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At least 127 records · Page 7Linked to original sources
PubMed1990

Potentiation of forskolin-induced increase of cAMP by diamide and N-ethylmaleimide in rat pancreatic islets.

In isolated rat pancreatic islets, the effect of diamide and N-ethylmaleimide (NEM) on forskolin- as well as on glucagon-induced elevation of cAMP was studied. Forskolin and glucagon increased cAMP levels in batch-incubated islets. Diamide and NEM further augmented forskolin-induced increase of cAMP levels, whereas glucagon-stimulated elevation of cAMP was not affected. From our data it is likely that under the conditions of the present study, the thiols related to the Ns-protein and the catalytic unit are insensitive to oxidation and alkylation. The potentiation of forskolin-induced cAMP production in intact islet cells by diamide and NEM may be due to inactivation of the thiols related to the Ni-protein.

Animals↗
PubMed1997

The Saccharomyces cerevisiae AP-1 protein discriminates between oxidative stress elicited by the oxidants H2O2 and diamide.

The Saccharomyces cerevisiae AP-1 protein (yAP-1) is a key mediator of oxidative stress tolerance. Transcriptional activation by yAP-1 has been shown to be inducible by exposure of cells to H2O2 and diamide, among other oxidative stress eliciting compounds. Here we define the segments of the yAP-1 protein that are required to respond to this environmental challenge. Western blotting analyses indicated that levels of yAP-1 do not change during oxidative stress. Deletion mutagenesis and gene fusion experiments indicate that two different segments of yAP-1 are required for oxidative stress inducibility. These two domains function differentially depending on the type of oxidant used to generate oxidative stress. Three repeated cysteine-serine-glutamate sequences located in the carboxyl terminus are required for normal regulation of yAP-1 function during oxidative stress. Replacement of these cysteine-serine-glutamate repeats by alanine residues does not similarly affect H2O2 and diamide regulation of yAP-1 function. While yAP-1 transactivation is enhanced by exposure to either H2O2 or diamide, the protein responds to the oxidative stress produced by these compounds in nonidentical ways.

Bacterial Proteins↗
PubMed1981

Modification of the action of misonidazole. III. The effect of diamide on toxicity and radiosensitization.

Diamide, a compound which depletes cells of reduced glutathione, decreases toxicity of misonidazole to hypoxic cells, as measured by single-strand break (SSB) production, cell inactivation, and uptake of radioactive misonidazole. As a radiosensitizer, the effect of diamide in cell pellets is additive to that of misonidazole. Individually, both misonidazole and diamide enhance SSB production in DNA of cells irradiated under hypoxia in dilute suspension, but a combination of the two drugs does not appear to produce a greater number of SSB than misonidazole alone.

Animals↗
PubMed1976

Effect of thiol-oxidation of glutathione with diamide on corneal endothelial function, junctional complexes, and microfilaments.

Intracellular-reduced glutathione (GSH) was removed by thiol-oxidation with diamide during in vitro perfusion of the corneal endothelium. By 15 min the normal mosaic-like pattern of the endothelial cells was disrupted by serpentine-like lines of cell separation at the cell juntions. After 45 min of perfusion, infividual clusters of cells formed cup-shaped islands. The resultant exposure of Descemet's membrane to the perfusion solution resulted in corneal swelling. Transmission electron microscopy revealed that the endothelial cells separated at the apical junctions and that the microfilaments in the apical cytoplasm of cells formed dense bands, whereas the other subcellular organelles were normal in appearance. The change in cellular shape may be due to loss of cellular adhesion which results in the condensation of the microfilaments or contraction of the microfilaments. The addition of glucose to the perfusate prevented the diamide effect, and the diamide effect could be reversed upon removal and perfusion of a glutathione bicarbonate Ringer's solution. These results suggest that the ratio of reduced to oxidized glutathione in the endothelial cells plays a role in the maintenance of the endothelial cell barrier function.

Animals↗
PubMed1998

Antioxidant therapy counteracts the disturbing effects of diamide and maternal ageing on meiotic division and chromosomal segregation in mouse oocytes.

This study aims (i) to ascertain whether oxidative-stress-induced disturbances in chromosomal distribution in the metaphase-II spindle of mouse oocytes can be counteracted by supplementing culture medium with antioxidants; and (ii) to determine whether supplemental intake of antioxidants neutralizes the disturbing effects of maternal ageing on segregation of chromosomes during the first meiotic division and distribution of chromosomes in the metaphase-II spindle. (i): Germinal vesicle oocytes from unstimulated 10-12 week old mice were matured in vitro in the presence or absence of diamide and/or dithiothreitol. Metaphase-II oocytes were fixed and stained with 4',6-diamidino-2-phenylindole (DAPI) to detect abnormalities in chromosomal distribution. The percentage of oocytes arrested in metaphase I (12.9% vs 28.4%; P < or = 0.05) or with a telophase-I chromosome configuration (0.0% vs 8.2%; P < or = 0.0005) was decreased in diamide-DTT-treated oocytes when compared to diamide-treated oocytes. (ii): Mice were fed, from the first day of weaning until their death, a diet supplemented or not with an antioxidant mixture of vitamin C and vitamin E. Ovulated oocytes were fixed and stained with DAPI or C-banded for chromosome analysis. The percentage of abnormal (chromosome scattering and nulloploidy) or asynchronous (anaphase I or telophase I) oocytes was 2.7-fold higher in controls than in females fed an antioxidant diet (24.4% vs 8.9%, P < or = 0.05). Furthermore, the percentage of aneuploidy (2.2% vs 0.0%; P < or = 0.01) and diploidy (5.8% vs 1.7%; P < or = 0.05) was significantly higher in controls than in females fed an antioxidant diet. These findings support Tarin's oxidative stress hypothesis of aneuploidy and have clinical implications for preventing both laboratory-induced and maternal-age-associated aneuploidy in human beings.

Aging↗
PubMed1998

Redox regulation of ubiquitin-conjugating enzymes: mechanistic insights using the thiol-specific oxidant diamide.

The ubiquitin-proteasome pathway (UPP) regulates critical cell processes, including the cell cycle, cytokine-induced gene expression, differentiation, and cell death. Recently we demonstrated that this pathway responds to oxidative stress in mammalian cells and proposed that activities of ubiquitin-activating enzyme (E1) and ubiquitin-conjugating enzymes (E2s) are regulated by cellular redox status (i.e., GSSG:GSH ratio). To test this hypothesis, we altered the GSSG:GSH ratio in retinal pigment epithelial cells with the thiol-specific oxidant, diamide, and assessed activities of the UPP. Treatment of cells with diamide resulted in a dose-dependent increase in the GSSG:GSH ratio resulting from loss of GSH and a coincident increase in GSSG. Increases in the GSSG:GSH ratio from 0.02 in untreated cells to > or = 0.5 in diamide-treated cells were accompanied by dose-dependent reductions in the levels of endogenous Ub-protein conjugates, endogenous E1-ubiquitin thiol esters, and de novo ubiquitin-conjugating activity. As determined by the ability to form E1-ubiquitin and E2s-ubiquitin thiol esters, E1 and E2s were both inhibited by elevated GSSG:GSH ratios. Inhibition of E1 was associated with the formation of E1-protein mixed disulfides. Activities of E1 and E2s gradually recovered to preoxidation levels, coincident with gradual recovery of the GSSG:GSH ratio. These data support S-thiolation/dethiolation as a mechanism regulating E1 and E2 activities in response to oxidant insult. Ubiquitin-dependent proteolytic capacity was regulated by the GSSG:GSH ratio in a manner consistent with altered ubiquitin-conjugating activity. However, ubiquitin-independent proteolysis was unaffected by changes in the GSSG:GSH ratio. Potential adaptive and pathological consequences of redox regulation of UPP activities are discussed.

Cell Line, Transformed↗
PubMed1983

Recovery of membrane potentials from diamide-induced depolarization in frog lens fibers.

Measurement of membrane potentials in isolated frog lens fibers was made by means of intracellular microelectrode techniques. The membrane potentials of lens fibers were depolarized to various degrees after exposure to diamide, an -SH inhibitor. When the degree of diamide-induced depolarization was less than 20 mV, the membrane potentials almost fully recovered to the control level within 12 h after immersion in a Ringer's solution containing dithiothreitol (DTT), a -SH protector. A similar tendency was also recognized in some lenses (57%) whose depolarization was 30 mV. When the degree of depolarization was 40 mV, the membrane potentials further depolarized in all cases tested in spite of treatment with DTT. From this study, it is considered that frog lens fibers could not recover their function if the damage was so severe as to produce a membrane depolarization of more 40 mV. Determination of ionic concentrations in lens fibers revealed a highly significant correlation between the degree of diamide-induced depolarization and changes in concentration ratio of Na+/K+.

Animals↗
PubMed2005

Comparison of gene expression signatures of diamide, H2O2 and menadione exposed Aspergillus nidulans cultures--linking genome-wide transcriptional changes to cellular physiology.

BACKGROUND: In addition to their cytotoxic nature, reactive oxygen species (ROS) are also signal molecules in diverse cellular processes in eukaryotic organisms. Linking genome-wide transcriptional changes to cellular physiology in oxidative stress-exposed Aspergillus nidulans cultures provides the opportunity to estimate the sizes of peroxide (O2(2-)), superoxide (O2*-) and glutathione/glutathione disulphide (GSH/GSSG) redox imbalance responses. RESULTS: Genome-wide transcriptional changes triggered by diamide, H2O2 and menadione in A. nidulans vegetative tissues were recorded using DNA microarrays containing 3533 unique PCR-amplified probes. Evaluation of LOESS-normalized data indicated that 2499 gene probes were affected by at least one stress-inducing agent. The stress induced by diamide and H2O2 were pulse-like, with recovery after 1 h exposure time while no recovery was observed with menadione. The distribution of stress-responsive gene probes among major physiological functional categories was approximately the same for each agent. The gene group sizes solely responsive to changes in intracellular O2(2-), O2*- concentrations or to GSH/GSSG redox imbalance were estimated at 7.7, 32.6 and 13.0 %, respectively. Gene groups responsive to diamide, H2O2 and menadione treatments and gene groups influenced by GSH/GSSG, O2(2-) and O2*- were only partly overlapping with distinct enrichment profiles within functional categories. Changes in the GSH/GSSG redox state influenced expression of genes coding for PBS2 like MAPK kinase homologue, PSK2 kinase homologue, AtfA transcription factor, and many elements of ubiquitin tagging, cell division cycle regulators, translation machinery proteins, defense and stress proteins, transport proteins as well as many enzymes of the primary and secondary metabolisms. Meanwhile, a separate set of genes encoding transport proteins, CpcA and JlbA amino acid starvation-responsive transcription factors, and some elements of sexual development and sporulation was ROS responsive. CONCLUSION: The existence of separate O2(2-), O2*- and GSH/GSSG responsive gene groups in a eukaryotic genome has been demonstrated. Oxidant-triggered, genome-wide transcriptional changes should be analyzed considering changes in oxidative stress-responsive physiological conditions and not correlating them directly to the chemistry and concentrations of the oxidative stress-inducing agent.

Aspergillus nidulans↗
PubMed1999

Further studies of the effects of diamide and hydrogen peroxide on calcium signaling in the human platelet.

Intracellular calcium levels were measured in Fura-2/AM loaded human platelets. Diamide (30 microM) reduced the Ca2+ response to serotonin (1 microM), but not to 4-bromo-A23187 (1 microM). The small reduction in the maximum fluorescence produced by diamide was not mirrored by a change in the sensitivity of Fura-2 pentapotassium salt to Ca2+. Changes in the spectral properties of Fura-2 thus cannot wholly explain the reduced serotonin response following diamide treatment. Hydrogen peroxide (500 microM) produced a steady increase in the observed fluorescence ratio that was partially inhibited by the phospholipase C inhibitor U-73122 (1.5 microM). In the absence of platelets, H2O2 reduced the Ca(2+)-sensitivity of Fura-2 pentapotassium salt, although it is not clear the extent to which this effect contributes to the reduction in the Ca2+ response to serotonin seen after H2O2. It is concluded that some caution may be warranted in the interpretation of the effects of H2O2 upon receptor-mediated Ca2+ signaling as measured using Fura-2.

Blood Platelets↗
PubMed1976

Radiation sensitizing effect of diamide on human cells cultivated in vitro.

Human cells of line NHIK 3025 were irradiated suspended in growth medium (E2a) in absence and presence of diamide under aerobic and extremely hypoxic (less than 4 ppm O2) conditions. A sensitizing effect of diamide was found for doses exceeding 8 Gy (800 rad) on cells irradiated under extremely hypoxic conditions in presence of diamide of concentration 200 micrometer, whereas no significant effect was observed for 20 micrometer.

Azo Compounds↗
PubMed1979

Diamide inhibited (Ca++ + Mg++) and (Mg++) dependent ATPase in erythrocyte membranes: activity at different temperatures.

After inhibition of the monovalent cation dependent ATPase, a (Ca++ + Mg++) and a (Mg++) dependent ATPase activity can be detected. The inhibition due to diamide on the (Mg++) ATPase, assayed in the 12.5 degrees C - 30 degrees C temperature range, is almost complete. On the contrary the diamide induced inhibition of (Ca++ + Mg++) ATPase, in the same temperature range, is not complete and the residual activity increases with temperature. The reported data indicate that the ATPase activity induced by calcium is much less diamide-sensitive and -SH-dependent than that elicited by Mg++ alone.

Adenosine Triphosphatases↗
PubMed1990

Effect of diamide on polyphosphoinositide metabolism in red blood cells.

In intact red cells diamide had no strong influence on monoester phosphate group turnover of phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP) and phosphatidic acid (PA) due to the potent GSH protection of these pathways in vivo. Following the complete GSH oxidation diamide impaired the turnover of PIP and PA dramatically. However that of PIP2 did not show any change. Indeed, a drastic irreversible decrease of PIP- and PA turnover was caused by a mild oxidative stress with diamide in G6PD deficient red cells.

Azo Compounds↗
PubMed1978

Glucose oxidation in the chick cornea: effect of diamide on the pentose shunt.

Chick embryo corneas (stages 38 and 45) have been used to study variations in pentose shunt activity following the use of a glutathione-specific oxidizing agent, diamide, and a sulfydryl blocking agent, N-ethylmaleimide (NEM). Shunt activity was measured by the ratio of radiolabeled carbon 1 (14C-1) of glucose to radiolabeled carbon 6 (14C-6) of glucose derived as expired 14CO2. Diamide and NEM were both found to increase pentose shunt activity relative to glycolysis, although by different means. Diamide appeared to exert its effect by oxidizing glutathione and creating a demand for higher shunt activity to facilitate glutathione reduction by NADPH. Both C-1 and C-6 oxidation were increased, but C-1 oxidation was increased to a much greater extent. In contrast, NEM decreased both C-1 and C-6 oxidation, with C-6 preferentially affected. Thus NEM appears to preferentially inhibit the enzymatic machinery of the glycolytic-tricarboxylic acid cycle pathway and acts as an effective metabolic stress on the cornea. Our data suggest that the pentose shunt in the cornea may serve as an important alternative pathway under conditions of metabolic stress for glucose utilization and the production of energy (ATP) in the corneal cells.

Animals↗
PubMed1987

The effect of glutathione depletion by diamide, diethyl maleate or buthione sulfoximine on the surface structure of mouse L-cells.

Radiosensitizers that act by reducing glutathione levels have been studied by many workers as agents to enhance the killing of hypoxic tumour cells. In this paper we describe a scanning electron microscope (SEM) study of the surface morphology of mouse L-cells after exposure to three of these: diazenedicarboxylic acid bis N,N-dimethylamide (diamide), DL-buthionine-S,R-sulfoximine (BSO) and diethyl maleate (DEM). Diamide at 0.1 to 0.6 mMol/L for 10 min produced large blebs on the cell surface as seen in the SEM. Transmission electron microscope (TEM) images show that these are clear, featureless regions of cytoplasm. BSO treatment for 24 h at 0.5 to 2.0 mMol/L, by contrast, left the surface similar to control cells. DEM at 0.5 mMol/L for 2 h produces small blebs over the cell surface and a reduction in the number of microvilli. A combined 24 h BSO treatment with 2 h of DEM produced large blebs, which were similar in TEM micrographs to those produced by diamide. Thus, although all three sensitizers reduce glutathione levels, they have different effects on cellular morphology and hence such secondary effects may account for the different degrees of radiation sensitization seen with these agents.

Animals↗
PubMed1985

Localization of phosphatidylethanolamine in the plasma membrane of diamide-treated human blood platelets.

In human blood platelet plasma membranes phosphatidylethanolamine (PE) is asymmetrically distributed between the two leaflets. The main part of this phospholipid is localized at the inner half of the lipid bilayer. Upon stimulation of the cell a substantial transbilayer movement of PE as well as phosphatidylserine occurs and the outer leaflet then provides a procoagulant surface. The thrombin-induced PE flip-flop is inhibited by pretreatment of platelets with diamide, whereas pretreatment of platelets with diamide alone up to 5 mM did not change considerably the localization of PE in the platelet membrane. Thus, cytoskeletal proteins, which are modified by diamide, are not involved in the maintenance of the PE asymmetry but are important for the realization of the agonist-induced events in the platelets.

Adenosine Diphosphate↗
PubMed1984

Effect of diamide (azodicarboxylic acid-bis-dimethylamide) on arachidonic acid release from human blood platelet phospholipids.

It was found that the thiol-oxidizing agent diamide inhibits the formation of arachidonic acid metabolites in thrombin-stimulated platelets. An inhibition of arachidonic acid release from membrane phospholipids in diamide-treated platelets is concluded since diamide does not diminish the formation of stable metabolites from exogenous arachidonic acid. The role of the cellular thiol-disulfide status for the activities of phospholipases involved in arachidonic acid release is discussed.

Arachidonic Acid↗
PubMed1984

Effects of diamide on cyclic nucleotide levels in rat retina.

The concentrations of guanosine 3',5' monophosphate (cyclic GMP) and adenosine 3',5' monophosphate (cyclic AMP) were measured in rat retinas incubated under control conditions and in retinas incubated with diamide, a relatively specific glutathione oxidant. Retinas incubated with either glucose or pyruvate as the substrate for 30 min in the dark contained about 50 picomoles cyclic GMP/mg protein and 7 picomoles cyclic AMP/mg protein. Light-exposed control retinas contained about 70% less cyclic GMP (14.4 picomoles/mg protein) and 20% less cyclic AMP (5.4 picomoles/mg protein). Addition of diamide to the incubation medium at concentrations between 0.1 and 1.0 mM produced a concentration-dependent decrease in the dark level of cyclic GMP, but did not affect its concentration in the light or the concentration of cyclic AMP in dark-maintained and light-exposed retinas. The major effect of diamide was to reduce the normal dark-light difference in the concentration of cyclic GMP. Oxidizing conditions thus appear to alter selectively the light-sensitive compartment of retinal cyclic GMP.

Animals↗