Diazenedicarboxylic Acid Bis Research Articles

Differential dielectroscopic data on the relation of erythrocyte membrane skeleton to erythrocyte deformability and flicker.

Two dielectric relaxations, βsp (1.5MHz) and γ1sp (7MHz), have been detected on spectrin-based membrane skeleton (MS) of red blood cells (RBCs) using the plot of admittance changes at the spectrin denaturation temperature (Ivanov and Paarvanova in Bioelectrochemistry 110: 59-68, 2016, Electrochim Acta 317: 289-300, 2019a). In this study, we treated RBCs and RBC ghost membranes with agents that make membranes rigid and suppress membrane flicker, and studied the effect on βsp and γ1sp relaxations. Diamide (diazene dicarboxylic acid bis-(N,N-dimethylamide)) (up to 0.85mM), taurine mustard (tris(2-chloroethyl)amine) (up to 2mM), known to specifically cross-link and stiffen spectrin, and glutaraldehyde (up to 0.044%) all inhibited the relaxations in RBC ghost membranes. Similar inhibition was obtained resealing RBC ghost membranes with 2,3-diphosphoglicerate (up to 15mM), binding WGA (wheat germ agglutinin) (up to 0.025mg/ml) to exofacial aspect of RBCs, incubating RBCs in hypotonic (200mOsm) and hypertonic (600-900mOsm) media and depleting RBCs of ATP. By contrast, concanavalin A (1mg/ml) and DIDS (4,4'-diiso-thiocyanato stilbene-2,2'-disulfonic acid) (75μM, pH 8.2), both known to bind specifically band 3 integral protein of RBCs without effect on RBC membrane rigidity, did not affect the relaxations. We conclude there might be a relation between the strength of dielectric relaxations on MS spectrin and the deformability and flicker of RBC membrane.

MexT Functions as a Redox-Responsive Regulator Modulating Disulfide Stress Resistance in Pseudomonas aeruginosa

MexT is a global LysR transcriptional regulator known to modulate antibiotic resistance and virulence in Pseudomonas aeruginosa. In this study, a novel role for MexT in mediating intrinsic disulfide stress resistance was demonstrated, representing the first identified phenotype associated with inactivation of this regulator in wild-type cells. Disruption of mexT resulted in increased susceptibility to the disulfide stress elicitor diamide [diazenedicarboxylic acid bis(N,N,-di-methylamide)]. This compound is known to elicit a specific stress response via depletion of reduced glutathione and alteration of the cellular redox environment, implicating MexT in redox control. In support of this, MexT-regulated targets, including the MexEF-OprN multidrug efflux system, were induced by subinhibitory concentrations of diamide. A mexF insertion mutant also exhibited increased diamide susceptibility, implicating the MexEF-OprN efflux system in MexT-associated disulfide stress resistance. Purified MexT protein was observed to form an oligomeric complex in the presence of oxidized glutathione, with a calculated redox potential of -189 mV. This value far exceeds the thiol-disulfide redox potential of the bacterial cytoplasm, ensuring that MexT remains reduced under normal physiological conditions. MexT is activated by mutational disruption of the predicted quinone oxidoreductase encoded by mexS. Alterations in the cellular redox state were observed in a mexS mutant (PA14nfxC), supporting a model whereby the perception of MexS-associated redox signals by MexT leads to the induction of the MexEF-OprN efflux system, which, in turn, may mediate disulfide stress resistance via efflux of electrophilic compounds.

Differential Effect of l-Cysteine in Isolated Whole-Bladder Preparations from Neonatal and Adult Rats

The present study was undertaken to compare the effects of the thiol reagents L-cysteine and (diazene dicarboxylic acid bis 5N,N-dimethylamide) diamide on contractile activity of neonatal and adult rat bladders. In vitro whole-bladder preparations from Wistar rats were used to study the modulation of spontaneous bladder contractions by thiol reagents. After blocking cholinergic and adrenergic transmission with atropine and guanethidine, L-cysteine facilitated spontaneous bladder contractions in neonatal rat bladders. The effect of L-cysteine was suppressed by diamide. Diamide alone did not change basal activity of the neonatal rat bladder. The facilitatory effects of L-cysteine were reduced by the L-type Ca2+ channel-blocking agent nifedipine and the calcium-activated K+ channel opener NS1619 [1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one]. ATP or suramin, a purinergic receptor antagonist, significantly inhibited the effect of L-cysteine in neonatal bladders, whereas the nitric-oxide synthase inhibitor N(omega)-nitro-L-arginine was ineffective. L-cysteine did not elicit any detectable effects in the adult rat bladder; whereas diamide caused a large-amplitude sustained tonic contraction. The contraction induced by diamide in adult bladder did not occur when the preparation was pretreated with L-cysteine. Also, L-Cysteine administered during the diamide-evoked contraction completely inhibited the contraction to diamide. In conclusion, our results suggest that L-cysteine has markedly different effects in isolated whole-bladder preparations from neonatal and adult rats. Thus thiol-sensitive mechanisms may modulate contractility by regulation of Ca2+ and K+ channels and/or purinergic transmission in the neonatal bladder. The effects of L-cysteine and diamide were reversed in adult bladders, indicating that the regulation of bladder contractility by thiols is markedly altered during postnatal development.

Enzymatic Mechanism Controls Redox-mediated Protein-DNA Interactions at the Replication Origin of Kinetoplast DNA Minicircles

Kinetoplast DNA (kDNA) is the mitochondrial DNA of trypanosomatids. Its major components are several thousand topologically interlocked DNA minicircles. Their replication origins are recognized by universal minicircle sequence-binding protein (UMSBP), a CCHC-type zinc finger protein, which has been implicated with minicircle replication initiation and kDNA segregation. Interactions of UMSBP with origin sequences in vitro have been found to be affected by the protein's redox state. Reduction of UMSBP activates its binding to the origin, whereas UMSBP oxidation impairs this activity. The role of redox in the regulation of UMSBP in vivo was studied here in synchronized cell cultures, monitoring both UMSBP origin binding activity and its redox state, throughout the trypanosomatid cell cycle. These studies indicated that UMSBP activity is regulated in vivo through the cell cycle dependent control of the protein's redox state. The hypothesis that UMSBP's redox state is controlled by an enzymatic mechanism, which mediates its direct reduction and oxidation, was challenged in a multienzyme reaction, reconstituted with pure enzymes of the trypanosomal major redox-regulating pathway. Coupling in vitro of this reaction with a UMSBP origin-binding reaction revealed the regulation of UMSBP activity through the opposing effects of tryparedoxin and tryparedoxin peroxidase. In the course of this reaction, tryparedoxin peroxidase directly oxidizes UMSBP, revealing a novel regulatory mechanism for the activation of an origin-binding protein, based on enzyme-mediated reversible modulation of the protein's redox state. This mode of regulation may represent a regulatory mechanism, functioning as an enzyme-mediated, redox-based biological switch.

Consequences of ERp57 Deletion on Oxidative Folding of Obligate and Facultative Clients of the Calnexin Cycle

Members of the protein-disulfide isomerase superfamily catalyze the formation of intra- and intermolecular disulfide bonds, a rate-limiting step of protein folding in the endoplasmic reticulum (ER). Here we compared maturation of one obligate and two facultative calnexin substrates in cells with and without ERp57, the calnexin-associated, glycoprotein-specific oxidoreductase. ERp57 deletion did not prevent the formation of disulfide bonds during co-translational translocation of nascent glycopolypeptides in the ER. It affected, however, the post-translational phases of oxidative influenza virus hemagglutinin (HA) folding, resulting in significant loss of folding efficiency for this obligate calnexin substrate. Without ERp57, HA also showed reduced capacity to recover from an artificially induced aberrant conformation, thus revealing a crucial role of ERp57 during post-translational reshuffling to the native set of HA disulfides. ERp57 deletion did not affect maturation of the model facultative calnexin substrates E1 and p62 (and of most cellular proteins, as shown by lack of induction of ER stress). ERp72 was identified as one of the ER-resident oxidoreductases associating with the orphan ERp57 substrates to maintain their folding competence.

Oxidation of a critical thiol residue of the adenine nucleotide translocator enforces Bcl-2-independent permeability transition pore opening and apoptosis.

Mitochondrial membrane permeabilization is a critical event in the process leading to physiological or chemotherapy-induced apoptosis. This permeabilization event is at least in part under the control of the permeability transition pore complex (PTPC), which interacts with oncoproteins from the Bcl-2 family as well as with tumor suppressor proteins from the Bax family, which inhibit or facilitate membrane permeabilization, respectively. Here we show that thiol crosslinking agents including diazenedicarboxylic acid bis 5N, N-dimethylamide (diamide), dithiodipyridine (DTDP), or bis-maleimido-hexane (BMH) can act on the adenine nucleotide translocator (ANT), one of the proteins within the PTPC. ANT alone reconstituted into artificial lipid bilayers suffices to confer a membrane permeabilization response to thiol crosslinking agents. Diamide, DTDP, and BMH but not tert-butylhydroperoxide or arsenite cause the oxidation of a critical cysteine residue (Cys 56) of ANT. Thiol modification within ANT is observed in intact cells, isolated mitochondria, and purified ANT. Recombinant Bcl-2 fails to prevent thiol modification of ANT. Concomitantly, a series of different thiol crosslinking agents (diamide, DTDP, and BMH, phenylarsine oxide) but not tert-butylhydroperoxide or arsenite induce mitochondrial membrane permeabilization and cell death irrespective of the expression level of Bcl-2. These data indicate that thiol crosslinkers cause a covalent modification of ANT which, beyond any control by Bcl-2, leads to mitochondrial membrane permeabilization and cell death.

Zinc Finger of Replication Protein A, a Non-DNA Binding Element, Regulates Its DNA Binding Activity through Redox

Eukaryotic replication protein A (RPA) is a single-stranded DNA-binding protein with multiple functions in DNA replication, repair, and genetic recombination. RPA contains an evolutionarily conserved 4-cysteine-type zinc finger motif (X(3)CX(2-4)CX(12-15)CX(2)C) that has a potential role in regulation of DNA replication and repair (Dong, J., Park, J-S., and Lee, S-H. (1999) Biochem. J. 337, 311-317 and Lin, Y.-L., Shivji, M. K. K., Chen, C., Kolodner, R., Wood, R. D., and Dutta, A. (1998) J. Biol. Chem. 273, 1453-1461), even though the zinc finger itself is not essential for its DNA binding activity (Kim, D. K., Stigger, E., and Lee, S.-H. (1996) J. Biol. Chem. 271, 15124-15129). Here, we show that RPA single-stranded DNA (ssDNA) binding activity is regulated by reduction-oxidation (redox) through its zinc finger domain. RPA-ssDNA interaction was stimulated 10-fold by the reducing agent, dithiothreitol (DTT), whereas treatment of RPA with oxidizing agent, diazene dicarboxylic acid bis[N,N-dimethylamide] (diamide), significantly reduced this interaction. The effect of diamide was reversed by the addition of excess DTT, suggesting that RPA ssDNA binding activity is regulated by redox. Redox regulation of RPA-ssDNA interaction was more effective in the presence of 0.2 M NaCl or higher. Cellular redox factor, thioredoxin, was able to replace DTT in stimulation of RPA DNA binding activity, suggesting that redox protein may be involved in RPA modulation in vivo. In contrast to wild-type RPA, zinc finger mutant (cysteine to alanine mutation at amino acid 486) did not require DTT for its ssDNA binding activity and is not affected by redox. Together, these results suggest a novel function for a putative zinc finger in the regulation of RPA DNA binding activity through cellular redox.

Erythrocyte flow and elasticity of microvessels evaluated by marginal cell-free layer and flow resistance.

Flow dynamics of human erythrocytes was compared in elastic (E) and hardened (H) microvessels with inner diameters of 10-40 microns. The thickness of the marginal cell-free layer and the overall flow resistance were measured with a vascular bed isolated from rabbit mesentery (E vascular bed) as well as with a 4% paraformaldehyde-fixed bed (H vascular bed). 1) In both E and H microvessels, the thickness of the cell-free layer increased with increasing inner diameter of the microvessels and with decreasing hematocrit accompanied by an overall decrease in the flow resistance. The hematocrit-dependent change of the cell-free layer thickness was greater in the E microvessels than in the H microvessels. The flow resistance was always greater in the H vascular beds than in the E vascular beds. 2) With decreasing erythrocyte deformability induced by treatment with 2 mM diazenedicar-boxylic acid bis(N,N-dimethylamide), the thickness of the cell-free layer decreased at a low hematocrit in the E microvessels and at a high hematocrit in the H microvessels, although the flow resistance was increased in both vascular beds. 3) Dextran of 70,400 average molecular weight accelerated the formation of the cell-free layer by inducing erythrocyte aggregation. A drastic increase in the cell-free layer thickness at 2-4 g/dl of dextran in the E microvessels and at 1-2 g/dl of dextran in the H microvessels was accompanied by a significantly lower increase in the flow resistance. This study concludes that the elasticity of microvessels may play an important role for reducing the overall flow resistance of a vascular bed, which is modulated by the marginal cell-free layer, itself a function of the rheological properties of the erythrocytes.

Deformation of erythrocytes in microvessels and glass capillaries: effects of erythrocyte deformability.

The deformation of erythrocytes in microvessels less than 15 microns in inner diameter was analyzed using a microvascular bed isolated from rabbit mesentery. The deformation was compared with that found in glass capillaries. Human erythrocytes were perfused through two media: first, a microvascular-bed section isolated from rabbit mesentery; and second, a set of glass capillaries. Images of deformed erythrocytes were recorded on videotape under strobe light and analyzed with an image processor. The flow velocity of the erythrocytes was determined from the difference of their positions between video frames or by a dual-spot cross-correlation technique. Erythrocyte deformability was modified with diamide, diazene dicarboxylic acid bis[N,N-dimethylamide], by crosslinking spectrins. Symmetrical (parachute-like or slipper-like) deformation of erythrocytes was observed only in microvessels smaller than 13 microns in inner diameter. Erythrocytes in microvessels were less deformed than those in glass capillaries with corresponding diameters, and the marginal cell-free layer was narrower. The deformation increased by increasing the flow velocity of erythrocytes, and the cell-free layer became wider. Diamide-treated cells in microvessels were less deformed than normal cells and showed slightly narrower cell-free layers. Stronger stress in narrower microvessels induced further deformation of cells. Erythrocyte deformation in microvessels was essentially different from that in glass capillaries, and the effect of erythrocyte deformability on the flow dynamics of erythrocytes in microvessels was properly evaluated using an isolated microvascular bed.

Preferential proteolysis and activation of oxidatively modified liver microsomal glutathione S-transferase of rat.

Proteolytic activation of oxidatively modified microsomal GSH S-transferase (GSTm) was investigated. When GSTm was incubated with diamide -diazenedicarboxylic acid bis(N,N-dimethylamide)- or hydrogen peroxide in the presence or absence of glutathione, a protein-gluathione mixed-disulfide and a dimer of the enzyme were formed with a concomitant increase in transferase activity. Although control GSTm was activated 3.4-fold by 3 micrograms/ml of trypsin, the monomeric form of the transferase in which the sulfhydryl group was modified by mixed-disulfide bond formation or by covalent binding with N-ethylmaleimide was further stimulated by lower concentrations of trypsin than that used in the control. In contrast, no activation of the dimeric transferase was observed with any concentration of trypsin. In immunoblot analysis, a proteolytic product (fragment A) from the dimer transferase was detected after treatment of oxidant-modified microsomes with low concentrations of trypsin, whereas the fragment (fragment B) from the unmodified-monomeric enzyme was observed by high concentrations of trypsin. These results show that oxidatively modified GSTm is sensitive to proteolytic attack by trypsin and that only monomeric transferase is further activated by limited proteolysis.

Glutathione removal reveals kinases as common targets for K-Cl cotransport stimulation in sheep erythrocytes.

K-Cl cotransport is activated by swelling, lowering of cellular free Mg (Mgi), and thiol modification of erythrocytes. Direct actions by thiol reagents on the K-Cl cotransport complex were separated from indirect effects through nonoxidative changes in cellular glutathione (GSH). We used 1-chloro-2,4-dinitrobenzene (CDNB), which, conjugated to GSH, is extruded from the erythrocyte as a thioether. CDNB caused a small biphasic effect (inhibition and stimulation) on K-Cl cotransport and, at 1 mM, abolished its stimulation by N-ethylmaleimide (NEM), diazenedicarboxylic acid bis[N,N-dimethylamide], methyl methanethiosulfonate, and staurosporine, a kinase inhibitor, independent of the order of treatment. Hence, NEM and other activating-thiol reagents, and perhaps GSH removal itself, target unidentified kinases involved in activation of K-Cl cotransport. CDNB also abrogated K-Cl cotransport stimulation by Mgi depletion independent of the order of treatment, indicating inhibition at a second site nearer to the transporter. Furthermore, CDNB treatment elevated and rendered K-Cl cotransport insensitive to osmotic shrinkage, suggesting uncoupling from the regulator.

Effects of membrane-permeant and -impermeant thiol reagents on Ca2+ and K+ channel currents of mouse pancreatic B cells

The membrane permeant thiol reagent diazene dicarboxylic acid bis-(N'-methylpiperazide) (DIP) has been shown to inhibit insulin secretion and Ca2+ uptake in pancreatic B cells in the presence of a stimulating glucose concentration (20 mM), whereas the nonpenetrating analog of DIP (bis-N'-methyliodide; DIP + 2) stimulates insulin release and Ca2+ uptake at a low glucose concentration (3 mM). The effects of DIP and DIP + 2 were tested on currents through ATP-sensitive K+ (K+ATP) channels and voltage-dependent Ca2+ channels (with Ba2+ as the charge carrier) in mouse pancreatic B cells in the whole-cell mode of the patch-clamp technique. DIP (0.1 mM) almost completely inhibited both the K+ATP and Ca2+ channel currents. In contrast, DIP + 2 (0.1 mM) did not affect the Ca2+ channel current but reduced the whole-cell K+ATP current by about 40%. The data strongly suggest that the suppression of insulin secretion previously observed with DIP is due to a reduction of the current through voltage-dependent Ca2+ channels, whereas the stimulation of hormone release induced by DIP + 2 is caused by the partial inhibition of K+ATP channel current.

Effects of membrane-permeant and -impermeant thiol reagents on Ca2+ and K+ channel currents of mouse pancreatic B cells.

The membrane permeant thiol reagent diazene dicarboxylic acid bis-(N'-methylpiperazide) (DIP) has been shown to inhibit insulin secretion and Ca2+ uptake in pancreatic B cells in the presence of a stimulating glucose concentration (20 mM), whereas the nonpenetrating analog of DIP (bis-N'-methyliodide; DIP + 2) stimulates insulin release and Ca2+ uptake at a low glucose concentration (3 mM). The effects of DIP and DIP + 2 were tested on currents through ATP-sensitive K+ (K+ATP) channels and voltage-dependent Ca2+ channels (with Ba2+ as the charge carrier) in mouse pancreatic B cells in the whole-cell mode of the patch-clamp technique. DIP (0.1 mM) almost completely inhibited both the K+ATP and Ca2+ channel currents. In contrast, DIP + 2 (0.1 mM) did not affect the Ca2+ channel current but reduced the whole-cell K+ATP current by about 40%. The data strongly suggest that the suppression of insulin secretion previously observed with DIP is due to a reduction of the current through voltage-dependent Ca2+ channels, whereas the stimulation of hormone release induced by DIP + 2 is caused by the partial inhibition of K+ATP channel current.

Simultaneous influence of erythrocyte deformability and macromolecules in the medium on erythrocyte aggregation: a kinetic study by a laser scattering technique

The aggregation and sedimentation kinetics of human erythrocytes was studied by modifying the cellular properties and medium compositions simultaneously. Dextrans of average molecular weight 70400 and 494000 were used to provide suspending medium modifications, while diamide (diazene dicarboxylic acid bis(N,N- dimethylamide) ) was used to alter the membrane structural properties. Laser scattering method was employed for this study, and it was compared with a kinetic method combined with a low-shear rheoscope and an image analyzer. From scattered light intensity profiles continuously obtained during aggregation of erythrocytes and sedimentation of the aggregates, characteristic kinetic parameters were computed. Kinetic parameters obtained from a phase of the one-dimensional aggregate formation and sedimentation corresponded well to the velocity of rouleaux formation obtained by the low-shear rheoscope technique. Dextrans accelerated the erythrocyte aggregation and the sedimentation, and diamide treatment suppressed the process by decreasing the erythrocyte deformability. The aggregating force by dextrans overcame the disaggregating force by the decreased deformability. However, the arrangement of erythrocytes as expressed in specific units for aggregates (i.e., rouleaux) became irregular by decreasing the erythrocyte deformability. In conclusion, the progression of erythrocyte aggregation and the structure of the aggregates were dependent on both erythrocyte properties and macromolecules in the medium.

Modifiers of Radiation-Induced Apoptosis

EL4 murine lymphoma cells and F9 murine teratocarcinoma cells undergo apoptosis-like cell death after exposure to ionizing radiation. Apoptosis differs in several ways from classical clonogenic cell killing by radiation. We have tested several modifiers and radiomimetic agents in an effort to determine if the mechanism of induction of apoptosis by radiation differs from the mechanism of classical clonogenic cell killing by radiation, and consequently that these two end points of radiation action might be differentially modifiable. We found that internucleosomal DNA fragmentation, characteristics of apoptosis, can result from treatment of EL4 and F9 cells with agents that have diverse modes of action: tert-butyl hydroperoxide, diazenedicarboxylic acid bis(N,N-piperidide), and etoposide. Hydrogen peroxide did not induce internucleosomal DNA fragmentation at concentrations expected to be produced by the doses of ionizing radiation that we used. Radiation-induced DNA fragmentation could be inhibited by 3-aminobenzamide, dibutryl cyclic AMP, or 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl, although in this respect there appear to be marked differences between the cell lines.

Thiol-Modulating Agents Increase Manganese Superoxide Dismutase Activity in Human Lung Fibroblasts

The effect of three thiol-modulating agents, buthionine sulfoximine (BSO), N-ethylmaleimide (NEM), and diazenedicarboxylic acid bis(N,N′-dimethylamide) (diamide), on cellular glutathione (GSH) level and manganese superoxide dismutase (MnSOD) activity was studied in a human lung fibroblast cell line, WI38. The results showed that treatment with BSO, NEM, or diamide can increase MnSOD activity in the human fibroblast cells. These findings are compatible with the view that alteration of intracellular GSH level may create a pro-oxidant environment in the cells and the MnSOD activity may increase as a cellular response to the oxidative stress.

Incorporation of 3H-N-ethylmaleimide into sheep red cell membrane thiol groups following protection by diamide-induced oxidation

The thiol oxidant diazene dicarboxylic acid bis [N,N-dimethylamide] (diamide) is known to reversibly activate K-Cl cotransport in sheep red blood cells. Although the detailed mechanism of activation is unknown, functional thiols at the membrane or at the cytoplasmic level are recognized as important. To search for membrane bound thiols involved in the regulation of K-Cl cotransport, sheep red cells were first exposed to diamide at concentrations activating K-Cl cotransport, and then to the alkylating agent N-ethylmaleimide (NEM) in order to block non-oxidized thiols. White ghosts, prepared by osmotic lysis from these cells, were again treated with NEM followed by reduction of the diamide-induced dithiols with dithiothreitol (DTT) concentrations known to reverse the diamide-induced K-Cl flux. Maximum 3H-NEM incorporation into the DTT-reduced thiols occurred at 50 microM DTT. Saturation labelling by 3H-NEM of about 2 x 10(4) diamide-protected thiols/cell occurred at 25 microM NEM. Diamide protected about 0.1% of all membrane thiols chemically determined earlier. Membranes from high K (HK) and low K (LK) sheep red cells did not differ significantly in the number of diamide-protected thiols, and polyacrylamide gels revealed a similar protein distribution of 3H-NEM-labelled thiols. Since diamide is known to stimulate K-Cl flux in LK cells ten times more than in HK cells this finding is consistent with the hypothesis of a cytoplasmic control effecting different K-Cl flux activities in the membranes of the two cation genotypic red blood cells.

Inhibition of biliary excretion of indocyanine green by the thiol-oxidizing agent, diazenedicarboxylic acid bis[ n, n′-dimethylamide

Biliary excretion of Indocyanine green (ICG) in Sprague-Dawley rats during constant intravenous infusion of the dye in vivo was inhibited by intraperitoneally administered diazenedicarboxylic acid bis[ N, N′-dimethylamide](diamide, 0.5 mmol/kg body wt), a glutathione-specific thiol-oxidizing agent. Significant inhibition of ICG excretion was observed also when ICG was injected rapidly 90 min after diamide administration. Disappearance of ICG from the plasma was not affected by diamide. Oxidized glutathione in bile increased transiently following diamide administration but returned to the basal level within 30 min. Hepatic concentrations of reduced and oxidized glutathione were not different from those of controls when determined 90 min after diamide administration. The inhibition of ICG excretion was completely prevented by subsequent administration of dithiothreitol (0.5 mmol/kg) 30 min after that of diamide. The results, therefore, suggest that the biliary excretion of ICG was inhibited by secondary changes in the redox status of thiols in hepatocytes caused by a transient increase in oxidized glutathione.

Diamide-induced cross-linking of the lens water-soluble proteins as a model of the early oxidative changes during senile cataract formation

This study deals with the effects of the SH oxidizing agent diamide (diazene dicarboxylic acid bis-( N, N-dimethyl-amide) on the water-soluble proteins from rabbit lenses. The dialyzed protein extracts were incubated for 0.5–1.5 h with various concentrations of diamide. Alterations in sulphydryl contents, gel filtration and gel eletrophoresis profiles of proteins were recorded. The response to 2 mM diamide treatment for 1 h consists of rapid oxidation (up to 40%) of protein-bound sulphydryl groups accompanied by appearance of polypeptides with apparent molecular weights in excess of 68 000. A protein with a molecular weight of 29 kDa was shown to be specially involved in cross-linking. The linkages in the dialyzed water-soluble lens protein fraction induced by diamide may be reduced by GSH (10 mM) treatment of the protein extract. The main target of oxidative insult induced by diamide in the water-soluble proteins of the lens is probably the superficially localized sulphydryl groups of crystallins. Our observations suggest that this oxidative system of proteins may be a useful tool for cataract research.

Anaerobic biosynthesis of the manganese-containing superoxide dismutase in Escherichia coli. Effects of diazenedicarboxylic acid bis(N,N'-dimethylamide) (diamide).

Anaerobically grown Escherichia coli accumulate active manganese-containing superoxide dismutase (MnSOD) upon exposure to diamide. This induction requires de novo biosynthesis of MnSOD. Catalase, glutathione disulfide reductase, and glucose-6-phosphate dehydrogenase were also induced by diamide in anaerobic E. coli. A GSH-negative strain of E. coli did not produce MnSOD under anaerobic conditions and was as responsive to diamide as was the wild type strain. Diamide which had been prereduced, by incubation with GSH, was ineffective. NO3- plus paraquat, which elicits increased anaerobic biosynthesis of the MnSOD polypeptide, but not of active MnSOD, synergized with diamide in the induction of active MnSOD. A similar increase in the ability of diamide to cause anaerobic biosynthesis of active MnSOD was seen when the production of the MnSOD polypeptide was increased by isopropyl-beta-D-thiogalactopyranoside, in a strain bearing the MnSOD gene under the control of the tac promoter. These results are explained in terms of a dual action of diamide, i.e. at both the transcriptional and the maturational levels of biosynthesis of MnSOD. Oxidative inactivation of an Fe(II)-containing repressor and oxidative facilitation of insertion of manganese, in place of iron, into the nascent MnSOD polypeptide, are the postulated bases of this dual action.