluorescence and confocal microscopy. After a 60-min incubation period at 37 degrees C, essentially all GSH was ... More
Use of monochlorobimane for glutathione measurements in hamster and human tumor cell lines.
AuthorsCook JA, Pass HI, Russo A, Iype S, Mitchell JB
JournalInt J Radiat Oncol Biol Phys
PubMed ID2715086
'The use of monochlorobimane (MCIB) as a fluorescence label for glutathione (GSH) quantitation was investigated in human tumor cell lines. When MCIB was used with a hamster fibroblast cell line under conditions where GSH was either depleted or elevated, an excellent correlation between bimane-GSH fluorescence and the standard cyclic GSH ... More
Regulation of glutathione in cardiac myocytes.
AuthorsLi S, Li X, Rozanski GJ
JournalJ Mol Cell Cardiol
PubMed ID12967637
'Reduced glutathione (GSH) is an essential, multifunctional tripepetide that controls redox-sensitive cellular processes, but its regulation in the heart is poorly understood. The present study used a pharmocological model of GSH depletion to examine cellular mechanisms controlling cardiac GSH. Inhibition of GSH metabolism was elicited in normal rats by daily ... More
Role of glutathione and dependent enzymes in anthracycline-resistant HL60/AR cells.
AuthorsLutzky J, Astor MB, Taub RN, Baker MA, Bhalla K, Gervasoni JE, Rosado M, Stewart V, Krishna S, Hindenburg AA
JournalCancer Res
PubMed ID2568167
'We studied the cellular enzymatic defenses against anthracycline-induced free radical damage in the HL60 human myelogenous leukemia cell line and in its anthracycline-resistant subline, HL60/AR. Intracellular glutathione (GSH) levels and gamma-glutamyl transpeptidase activity were lower in HL60/AR than in HL60 cells. Glutathione-S-transferase (GST) and glutathione peroxidase activities were similar in ... More
Role of glutathione in determining the differential sensitivity between the cortical and cerebellar regions towards mercury-induced oxidative stress.
AuthorsKaur P, Aschner M, Syversen T
JournalToxicology
PubMed ID17169475
'Certain discrete areas of the CNS exhibit enhanced sensitivity towards MeHg. To determine whether GSH is responsible for this particular sensitivity, we investigated its role in MeHg-induced oxidative insult in primary neuronal and astroglial cell cultures of both cerebellar and cortical origins. For this purpose, ROS and GSH were measured ... More
Glutathione levels in primary glial cultures: monochlorobimane provides evidence of cell type-specific distribution.
AuthorsChatterjee S, Noack H, Possel H, Keilhoff G, Wolf G
JournalGlia
PubMed ID10417814
'Because glutathione (GSH) levels in glia play an important role in cellular defense against oxidative and nitrosative stress, the present study was designed to study GSH levels in the primary glial cell cultures. Here we used fluorescence microscopy and spectroscopy with monochlorobimane for measurement of intracellular glutathione content. Monochlorobimane showed ... More
Evidence for redox regulation of cytochrome C release during programmed neuronal death: antioxidant effects of protein synthesis and caspase inhibition.
AuthorsKirkland RA, Franklin JL,
JournalJ Neurosci
PubMed ID11245680
'Sympathetic neurons die by apoptosis when they are deprived of nerve growth factor (NGF). Activation of caspases by cytochrome c released from mitochondria is central to this death. In this report we present evidence that cellular redox state regulates cytochrome c redistribution in these neurons. An increase of mitochondrial-produced reactive ... More
Direct measurement of glutathione in epidermal cells of intact Arabidopsis roots by two-photon laser scanning microscopy.
AuthorsMeyer AJ, Fricker MD
JournalJ Microsc
PubMed ID10849195
'Two-photon laser scanning microscopy (TPLSM) was used to directly measure glutathione (GSH) as its fluorescent glutathione S-bimane conjugate (GSB) in developing root hair cells (trichoblasts) and non-root hair cells (atrichoblasts) of intact Arabidopsis roots. In comparison to confocal microscopy, TPLSM showed more detail deep within the tissue with less signal ... More
Control of demand-driven biosynthesis of glutathione in green Arabidopsis suspension culture cells.
AuthorsMeyer AJ, Fricker MD
JournalPlant Physiol
PubMed ID12481075
'We have investigated what limits demand-driven de novo glutathione (GSH) biosynthesis in green Arabidopsis suspension culture cells. GSH is the most abundant low-molecular weight thiol in most plants and can be quantified using monochlorobimane to fluorescently label GSH in live cells. Progress curves for labeling reached a plateau as all ... More
Measurement of glutathione levels in intact roots of Arabidopsis.
AuthorsFricker MD, May M, Meyer AJ, Sheard N, White NS
JournalJ Microsc
PubMed ID10849194
'Levels of glutathione were measured for different cell types in roots of intact Arabidopsis seedlings after labelling with monochlorobimane to give fluorescent glutathione S-bimane (GSB) and imaging using confocal laser scanning microscopy with excitation at 442 nm. Labelling increased to a plateau in most cell types after about 15-20 min ... More
The use of monochlorobimane to determine hepatic GSH levels and synthesis.
AuthorsFernández-Checa JC, Kaplowitz N
JournalAnal Biochem
PubMed ID2291468
'We have used the specific reaction of monochlorobimane (mBCI) with GSH to analyze hepatic GSH, mBCI, itself nonfluorescent, forms a stable, fluorescent adduct with GSH in a reaction catalyzed by the GSH S-transferases (GST). When hepatocytes were labeled with mBC1 (100 microM) in Krebs-Henseleit buffer, the fluorescent signal recorded over ... More
Flow cytometric analysis of tracheary element differentiation in Zinnia elegans cells.
AuthorsWeir IE, Maddumage R, Allan AC, Ferguson IB
JournalCytometry A
PubMed ID16228979
'BACKGROUND: Tracheary element (TE) differentiation in single cells in culture isolated from Zinnia elegans leaves involves programmed cell death (PCD) co-ordinated with key morphological developments. We have used flow cytometry to analyze physiological and nuclear changes in the differentiating cells. Flow cytometry allows the identification of subpopulations, thereby removing the ... More
Flow cytometric monitoring of glutathione content and anthracycline retention in tumor cells.
AuthorsNair S, Singh SV, Krishan A
JournalCytometry
PubMed ID1648468
'We have used an enzymatic (spectro-photometric) and a flow cytometric (GSH-MBCL) method to compare the glutathione (GSH) content of doxorubicin sensitive (P388) and resistant (P388/R-84) murine leukemic and human lung cancer cells. The flow cytometric analysis revealed that GSH-MBCL conjugate formation was dependent on glutathione-S-transferase (GST) activity. The human solid ... More
Thiol levels in CD134-defined subsets of rat T lymphocytes: possible implications for HgCl2-induced immune dysregulation.
AuthorsRoos A, Claessen N, Schilder-Tol EJ, Chand MA, Weening JJ, Aten J
JournalBiochem Biophys Res Commun
PubMed ID9388500
'CD134 (OX40), a member of the tumour necrosis factor receptor family, is expressed on activated T cells and mediates T and B cell costimulation. Its expression is increased after exposure to the thiol-binding compound HgCl2 in BN rats, but not in Lewis rats, in association with induction of a T ... More
Benzo[a]pyrene-resistant MCF-7 human breast cancer cells. A unique aryl hydrocarbon-nonresponsive clone.
AuthorsMoore M, Wang X, Lu YF, Wormke M, Craig A, Gerlach JH, Burghardt R, Barhoumi R, Safe S
JournalJ Biol Chem
PubMed ID7909315
'Wild-type MCF-7 human breast cancer cells were cultured for 3 months in 1 microM benzo[a]pyrene (BaP), and resistant clones were screened for inducibility of CYP1A1 gene expression by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). One of the BaP-resistant (BaPR) clones exhibited unique genotypic expression which distinguished it from both wild-type and drug-resistant (AdrR) variant ... More
Culturing at atmospheric oxygen levels impacts lymphocyte function.
AuthorsAtkuri KR, Herzenberg LA, Herzenberg LA
JournalProc Natl Acad Sci U S A
PubMed ID15738407
'To determine whether culturing peripheral blood mononuclear cells at atmospheric oxygen levels skews responses in comparison with culturing lymphocytes at physiologic oxygen levels, we cultured peripheral blood mononuclear cells at 5%, 10%, and atmospheric (20%) gas-phase oxygen for 5 days. We found that incubator oxygen levels influenced lymphocyte proliferation stimulated ... More
Effects of an oxidizing agent (hydrogen peroxide) on the glutathione system in epidermal cells of Allium cepa L. investigated by histochemical staining.
AuthorsMüller M, Tausz M, Wonisch A, Grill D
JournalFree Radic Res
PubMed ID10694050
'The glutathione system in epidermal cells of Allium cepa L. was measured by a quantitative image analysis method, using histochemical staining by monochlorobimane. Blockage of thiol groups with N-ethylmaleimide decreased fluorescence to a small rest fluorescence of maximum 5%. Pre-treatment with dithiothreitol increased fluorescence yield in all cells compared to ... More
Applications of cytotoxicity assays and pre-lethal mechanistic assays for assessment of human hepatotoxicity potential.
AuthorsXu JJ, Diaz D, O'Brien PJ
JournalChem Biol Interact
PubMed ID15522265
'While drug toxicity (especially hepatotoxicity) is the most frequent reason cited for withdrawal of an approved drug, no simple solution exists to adequately predict such adverse events. Simple cytotoxicity assays in HepG2 cells are relatively insensitive to human hepatotoxic drugs in a retrospective analysis of marketed pharmaceuticals. In comparison, a ... More
Expression of tandem glutathione S-transferase recombinant genes in COS cells for analysis of efficiency of protein expression and associated drug resistance.
'Expression vectors were designed and constructed to achieve optimum production of two different isozymes of rat glutathione S-transferase (GST) (EC 2.5.1.18) in COS cells, for studies of drug resistance. Promoter-enhancer elements from the simian virus 40 (SV40) early-region or the mouse alpha 2(I)-collagen gene, GST cDNAs encoding the rat Ya ... More
Cytotoxic effects of 110 reference compounds on HepG2 cells and for 60 compounds on HeLa, ECC-1 and CHO cells. II mechanistic assays on NAD(P)H, ATP and DNA contents.
AuthorsSchoonen WG, de Roos JA, Westerink WM, Débiton E,
JournalToxicol In Vitro
PubMed ID15826807
'In this study the focus is on the comparison of fluorometric assays, using Alamar Blue (AB) and Hoechst 33342 coloration, and luminometric assays, using Cyto-Lite and ATP-Lite, for toxicity measurements. With AB, ATP-Lite and Cyto-Lite the energy status of the cell is measured and with Hoechst 33342 the amount of ... More
Quantification of sulfur and sulfur-containing compounds in wastewaters by means of a combination of liquid chromatographic methods.
AuthorsHurse TJ, Abeydeer WP
JournalJ Chromatogr A
PubMed ID11826872
'Low-micromolar concentrations of sulfite, thiosulfate and sulfide, present in synthetic wastewater or anaerobic digester effluent, were quantified by means of derivatization with monobromobimane, followed by HPLC separation with fluorescence detection. The concentration of elemental sulfur was determined, after its extraction with chloroform from the derivatized sample. by HPLC with UV ... More
Flow cytometric assays of anticancer drug resistance.
AuthorsHedley DW
JournalAnn N Y Acad Sci
PubMed ID8098593
A brief survey of methods for preparing protein conjugates with dyes, haptens, and cross-linking reagents.
AuthorsBrinkley M
JournalBioconjug Chem
PubMed ID1616945
Bcl-2 inhibition of neural death: decreased generation of reactive oxygen species.
AuthorsKane DJ, Sarafian TA, Anton R, Hahn H, Gralla EB, Valentine JS, Ord T, Bredesen DE
JournalScience
PubMed ID8235659
'The proto-oncogene bcl-2 inhibits apoptotic and necrotic neural cell death. Expression of Bcl-2 in the GT1-7 neural cell line prevented death as a result of glutathione depletion. Intracellular reactive oxygen species and lipid peroxides rose rapidly in control cells depleted of glutathione, whereas cells expressing Bcl-2 displayed a blunted increase ... More
Thallium induces hydrogen peroxide generation by impairing mitochondrial function.
AuthorsHanzel CE, Verstraeten SV,
JournalToxicol Appl Pharmacol
PubMed ID16934846
'Thallium (Tl) is highly toxic through yet poorly understood mechanisms. In this study, we comparatively investigated the effects of thallic (Tl(III)) cations on mitochondrial functionality and oxidative stress promotion, and results were compared to those obtained for thallous (Tl(I)) cation. PC12 cells were incubated between 1 and 72 h in ... More
The yeast cadmium factor protein (YCF1) is a vacuolar glutathione S-conjugate pump.
AuthorsLi ZS, Szczypka M, Lu YP, Thiele DJ, Rea PA
JournalJ Biol Chem
PubMed ID8626454
'The yeast cadmium factor gene (YCF1) from Saccharomyces cerevisiae, which was isolated according to its ability to confer cadmium resistance, encodes a 1,515 amino acid ATP-binding cassette (ABC) protein with extensive sequence homology to the human multidrug resistance-associated protein (MRP1) (Szczypka, M., Wemmie, J. A., Moye-Rowley, W. S., and Thiele, ... More
Rapid activation of antioxidant defenses by nerve growth factor suppresses reactive oxygen species during neuronal apoptosis: evidence for a role in cytochrome c redistribution.
AuthorsKirkland RA, Saavedra GM, Franklin JL,
JournalJ Neurosci
PubMed ID17942726
'Depriving mouse sympathetic neurons of nerve growth factor (NGF) causes their apoptotic death. A Bax-dependent increase of mitochondrial-derived reactive oxygen species (ROS) begins in these cells soon after NGF withdrawal. We investigated the effects on these ROS of adding NGF to cultures of NGF-deprived neurons. ROS levels were monitored with ... More
Synergistic depletion of astrocytic glutathione by glucose deprivation and peroxynitrite: correlation with mitochondrial dysfunction and subsequent cell death.
AuthorsJu C, Yoon KN, Oh YK, Kim HC, Shin CY, Ryu JR, Ko KH, Kim WK
JournalJ Neurochem
PubMed ID10800942
'Previously we reported that immunostimulated astrocytes were highly vulnerable to glucose deprivation. The augmented death was mimicked by the peroxynitrite (ONOO )-producing reagent 3-morpholinosydnonimine (SIN-1). Here we show that glucose deprivation and ONOO- synergistically deplete intracellular reduced glutathione (GSH) and augment the death of astrocytes via formation of cyclosporin A-sensitive ... More
Internal thiols and reactive oxygen species in candidacidal activity exerted by an N-terminal peptide of human lactoferrin.
AuthorsLupetti A, Paulusma-Annema A, Senesi S, Campa M, Van Dissel JT, Nibbering PH,
JournalAntimicrob Agents Chemother
PubMed ID12019068
'We previously showed that the energized mitochondrion and extracellular ATP are essential for the candidacidal activity of the N-terminal peptide of human lactoferrin, subsequently referred to as hLF(1-11). The present study focuses on the involvement of internal thiols and reactive oxygen species (ROS) in the candidacidal activity exerted by hLF(1-11). ... More
Oxygen radical production and thiol depletion are required for Ca(2+)-mediated endogenous endonuclease activation in apoptotic thymocytes.
AuthorsFernandez A, Kiefer J, Fosdick L, McConkey DJ
JournalJ Immunol
PubMed ID7594522
'Glucocorticoid hormones stimulate apoptosis in thymocytes via a mechanism that involves changes in intracellular Ca2+, and exogenous Ca2+ can also directly promote the nuclear alterations of apoptosis (lamin degradation and chromatin cleavage) in isolated nuclei. Here we report that glucocorticoid treatment resulted in the production of reactive oxygen species and ... More
High-performance liquid chromatographic measurement of exogenous thiosulfate in urine and plasma.
AuthorsShea M, Howell S
JournalAnal Biochem
PubMed ID6486442
'A simple technique using reverse-phase ion-pair liquid chromatography for measurement of exogenous thiosulfate is described. Accurate measurement of thiosulfate in plasma and urine was permitted by precolumn derivatization with monobromobimane, a substance that readily yields fluorescent compounds upon reaction with a variety of biologically important nucleophiles including glutathione, cysteine, and ... More
Multi-photon microscopy: seeing more by imaging less.
AuthorsWhite N, Errington R
JournalBiotechniques
PubMed ID12188180
Glutathione and cellular resistance to anti-cancer drugs.
AuthorsHedley D, Chow S
JournalMethods Cell Biol
PubMed ID7533246
Monitoring simultaneous subcellular events in vitro by means of coherent multiprobe fluorescence.
Peroxynitrite was demonstrated to inhibit the active efflux of glutathione S-conjugates (2,4-dinitrophenyl-S-glutathione and bimane-S-glutathione) from human erythrocytes and the erythrocyte membrane ATPase activity stimulated by glutathione S-conjugates. As the multidrug resistance-associated protein (MRP) is responsible for the transport of glutathione S-conjugates in mammalian cells, these results point to the possibility ... More
Simple modification of a commercial flow cytometer to triple laser excitation. Simultaneous five-color fluorescence detection.
AuthorsWoronicz JD, Rice GC
JournalJ Immunol Methods
PubMed ID2661695
We describe a simple and inexpensive modification to the Coulter 753 flow cytometer which enables simultaneous triple laser excitation for advanced multi-color analysis and sorting applications. The salient feature of the modification was to split the rear laser operating in the all lines mode for use in pumping the rhodamine ... More
S-nitrosothiol inhibition of mitochondrial complex I causes a reversible increase in mitochondrial hydrogen peroxide production.
AuthorsBorutaite V, Brown GC
JournalBiochim Biophys Acta
PubMed ID16584707
We found that reversible inactivation of mitochondrial complex I by S-nitroso-N-acetyl-D,L-penicillamine (SNAP) in isolated rat heart mitochondria resulted in a three-fold increase in H2O2 production, when mitochondria were respiring on pyruvate and malate, (but not when respiring on succinate or in the absence of added respiratory substrate). The inactivation of ... More
Assessment of fluorochromes for cellular structure and function studies by flow cytometry.
AuthorsPetit JM, Denis-Gay M, Ratinaud MH
JournalBiol Cell
PubMed ID7693118
Because flow cytometry permits the analysis of individual whole cells, one of the key requirements in selecting a probe is its ability to target the site of interest into cells. In addition, dyes must possess ideal properties (ie extinction coefficient, Stoke's shift) rendering them appropriate for this methodology. Other characteristics, ... More
Troglitazone-induced hepatic necrosis in an animal model of silent genetic mitochondrial abnormalities.
AuthorsOng MM, Latchoumycandane C, Boelsterli UA,
JournalToxicol Sci
PubMed ID17150972
Troglitazone, a first-generation thiazolidinedione antidiabetic drug, was withdrawn from the market due to an unacceptable risk of idiosyncratic hepatotoxicity. Troglitazone does not cause hepatotoxicity in normal healthy rodents, but it produces mitochondrial injury in vitro at high concentrations. The aim of this study was to explore whether genetic mitochondrial abnormalities ... More
Glutathione depletion and disruption of intracellular ionic homeostasis regulate lymphoid cell apoptosis.
Intracellular glutathione (GSH) depletion is an important hallmark of apoptosis. We have recently shown that GSH depletion by its extrusion regulates apoptosis independently of excessive reactive oxygen species accumulation. However, the mechanisms by which GSH depletion regulates apoptosis are still unclear. Because disruption of intracellular ionic homeostasis, associated with apoptotic ... More
Cyclic mechanical strain increases reactive oxygen species production in pulmonary epithelial cells.
AuthorsChapman KE, Sinclair SE, Zhuang D, Hassid A, Desai LP, Waters CM,
JournalAm J Physiol Lung Cell Mol Physiol
PubMed ID15964900
Overdistention of lung tissue during mechanical ventilation may be one of the factors that initiates ventilator-induced lung injury (VILI). We hypothesized that cyclic mechanical stretch (CMS) of the lung epithelium is involved in the early events of VILI through the production of reactive oxygen species (ROS). Cultures of an immortalized ... More
Nrf2 signaling, a mechanism for cellular stress resistance in long-lived mice.
AuthorsLeiser SF, Miller RA,
JournalMol Cell Biol
PubMed ID19933842
Transcriptional regulation of the antioxidant response element (ARE) by Nrf2 is important for the cellular adaptive response to toxic insults. New data show that primary skin-derived fibroblasts from the long-lived Snell dwarf mutant mouse, previously shown to be resistant to many toxic stresses, have elevated levels of Nrf2 and of ... More
In vitro assessment of mitochondrial dysfunction and cytotoxicity of nefazodone, trazodone, and buspirone.
AuthorsDykens JA, Jamieson JD, Marroquin LD, Nadanaciva S, Xu JJ, Dunn MC, Smith AR, Will Y,
JournalToxicol Sci
PubMed ID18344530
Mitochondrial toxicity is increasingly implicated in a host of drug-induced organ toxicities, including hepatotoxicity. Nefazodone was withdrawn from the U.S. market in 2004 due to hepatotoxicity. Accordingly, we evaluated nefazodone, another triazolopyridine trazodone, plus the azaspirodecanedione buspirone, for cytotoxicity and effects on mitochondrial function. In accord with its clinical disposition, ... More
Enhanced mitochondrial superoxide in hyperglycemic endothelial cells: direct measurements and formation of hydrogen peroxide and peroxynitrite.
AuthorsQuijano C, Castro L, Peluffo G, Valez V, Radi R,
JournalAm J Physiol Heart Circ Physiol
PubMed ID17906108
Hyperglycemic challenge to bovine aortic endothelial cells (BAECs) increases oxidant formation and cell damage that are abolished by MnSOD overexpression, implying mitochondrial superoxide (O(2)(.-)) as a central mediator. However, mitochondrial O(2)(.-) and its steady-state concentrations have not been measured directly yet. Therefore, we aimed to detect and quantify O(2)(.-) through ... More
Glutathione depletion is necessary for apoptosis in lymphoid cells independent of reactive oxygen species formation.
AuthorsFranco R, Panayiotidis MI, Cidlowski JA,
JournalJ Biol Chem
PubMed ID17724027
Changes in the intracellular redox environment of cells have been reported to be critical for the activation of apoptotic enzymes and the progression of programmed cell death. Glutathione (GSH) depletion is an early hallmark observed in apoptosis, and we have demonstrated that GSH efflux during death receptor-mediated apoptosis occurs via ... More
Mechanism of neurodegeneration of neurons with mitochondrial DNA mutations.
Mutations of mitochondrial DNA are associated with a wide spectrum of disorders, primarily affecting the central nervous system and muscle function. The specific consequences of mitochondrial DNA mutations for neuronal pathophysiology are not understood. In order to explore the impact of mitochondrial mutations on neuronal biochemistry and physiology, we have ... More
Simultaneous measurement of neutrophil, lymphocyte, and monocyte glutathione by flow cytometry.
AuthorsScott RB, Collins JM, Matin S, White F, Swerdlow PS
JournalJ Clin Lab Anal
PubMed ID1700090
A flow cytometric method for quantitation of glutathione (GSH) was applied to simultaneous analysis of the major leukocyte types in peripheral blood. Cellular thiols (predominantly GSH) were stained with monochlorobimane (MCIB), and thiol fluorescence was measured with a flow cytometer. The fluorescence of the thiols closely reflected the GSH content, ... More
Macromolecular synthesis inhibitors prevent oxidative stress-induced apoptosis in embryonic cortical neurons by shunting cysteine from protein synthesis to glutathione.
AuthorsRatan RR, Murphy TH, Baraban JM
JournalJ Neurosci
PubMed ID8027786
Although macromolecular synthesis inhibitors have been demonstrated to prevent neuronal apoptosis in a number of paradigms, their mechanisms of protection remains unclear. Recently, we found that neuronal death resulting from cystine deprivation, glutathione loss, and oxidative stress is apoptotic and is prevented by inhibitors of macromolecular synthesis. We now report ... More
Fast kinetic measurements and on-line dilution by flow injection cytometry.
AuthorsLindberg W, Scampavia LD, Ruzicka J, Christian GD
JournalCytometry
PubMed ID7527315
An improved flow injection cytometry (FIC) system suitable for fast cellular kinetic measurements and on-line dilution is described. The instrument allows for measurements from 1.2 s (+/- 0.05 s) after the initiation of mixing, up to any time thereafter. Crucial factors in determining fast kinetic measurements, such as the displacement ... More
Glutathione metabolism in primary astrocyte cultures: flow cytometric evidence of heterogeneous distribution of GSH content.
AuthorsDevesa A, O'Connor JE, Garciá C, Puertes IR, Viña JR
JournalBrain Res
PubMed ID8374750
The time-course of intracellular glutathione (GSH) values after incubation with L-buthionine-(S,R)-sulfoximine (BSO), a selective inhibitor of gamma-glutamylcysteine synthetase, showed that glutathione turns over with a half-life of 5 h. Intracellular GSH was assayed by flow cytometry using three different methods. Astrocytes showed a narrow range of cellular size but a ... More
Distribution of the monochlorobimane-glutathione conjugate between nucleus and cytosol in isolated hepatocytes.
AuthorsBriviba K, Fraser G, Sies H, Ketterer B
JournalBiochem J
PubMed ID8379916
The nuclear/cytoplasmic distributions of the GSH and N-acetylcysteine conjugates of monochlorobimane (MCB) have been studied in rat hepatocytes in primary culture after microinjection into the cytoplasm. The immediately occurring fluorescence (within 1-2 s) in the nucleus was much greater than that in the cytoplasm. Other water-soluble fluorescent markers such as ... More
Membrane topography of cardiac triadin.
AuthorsCaswell AH, Brandt NR
JournalArch Biochem Biophys
PubMed ID11811949
Fusion constructs of partial sequences of triadin that contain green fluorescent protein at the N-terminus and glutathione transferase at the C-terminus have been expressed in human embryonic kidney -293 cells. A comparison of the subcellular disposition of a range of triadin fusion peptides indicates localization either to a few large ... More
Intracellular thiols regulate activation of nuclear factor kappa B and transcription of human immunodeficiency virus.
AuthorsStaal FJ, Roederer M, Herzenberg LA, Herzenberg LA
JournalProc Natl Acad Sci U S A
PubMed ID2263644
The activation of nuclear factor kappa B (NF-kappa B) has been implicated in the regulation of transcription of a variety of genes and has been shown to be essential for the expression of genes controlled by the long terminal repeat of human immunodeficiency virus (HIV LTR). We show here that ... More
Active transport of glutathione S-conjugate in human colon adenocarcinoma cells.
AuthorsZhang K, Wong KP
JournalCancer Lett
PubMed ID8950221
The formation of the glutathione S-conjugate of monochlorobimane (GSH-bimane) in human colon adenocarcinoma cells was identified by HPLC-fluorimetry and its transport from the cells was found to be temperature-sensitive, saturable and ATP-dependent. The apparent K(m) and Vmax values were 2.4 +/- 0.5 nmol GSH-bimane/10(6) cells and 0.5 +/- 0.1 nmol ... More
Barley aleurone cells contain two types of vacuoles. Characterization Of lytic organelles by use of fluorescent probes
AuthorsSwanson SJ, Bethke PC, Jones RL
JournalPlant Cell
PubMed ID9596630
Light microscopy was used to study the structure and function of vacuoles in living protoplasts of barley (Hordeum vulgare cv Himalaya) aleurone. Light microscopy showed that aleurone protoplasts contain two distinct types of vacuole: the protein storage vacuole and a lysosome-like organelle, which we have called the secondary vacuole. Fluorescence ... More
Selective staining by vital dyes of Müller glial cells in retinal wholemounts.
AuthorsUckermann O, Iandiev I, Francke M, Franze K, Grosche J, Wolf S, Kohen L, Wiedemann P, Reichenbach A, Bringmann A
JournalGlia
PubMed ID14648546
Müller glial cells within the retina may respond to different signaling molecules with an elevation of their intracellular free calcium. To prove the localization of the recorded calcium responses in Müller cells within acutely isolated retinal wholemounts, retinal pieces from adult animals and humans were exposed to different vital dyes ... More
Demonstration of nuclear compartmentalization of glutathione in hepatocytes.
AuthorsBellomo G, Vairetti M, Stivala L, Mirabelli F, Richelmi P, Orrenius S
JournalProc Natl Acad Sci U S A
PubMed ID1584774
The intracellular distribution of glutathione (GSH) in cultured hepatocytes has been investigated by using the compound monochlorobimane (BmCl), which interacts specifically with GSH to form a highly fluorescent adduct. Image analysis of BmCl-labeled hepatocytes predominantly localized the fluorescence in the nucleus; the nuclear/cytoplasmic concentration gradient was approximately three. This concentration ... More
Kinetic analysis of the intracellular conjugation of monochlorobimane by IC-21 murine macrophage glutathione-S-transferase.
AuthorsYoung PR, ConnorsWhite AL, Dzido GA
JournalBiochim Biophys Acta
PubMed ID7803478
Monochlorobimane (MCB) reacts with glutathione (GSH) in a reaction catalyzed by the glutathione-S-transferase (GST) isozymes. The diffusion of MCB through cell membranes is rapid and the fluorescence conjugates are relatively insensitive to quenching and to pH effects, and are expelled slowly from the cell, allowing the rate of fluorescence increase ... More
Transport of glutathione conjugates into secretory vesicles is mediated by the multidrug-resistance protein 1.
AuthorsVan Luyn MJ, Müller M, Renes J, Meijer C, Scheper RJ, Nienhuis EF, Mulder NH, Jansen PL, De Vries EG
JournalInt J Cancer
PubMed ID9533762
Intracellular glutathione-conjugate transport was evaluated in the human small cell lung carcinoma cell line GLC4 with low multidrug resistance protein (MRP1) expression and its 300x doxorubicin-resistant, MRP1-over-expressing, GLC4-Adr subline. Transport of non-toxic concentrations of monochlorobimane and 5-chloro-methyl fluorescein diacetate was evaluated using fluorescence microscopy. After exposure to these compounds, fluorescence ... More
Transfected rat cMOAT is functionally expressed on the apical membrane in Madin-Darby canine kidney (MDCK) cells.
AuthorsKinoshita S, Suzuki H, Ito K, Kume K, Shimizu T, Sugiyama Y
JournalPharm Res
PubMed ID9892468
PURPOSE: The purpose of the present study is to investigate the expression of canalicular multispecific organic anion transporter (cMOAT) by its cDNA transfection in polarized Madin-Darby canine kidney cells (MDCK). METHODS: MDCK cells were transfected with an expression vector (pCXN2) containing the rat cMOAT cDNA with lipofectamine to obtain the ... More
Monochlorobimane fluorometric method to measure tissue glutathione.
AuthorsKamencic H, Lyon A, Paterson PG, Juurlink BH
JournalAnal Biochem
PubMed ID11038270
Glutathione (GSH) is the principal intracellular low-molecular-weight thiol and plays a critical role in the cellular defense against agents that impose oxidative stress. A common technique to measure GSH uses reversed-phase high-performance liquid chromatography (HPLC) following derivatization with 5, 5'-dithiobis(2-nitrobenzoic acid), a technique, although reliable and sensitive, that is time ... More
Low-molecular-mass thiol compounds from a free-living highly pathogenic amoeba, Naegleria fowleri.
AuthorsOndarza RN, Iturbe A, Hernández E, Hurtado G
JournalBiotechnol Appl Biochem
PubMed ID12630909
Acid extracts labelled with the fluorescent reagent monobromobimane and separated by HPLC have enabled the detection of low-molecular-mass thiol compounds in Naegleria fowleri for the first time. The amounts detected are expressed in nmol/1 x 10(6) trophozoites cultivated at various stages of growth in the appropriate culture medium. N. fowleri ... More
Identification of basic residues involved in drug export function of human multidrug resistance-associated protein 2.
AuthorsRyu S, Kawabe T, Nada S, Yamaguchi A
JournalJ Biol Chem
PubMed ID10978330
Multidrurg resistance-associated protein 2 (MRP2)/canalicular multispecific organic anion transporter (cMOAT) is involved in the ATP-dependent export of organic anions across the bile canalicular membrane. To identify functional amino acid residues that play essential roles in the substrate transport, each of 13 basic residues around transmembrane regions (TMs) 6-17 were replaced ... More
Diminished glutathione levels cause spontaneous and mitochondria-mediated cell death in neurons from trisomy 16 mice: a model of Down's syndrome.
AuthorsSchuchmann S, Heinemann U
JournalJ Neurochem
PubMed ID10693953
It has been suggested that the increased neuronal death in cultures from trisomy 16 (Ts16) mice, a model of Down's syndrome, might result from a diminished concentration of reduced glutathione (GSH). In this study we used microfluorometric techniques to investigate the effect of GSH levels on neuronal survival in diploid ... More
Low glutathione and high iron govern the susceptibility of oligodendroglial precursors to oxidative stress.
AuthorsThorburne SK, Juurlink BH
JournalJ Neurochem
PubMed ID8752107
We have previously shown, using qualitative approaches, that oligodendroglial precursors are more readily damaged by free radicals than are astrocytes. In the present investigation we quantified the oxidative stress experienced by the cells using oxidation of dichlorofluorescin diacetate to dichlorofluorescein as a measure of oxidative stress; furthermore, we have delineated ... More
UV lasers for flow cytometric analysis: HeCd versus argon laser excitation.
AuthorsGoller B, Kubbies M
JournalJ Histochem Cytochem
PubMed ID1372632
Applying flow cytometric single cell analysis, we compared the performance of UV excitation from argon ion and HeCd lasers using various UV-excitable fluorochromes of cell kinetic and cell physiological relevance. The AT-specific DNA fluorochromes DAPI, Hoechst 33258, and Hoechst 33342 showed no significant differences of G1-phase resolution and cell cycle ... More
T cell surface redox levels determine T cell reactivity and arthritis susceptibility.
AuthorsGelderman KA, Hultqvist M, Holmberg J, Olofsson P, Holmdahl R
JournalProc Natl Acad Sci U S A
PubMed ID16908843
Rats and mice with a lower capacity to produce reactive oxygen species (ROS) because of allelic polymorphisms in the Ncf1 gene (which encodes neutrophil cytosolic factor 1) are more susceptible to develop severe arthritis. These data suggest that ROS are involved in regulating the immune response. We now show that ... More
Concurrent analysis of intracellular glutathione content and gap junctional intercellular communication.
The potential for performing dual analysis of intracellular glutathione levels and assessment of gap junctional intercellular communication with thiol-specific fluorescent probes in anchored cells was evaluated. Gap junction-mediated diffusion of monochlorobimane and 5-chloromethylfluorescein diacetate following intracellular loading and conjugation with glutathione was compared with 5-carboxyfluorescein diacetate (which is routinely used ... More
Human T cell leukemia virus-I (HTLV-I) Tax-mediated apoptosis in activated T cells requires an enhanced intracellular prooxidant state.
AuthorsLos M, Khazaie K, Schulze-Osthoff K, Baeuerle PA, Schirrmacher V, Chlichlia K
JournalJ Immunol
PubMed ID9743370
We have shown that an estradiol-dependent activation of human T cell leukemia virus-I Tax leads to the inhibition of cell proliferation and to the induction of apoptosis. The present study demonstrates that a hormone-dependent activation of Tax promotes an enhanced prooxidant state in stably transfected Jurkat cells as measured by ... More
Gene transfer by electroporation, lipofection, and DEAE-dextran transfection: compatibility with cell-sorting by flow cytometry.
Authors Puchalski R B; Fahl W E;
JournalCytometry
PubMed ID1547655
The aim of this work was to define a transfection procedure that is compatible with the sorting and propagation of cells that transiently express a heterologous gene. Three requirements were established for the procedure and were met with COS monkey kidney cells that express a recombinant glutathione S-transferase (GST) gene. ... More
Measurement of the intracellular distribution of reduced glutathione in cultured rat hepatocytes using monochlorobimane and confocal laser scanning microscopy.
AuthorsStevenson D, Wokosin D, Girkin J, Grant MH
JournalToxicol In Vitro
PubMed ID12206828
Intracellular reduced glutathione (GSH) plays a key role in protecting cells from toxicity by maintaining intracellular redox status, conjugating with electrophilic xenobiotics and free radicals, and detoxifying reactive peroxides. Several toxic chemicals interact with GSH during their metabolism, and in many cases it would be advantageous to monitor intracellular GSH ... More
The APG8/12-activating enzyme APG7 is required for proper nutrient recycling and senescence in Arabidopsis thaliana.
Authors Doelling Jed H; Walker Joseph M; Friedman Eric M; Thompson Allison R; Vierstra Richard D;
JournalJ Biol Chem
PubMed ID12070171
The vacuole/lysosome serves an important recycling function during starvation and senescence in eukaryotes via a process called autophagy. Here bulk cytosolic constituents and organelles become sequestered in specialized autophagic vesicles, which then deliver their cargo to the vacuole for degradation. In yeasts, genetic screens have identified two novel post-translational modification ... More
Fluorescence imaging study of organic anion transport from renal proximal tubule cell to lumen.
AuthorsMiller DS, Letcher S, Barnes DM
JournalAm J Physiol
PubMed ID8853412
The mechanisms driving organic anion transport from cell to lumen were studied in intact killifish proximal tubules using fluorescence microscopy. Three fluorescent substrates were used as follows: 1) fluorescein (FL); 2) carboxyfluorescein (CF), generated intracellularly from carboxyfluorescein diacetate (CFDA); and 3) bimane-S conjugates, generated intracellularly by conjugation of monochlorobimane (MCB) ... More
Fluorescence-activated cell sorting (FACS) of Drosophila hemocytes reveals important functional similarities to mammalian leukocytes.
AuthorsTirouvanziam R, Davidson CJ, Lipsick JS, Herzenberg LA
JournalProc Natl Acad Sci U S A
PubMed ID14976247
Drosophila is a powerful model for molecular studies of hematopoiesis and innate immunity. However, its use for functional cellular studies remains hampered by the lack of single-cell assays for hemocytes (blood cells). Here we introduce a generic method combining fluorescence-activated cell sorting and nonantibody probes that enables the selective gating ... More
Fluorometric microplate assay to measure glutathione S-transferase activity in insects and mites using monochlorobimane.
AuthorsNauen R, Stumpf N
JournalAnal Biochem
PubMed ID11950219
Elevated levels of glutathione S-transferases (GSTs) play a major role as a mechanism of resistance to insecticides and acaricides in resistant pest insects and mites, respectively. Such compounds are either detoxicated directly via phase I metabolism or detoxicated by phase II metabolism of metabolites as formed by microsomal monooxygenases. Here ... More
Blood glutathione disulfide: in vivo factor or in vitro artifact?
AuthorsRossi R, Milzani A, Dalle-Donne I, Giustarini D, Lusini L, Colombo R, Di Simplicio P
JournalClin Chem
PubMed ID11978601
BACKGROUND: The reported mean concentration of glutathione disulfide (GSSG) in human blood/erythrocytes varies widely (1 to >500 micromol/L), as does that of reduced glutathione (GSH) to a lesser extent. We have identified and investigated possible pitfalls in measurement of both GSH and GSSG. METHODS: We measured GSH and GSSG using ... More
Synthesis and characterization of 6-chloroacetyl-2-dimethylaminonaphthalene as a fluorogenic substrate and a mechanistic probe for glutathione transferases.
AuthorsSvensson R, Grenö C, Johansson AS, Mannervik B, Morgenstern R
JournalAnal Biochem
PubMed ID12470677
Here we demonstrate that the thiol-reactive, environmentally sensitive fluorogenic molecules 6-bromoacetyl-2-dimethylaminonaphthalene and 6-acryloyl-2-dimethylaminonaphthalene are substrates for glutathione transferases (GSTs). Product formation can be measured by strong increase in fluorescence of the glutathione conjugate. As these substances display a high nonenzymatic background reaction rate, we have synthesized and characterized 6-chloroacetyl-2-dimethylaminonaphthalene, which ... More
High-affinity efflux transport system for glutathione conjugates on the luminal membrane of a mouse brain capillary endothelial cell line (MBEC4).
AuthorsHomma M, Suzuki H, Kusuhara H, Naito M, Tsuruo T, Sugiyama Y
JournalJ Pharmacol Exp Ther
PubMed ID9862771
Cumulative evidence suggests that several organic anions are excreted from the brain to the blood across the blood-brain barrier. In the present study, we carried out a kinetic investigation of the transport activity in MBEC4, an immortalized cell line established from BALB/c mouse cerebral microvessel endothelial cells. The presence of ... More
Monochlorobimane does not selectively label glutathione in peripheral blood mononuclear cells.
Authorsvan der Ven AJ, Mier P, Peters WH, Dolstra H, van Erp PE, Koopmans PP, van der Meer JW
JournalAnal Biochem
PubMed ID7515598
Monochlorobimane (MCB) has been used by several investigators as a fluorescent label for quantifying glutathione (GSH) levels in human peripheral blood mononuclear cells (PBMC). This paper describes a biochemical evaluation of this approach. PBMC were incubated with MCB (10-100 microM) and the fluorescence in extracellular medium and cell lysates was ... More
Excretion of fluorescent substrates of mammalian multidrug resistance-associated protein (MRP) in the Schistosoma mansoni excretory system.
AuthorsSato H, Kusel JR, Thornhill J
JournalParasitology
PubMed ID15002903
The protonephridium of platyhelminths including Schistosoma mansoni plays a pivotal role in their survival by excretion of metabolic wastes as well as xenobiotics, and can be revealed in the living adult parasite by certain fluorescent compounds which are concentrated in excretory tubules and collecting ducts. To determine the presence of ... More
Recombinant glutathione S-transferase (GST) expressing cells purified by flow cytometry on the basis of a GST-catalyzed intracellular conjugation of glutathione to monochlorobimane.
AuthorsPuchalski RB, Manoharan TH, Lathrop AL, Fahl WE
JournalCytometry
PubMed ID1664313
COS cells transiently expressing glutathione S-transferase (GST) pi, Ya, or Yb1 (human Pi, rat Alpha or Mu, cytosolic classes) were purified by flow cytometry and used in colony-forming assays to show that GST confers cellular resistance to the carcinogen benzo[a]pyrene (+/-)-anti-diol epoxide (anti-BPDE). We developed a sorting technique to viably ... More
The role of cysteinylglycine S-conjugate dipeptidases in the intrahepatic mercapturic acid pathway was investigated in rat liver. Subcellular compartmentation studies and liver perfusions were performed using monochlorobimane and bimane S-conjugates as model compounds. The major part (over 95%) of total hepatic cysteinylglycine S-conjugate dipeptidase activity was located in the cytosol. ... More
Isolation by flow cytometry of a human ovarian tumor cell subpopulation exhibiting a high glutathione content phenotype and increased resistance to adriamycin.
AuthorsLee FY, Siemann DW
JournalInt J Radiat Oncol Biol Phys
PubMed ID2715085
We have used monochlorobimane as a quantitative marker by which cells of naturally high or low GSH contents were purified by fluorescence-activated cell sorting (FACS). The cell line chosen for this purpose, MLS, was a human ovarian tumor cell line established from a patient who had received extensive chemotherapy and ... More
Cell-specific measurement of cytosolic glutathione in poplar leaves.
AuthorsHartmann TN, Fricker MD, Rennenberg H, Meyer AJ
JournalPlant Cell Environ
PubMed ID12803623
The level of glutathione (GSH) in plants is important in defence reactions against biotic and abiotic stresses and can place considerable demand of the sulphur assimilation pathway. Enzymes involved in sulphur assimilation and GSH metabolism are not evenly distributed between different subcellular compartments or between different cell types in leaves ... More
Proliferative capacity of human peripheral blood lymphocytes sorted on the basis of glutathione content.
AuthorsKavanagh TJ, Grossmann A, Jaecks EP, Jinneman JC, Eaton DL, Martin GM, Rabinovitch PS
JournalJ Cell Physiol
PubMed ID1703168
Glutathione (GSH) is important in defense against oxygen free radical damage, in detoxification of xenobiotics, and in mitogenesis. The reducing conditions provided by low molecular weight thiols such as 2-mercaptoethanol (ME) have been shown to promote the growth of lymphocytes in culture. We wished to determine the effects of 2-ME ... More
A flow cytometric comparison of DNA content and glutathione levels in hepatocytes of English sole (Parophyrs vetulus) from areas of differing water quality.
English sole (Parophyrs vetulus) in Puget Sound, Washington, USA are at risk of hepatocarcinogenesis specifically in areas adjacent to polluting industrial effluents. A question concerning population and ecosystem survival is whether any of the effects of etiopathologic change are reversible. This has been approached by looking for evidence of tumor ... More
Role of multidrug resistance protein 2 (MRP2) in glutathione-bimane efflux from Caco-2 and rat renal proximal tubule cells.
AuthorsTerlouw SA, Masereeuw R, van den Broek PH, Notenboom S, Russel FG
JournalBr J Pharmacol
PubMed ID11682439
1. The multidrug resistance protein 2 (MRP2) has been shown to play an important role in the transport of glutathione conjugates in the liver. Its importance in renal excretion, however, is still uncertain and other organic anion transporters may be involved. The objective of the present study was to characterize ... More
Cell proliferation and hair tip growth in the Arabidopsis root are under mechanistically different forms of redox control.
AuthorsSánchez-Fernández R, Fricker M, Corben LB, White NS, Sheard N, Leaver CJ, Van Montagu M Inzé D, May MJ
JournalProc Natl Acad Sci U S A
PubMed ID11038608
We provide evidence that the tripeptide thiol glutathione (GSH) participates in the regulation of cell division in the apical meristem of Arabidopsis roots. Exogenous application of micromolar concentrations of GSH raised the number of meristematic cells undergoing mitosis, while depletion of GSH had the opposite effect. A role for endogenous ... More
Sequential opening of mitochondrial ion channels as a function of glutathione redox thiol status.
AuthorsAon MA, Cortassa S, Maack C, O'Rourke B
JournalJ Biol Chem
PubMed ID17540766
Mitochondrial membrane potential (DeltaPsi(m)) depolarization contributes to cell death and electrical and contractile dysfunction in the post-ischemic heart. An imbalance between mitochondrial reactive oxygen species production and scavenging was previously implicated in the activation of an inner membrane anion channel (IMAC), distinct from the permeability transition pore (PTP), as the ... More
The apoptosis-necrosis paradox. Apoptogenic proteases activated after mitochondrial permeability transition determine the mode of cell death.
AuthorsHirsch T, Marchetti P, Susin SA, Dallaporta B, Zamzami N, Marzo I, Geuskens M, Kroemer G
JournalOncogene
PubMed ID9380409
Mitochondrial alterations including permeability transition (PT) constitute critical events of the apoptotic cascade and are under the control of Bcl-2 related gene products. Here we show that induction of PT is sufficient to activate CPP32-like proteases with DEVDase activity and the associated cleavage of the nuclear DEVDase substrate poly(ADP-ribose) polymerase ... More
Patulin-induced cellular toxicity: a vital fluorescence study.
AuthorsBurghardt RC, Barhoumi R, Lewis EH, Bailey RH, Pyle KA, Clement BA, Phillips TD
JournalToxicol Appl Pharmacol
PubMed ID1539161
The mechanisms of patulin-induced cellular toxicity in an immortalized rat granulosa cell line were examined using several vital fluorescence bioassays. Monochlorobimane and 5-chloromethylfluorescein diacetate were used to monitor cellular glutathione (GSH) levels and revealed dose- and time-dependent depletion of GSH by patulin. A significant reduction in the fluorescence of the ... More
Quantitative analysis of cellular glutathione by flow cytometry utilizing monochlorobimane: some applications to radiation and drug resistance in vitro and in vivo.
AuthorsRice GC, Bump EA, Shrieve DC, Lee W, Kovacs M
JournalCancer Res
PubMed ID3779630
An assay using a bimane derivative has been developed to detect free glutathione (GSH) in individual viable cells by flow cytometry. Monochlorobimane [syn-(ClCH2CH3)-1,5-diazabicycla[3.30]acta-3,6-diene-2,8-dio ne], itself nonfluorescent, reacts with GSH to form a highly fluorescent derivative. High pressure liquid chromatography analysis showed that, using specific staining conditions, the only low molecular ... More
Influence of glutathione S-transferases on cellular glutathione determination by flow cytometry using monochlorobimane.
AuthorsUblacker GA, Johnson JA, Siegel FL, Mulcahy RT
JournalCancer Res
PubMed ID2004362
Intracellular glutathione (GSH) levels for seven mammalian cell lines (four human tumors, two rodent, one monkey) were determined by flow cytometry following staining with monochlorobimane (MBCl), and the results were compared with GSH levels measured by the Tietze assay. The mean fluorescence intensity for all but the two rodent lines ... More
CYP2E1 overexpression in HepG2 cells induces glutathione synthesis by transcriptional activation of gamma-glutamylcysteine synthetase.
AuthorsMarí M, Cederbaum AI
JournalJ Biol Chem
PubMed ID10748080
Induction of CYP2E1 (cytochrome P450 2E1) by ethanol appears to be one of the central pathways by which ethanol generates a state of oxidative stress. CYP2E1 is a loosely coupled enzyme; formation of reactive oxygen species occurs even in the absence of added substrate. GSH is critical for preserving the ... More