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Glutathione (GSH) plays a key role in defense against oxidative stress. The availability of GSH is ensured in tissues by systems devoted to its maintenance in the reduced state and by the flux of GSH and cysteine between sites of biosynthesis and sites of utilization. Little is known about the effect of oxidative stress on the distribution of low-molecular-mass thiols and their exchange rate between tissues. In this study, we found that a slow infusion of diamide (a specific thiol-oxidizing compound) evoked a dramatic increase in blood cysteine in rats. Our data suggest that inter-organ exchange of cysteine occurs, that cysteine derives from both glutathione via γ-glutamyl transpeptidase and methionine via homocysteine and the trans-sulfuration pathway, and that these pathways are considerably influenced by oxidative stress. [ABSTRACT FROM AUTHOR]

The effect of the cryoprotectants DMSO and PEG-1500 as well as freezing-thawing on the proteins of the canine erythrocyte membrane-cytoskeleton complex was studied using the cross-linking agent diamide. It was shown that the intensity of disturbances in the protein network structure correlated with the increased SH-group accessibility for oxidative bridging by this compound and accordingly, enhanced formation of high-molecular-weight protein aggregates. The maximum level of diamide-induced aggregability was revealed upon freezing of erythrocytes in liquid nitrogen without cryoprotectant. Electrophoretic analysis of the ghosts of erythrocytes incubated with cryoprotectants showed a significant increase in the aggregation level only for the cells in the polymer solution. After the freezing-thawing cycle, the diamide-induced protein aggregability in erythrocytes cryopreserved with PEG-1500 strongly increased; when DMSO was used for cell protection, the aggregation was much less pronounced than in the unprotected cells. One can suppose that the exocellular cryoprotectant PEG-1500, as distinct from the endocellular cryoprotectant DMSO, is unable to provide for preservation of the structure of the membrane-cytoskeleton protein complex at a level necessary for the maintenance of cell integrity after the return to physiological conditions. [ABSTRACT FROM AUTHOR]

2,2'-(Methylimino)bis(N,N-dioctylacetamide) (MIDOA) was developed as a new extractant for technetium. MIDOA has a similar backbone to TODGA, N,N,N',N'-tetraoctyldiglycolamide, where the nitrogen atom bearing a methyl group replaces the ether oxygen in TODGA. MIDOA is highly lipophilic and ready to use in the HNO3-n-dodecane extraction system. The distribution ratio (D) for Tc(VII) is extremely high. In addition, Cr(VI), Re(VII), Mo(VI), W(VI), Pd(II), and Pu(IV) are well extracted by MIDOA. MIDOA has high selectivity toward certain oxometallates. The D(Tc) values decrease gradually with HNO3, H+, and NO3 - concentrations, and the log D vs log [MIDOA] dependence indicates the species extracted to be the 1:1 metal-ligand complex. It is clear that MIDDA [2,2'-(methylimino)bis(N,N-didodecylacetamide)] and IDDA [2,2'-(imino)bis(N,N-didodecylacetamide)], which have structures analogous to MIDOA, have similar extraction behavior to that of MIDOA. [ABSTRACT FROM AUTHOR]

A cDNA expression library from Brassica juncea was introduced into the fission yeast Schizosaccharomyces pombe to select for transformants tolerant to cadmium. Transformants expressing OXIDATIVE STRESS 3 ( OXS3) or OXS3- Like cDNA exhibited enhanced tolerance to a range of metals and oxidizing chemicals. OXS3 belongs to a family of proteins that share a highly conserved domain corresponding to a putative N-acetyltransferase or thioltransferase catalytic site. Mutations within this conserved domain abolished the ability of Arabidopsis thaliana OXS3 to enhance stress tolerance in S. pombe, indicating a role in stress tolerance for the presumptive catalytic domain. A stress-sensitive mutant of AtOXS3 and enhanced tolerance of overexpression lines support the role of OXS3 in stress tolerance. The expression of tagged B. juncea and A. thaliana OXS3 proteins in plant cells revealed a subnuclear speckling pattern related to the nucleosome in discrete parts of the chromatin. It is possible that OXS3 might act as a chromatin remodeling factor for the stress response. [ABSTRACT FROM AUTHOR]

Abstract: Chromium(VI) compounds (e.g. chromates) are cytotoxic, mutagenic, and potentially carcinogenic. The reduction of Cr(VI) can yield reactive intermediates such as Cr(V) and reactive oxygen species. Bronchial epithelial cells are the primary site of pulmonary exposure to inhaled Cr(VI) and are the primary cells from which Cr(VI)-associated human cancers arise. BEAS-2B cells were used here as a model of normal human bronchial epithelium for studies on the reductive activation of Cr(VI). Cells incubated with Na2CrO4 exhibited two Cr(V) ESR signals, g =1.979 and 1.985, which persisted for at least 1h. The g =1.979 signal is similar to that generated in vitro by human microsomes and by proteoliposomes containing P450 reductase and cytochrome b 5. Unlike many cells in culture, these cells continued to express P450 reductase and cytochrome b 5. Studies with the non-selective thiol oxidant diamide indicated that the g =1.985 signal was thiol-dependent whereas the g =1.979 signal was not. Pretreatment with phenazine methosulfate eliminated both Cr(V) signals suggesting that Cr(V) generation is largely NAD(P)H-dependent. ESR spectra indicated that a portion of the Cr(VI) was rapidly reduced to Cr(III). Cells incubated with an insoluble chromate, ZnCrO4, also generated both Cr(V) signals, whereas Cr(V) was not detected with insoluble PbCrO4. In clonogenic assays, the cells were very sensitive to Na2CrO4 and ZnCrO4, but considerably less sensitive to PbCrO4. [Copyright &y& Elsevier]

Abstract: Changes of the redox balance in cells alter the availability of intracellular free Zn2+. Here, cells were exposed to ultraviolet (UV)-A, UV-B, or infrared (IR)-A light irradiation, and the intracellular free zinc pool was monitored. Under sublethal conditions only UV-A irradiation resulted in a transient cytoplasmic and nuclear increase of intracellular free Zn2+. Likewise, tert-butyl hydroperoxide and singlet oxygen, but not H2O2 or intracellular generation of O2˙ˉ by redox cyclers, mimicked the effects of UV-A irradiation, while disulfide stress by diamide only led to a transient cytoplasmic zinc release. These results show that only certain types of subtoxic cellular stress massively disturb the zinc homeostasis in cells. [Copyright &y& Elsevier]

Abstract: Cystein residues within metallothionein (MT) structure have been shown to be particularly prone to S-nitrosylation. The objective of this study was to examine the possibility that MTs undergo S-glutathionylation under nitrosative/oxidative stress. MT from rabbit liver was treated with different concentrations of GSNO, diamide plus GSH or H2O2 plus GSH. Parallel sets of samples were treated with 10mM DTT for 30min at 37°C to reduce mixed disulphides. Incubations were then processed for Western blot or dot-immunobinding assay. Western blot with anti-MT or anti-GSH were also performed on peripheral blood mononuclear cell extracts. Structural aspects of S-glutathionylation of MTs were also examined. Treatment with GSNO, diamide/GSH or H2O2/GSH induced a dose-dependent increase in the levels of MT S-glutathionylation. This effect was completely reversed by treatment with the reducing agent DTT, indicating that S-glutathionylation of MT protein was related to formation of protein-mixed disulphides. Structural analysis of rat MT indicated that Cys residues located in the N-terminal domain of the protein are the likely targets for S-glutathionylation, both for their solvent accessibility and electrostatics induced reactivity. S-Glutathionylation of MT, given its reversibility, would provide protection from irreversible oxidation of Cys residues, thus representing a mechanism of high potential biological relevance. [Copyright &y& Elsevier]

The effect of chloride and diamide on angiotensin converting enzyme (ACE) activity from sheep kidney, lung and serum was investigated by using Hip-His-Leu as substrate. Optimum chloride concentrations were 400–1000 mM for kidney, 700 mM for lung and 1000 mM for serum. Optimum chloride concentration increased ACE activity of serum and lung 1.70 folds and 2.73 folds respectively of the activity at physiological chloride concentrations, suggesting that the effect of salt on blood pressure may be due to the chloride sensitivity of ACE. The difference in effect of chloride on lung, kidney and serum ACE suggest that each tissue ACE has unique three dimentional structure. Increased pulmonary and serum ACE activity on pretreatment with diamide indicates that tissue oxidation may alter blood pressure. [ABSTRACT FROM AUTHOR]

New dinaphthosulfide podands [3(a, b, and c)] were synthesized. The structures of these compounds were confirmed by IR, 1H NMR, 13C NMR, and Mass spectra. A simple, efficient, and sensitive procedure is described for the spectrophotometric determination of a trace amount of Ag+, Hg2+, Cr3+, Pb2+, and Ni2+ cations in the presence of Mn2+, Co2+, Cd2+ cations using the podand (3a). Formation constants of 1:1 complexes were determined from the absorbance-mole ratio data and found to vary in the order Pb2+ > Ni2+ > Hg2+ > Cr3+ > Ag+. [ABSTRACT FROM AUTHOR]

Abstract: The article describes the synthesis and evaluation of the dichromate anion (Cr2O7 2−/HCr2O7 −) extraction properties of p-tert-butylcalix[4]arene diamide derivatives (5–7) containing different binding sites. Among these compounds, 6 and 7 have been synthesized via aminolysis in a toluene–methanol solvent system with 3-aminomethylpyridine and 3,6-dioxa-1,8-diamino octane, respectively. On the other hand, compound 5 has been synthesized via an acid chloride method due to its inefficiency under aminolysis. The extraction properties of these diamides toward dichromate anions are studied by liquid–liquid extraction. The results show that p-tert-butylcalix[4]arene diamide derivative 7 exhibited a much higher affinity toward dichromate anions than that of 6 due to its special structure, while 5 was an ineffective ligand for these anions. [Copyright &y& Elsevier]

A series of 4-acylated derivatives of piperazine-2,6-dione were synthesized. Ammonolysis of the synthesized piperazine-2,6-diones with aqueous ammonia led to a ring-opening reaction and the formation of corresponding iminodiacetic acid diamides with a good yield. The optimal conditions of ammonolysis were analysed. [ABSTRACT FROM AUTHOR]

The synthesis of novel diamide derivatives of calix[4]arene by aminolysis of calix[4]arene diesters was reported. The 1H and 13C NMR, data showed that the synthesized compounds exist in the cone conformation. The complexation properties of these calix[4]arenes have been studied towards [image omitted] anions and it has been found that the protonated alkyl ammonium forms of calix[4]arene diamide derivatives (5,11,17,23-tetra-tert-butyl-25,27-(diethylpiperidineacetamido)26,28-dihydroxy-calix[4]arene 3 and 25,27-(diethyl-piperidineacetamido)26,28-dihydroxycalix[4]arene 4) are effective extractants for transferring [image omitted] anions. [ABSTRACT FROM AUTHOR]

Cellular redox change regulates pulmonary vascular tone by affecting function of membrane and cytoplasmic proteins, enzymes, and second messengers. This study was designed to test the hypothesis that functional modulation of ion channels by thiol oxidation contributes to regulation of excitation-contraction coupling in isolated pulmonary artery (PA) rings. Acute treatment with the thiol oxidant diamide produced a dose-dependent relaxation in PA rings; the IC50 was 335 and 58 µM for 40 mM K+- and 2 µM phenylephrine-induced PA contraction, respectively. The diamide-mediated pulmonary vasodilation was affected by neither functional removal of endothelium nor 8-bromoguanosine-3′-5′-cyclic monophosphate (50 µM) and HA-1004 (30 µM). A rise in extracellular K+ concentration (from 20 to 80 mM) attenuated the thiol oxidant-induced PA relaxation. Passive store depletion by cyclopiazonic acid (50 µM) and active store depletion by phenylephrine (in the absence of external Ca2+) both induced PA contraction due to capacitative Ca2+ entry. Thiol oxidation by diamide significantly attenuated capacitative Ca2+ entry-induced PA contraction due to active and passive store depletion. The PA rings isolated from left and right PA branches appeared to respond differently to store depletion. Although the active tension induced by passive store depletion was comparable, the active tension induced by active store depletion was 3.5-fold greater in right branches than in left branches. These data indicate that thiol oxidation causes pulmonary vasodilation by activating K+ channels and inhibiting store-operated Ca2+ channels, which subsequently attenuate Ca2+ influx and decrease cytosolic free Ca2+ concentration in pulmonary artery smooth muscle cells. The mechanisms involved in thiol oxidation-mediated pulmonary vasodilation or activation of K+ channels and inhibition of store-operated Ca2+ channels appear to be independent of functional endothelium and of the cGMP-dependent protein kinase pathway. [ABSTRACT FROM AUTHOR]

The synthesis of mono and diamide derivatives of the p‐tert‐butylcalix[4]arenes/calix[4]arenes from the reaction of 5,11,17,23‐tetra‐tert‐butyl‐25,27‐diethoxycarbonyl‐methoxy‐26,28‐dihydroxycalix[4]arene 1 and 25,27‐diethoxycarbonyl‐methoxy‐26,28‐dihydroxycalix[4]arene 2 with various primary amines were reported. The effect of reaction time and steric hindrance of the primary amines used on the reactions have been investigated. All the amide derivatives of calix[4]arene are in a cone conformation according to the 1H‐NMR doublet–doublet pattern of the protons of the methylene groups between the aromatic rings. The complexing properties of these compounds toward selected alkali and alkaline earth metal cations are also studied. It has been observed that receptor 8 is a selective extractant for Cs+ and Sr2+ cations. [ABSTRACT FROM AUTHOR]

Extraction of nitric acid, perchloric acid, americium, and europium with dialkyldiarylamides of 2,6-pyridinecarboxylic (dipicolinic) acid in polar fluorinated solvents (diluents) was analysed. Among the extractants studied N, N′-diethyl-N, N′-ditolylamide dipicolinate is characterized by the best extraction properties. On passing from nitric to perchloric acid solutions extraction of metals sharply increases. Extraction of americium from nitric acid solutions is significantly greater as compared with europium (separation coefficient reaches 6), whereas extraction of americium and europium from perchloric acid solutions is nearly similar. The effect of diluent on the extraction was analyzed; for different diluents the maximum of extraction from perchloric acid solutions shifts form 3 to 6 M. [ABSTRACT FROM AUTHOR]

Abstract: In this work, protein–glutathione mixed disulfide formation in human red blood cells (RBCs) was evaluated in vitro by using the thiol-specific reagent diamide. We investigated what mechanism could lead to S-glutathionylation of membrane skeletal proteins, what are the main target proteins, and the correlation between protein S-glutathionylation and RBC hemolysis. Diamide caused a decrease in the reduced form of glutathione (GSH), which was accompanied by an increase in the basal level of glutathione disulfide (GSSG) and in S-glutathionylation of protein 4.2 and spectrin. The increase in membrane skeletal protein S-glutathionylation was correlated with a lower susceptibility of RBCs to osmotic hemolysis, suggesting that S-glutathionylation of protein 4.2 and spectrin could contribute to regulate RBC membrane stability. [Copyright &y& Elsevier]

Abstract: A cysteine-rich metal binding protein MT (metallothionein) (named BmtA) is induced upon exposure to multiple heavy metal ions in the freshwater cyanobacterium Oscillatoria brevis. The SmtB/ArsR family repressor BxmR from O. brevis represses the expression of an operon encoding bmtA and bxmR. In the present study, the expression of bmtA was induced in vivo by diamide, a specific thiol oxidant, in O. brevis cells. In vitro electrophoretic gel mobility shift experiments revealed that the incubation with diamide induces disassembly of the BxmR-bxmR/bmtA operator (O)/promoter (P) complex [multiple resolvable complexes of BxmR with oligonucleotide (named P5) containing a single 12-2-12 inverted repeat derived from the O/P region of bxmR/bmtA]. Thus, the exposure to diamide induces MT mRNA in O. brevis, and this induction is associated with diamide-mediated inhibition of BxmR-P5 complex. BxmR is more sensitive to diamide than to H2O2. Furthermore, pretreatment of O. brevis with Zn decreased intracellular peroxidation products caused by diamide. Thus, these results imply that MT induced by Zn-pretreatment functions to protect O. brevis cells against diamide stress. [Copyright &y& Elsevier]

Glucose transport is stimulated in a variety of cells and tissues in response to inhibition of oxidative phosphorylation. However, the underlying mechanisms and mediating steps remain largely unknown. In the present study we first tested whether a decrease in the redox state of the cell per se and the resultant increase in generation of reactive oxygen species (ROS) lead to stimulation of glucose transport. Clone 9 cells (expressing the Glut1 isoform of facilitative glucose transporters) were exposed to azide, lactate, and ethanol for 1 h. Although all three agents stimulated glucose transport and increased cell NADH-to-NAD+ ratio and phospho-ERK1/2, signifying increased ROS generation, the response to the stimuli was not blocked by N-acetyl-L-cysteine (an agent that counteracts ROS); moreover, the response to azide was not blocked by diamide (an intracellular sulfhydryl oxidizing agent). We then found that cell AMP-to-ATP and ADP-to-ATP ratios were increased and 5′-AMP-activated protein kinase (AMPK) was stimulated by all three agents, as evidenced by increased phosphorylation of AMPK and acetyl-CoA carboxylase. We conclude that although azide, lactate, and ethanol increase NADH-to-NAD+ ratios and ROS production, their stimulatory effect on glucose transport is not mediated by increased ROS generation. However, all three agents increased cell AMP-to-ATP ratio and stimulated AMPK, making it likely that the latter pathway plays an important role in the glucose transport response. [ABSTRACT FROM AUTHOR]

Abstract: The combination of proteomics with highly specific and sensitive affinity techniques is important for the identification of posttranslational modifications by reactive oxygen and nitrogen species (ROS/RNS). One of the most pressing problems with this approach is to determine accurately the extent of modification of specific amino acids, such as cysteine residues, in a complex protein sample. A number of techniques relevant to free radical biology use biotin tagging as a method to follow protein modification with high sensitivity and specificity. To realize the potential of this approach to provide quantitative data, we have prepared a series of biotinylated proteins through the modification of lysine residues. These proteins were then used as quantitative standards in electrophoretic separation of protein samples labeled with biotin-conjugated iodoacetamide. The utility of the approach was assessed by measuring modification of thiols in response to exposure to thiol oxidants, as well as the amount of protein adduct formation with a biotin-tagged electrophilic lipid. Furthermore, using a combination of native and biotin-tagged cytochrome c, this method was used to quantitate the amount of thiol relative to the amount of protein in a given spot on a two-dimensional gel. Thus, we have developed a versatile, cost-effective standard that can be used in proteomic methods to quantitate biotin tags in response to oxidative stress. [Copyright &y& Elsevier]

Oxidant-mediated modulation of the intracellular redox state affects the apoptotic cascade by altering the balance between cellular signals for survival and suicide. Apolipoprotein A-IV (Apo A-IV) is known to possess antioxidant-like activity. In the present study, we tested 1) whether Apo A-IV could influence redox-dependent apoptosis and, if so, 2). Whether such an effect could be mediated by modulation of intracellular redox balance. Mitotic competent, undifferentiated PC-12 cells were incubated with either tert-butyl hydroperoxide (TBH) or diamide with or without preincubation with human Apo A-IV. Apo A-IV significantly decreased apoptosis produced by both TBH and diamide, and washout of A-IV before incubation with TBH and diamide did not eliminate its protective effect. Apo A-I had no such protective effect. The Apo A-IV effect was not blocked by D,L-buthionine-[S,R]-sulfoximine, but it was reversed by both dehydroisoandrosterone and transfection with an anti sense oligode-oxynucleotide to glucose-6-phosphate dehydrogenase (G6PD). Apo A-IV abolished the transient, oxidant-induced rise in glutathione disulfide (GSSG) and cellular redox imbalance previously shown to initiate the apoptotic cascade. Apo A-IV had no effect on GSSG reductase activity, but it stimulated G6PD activity 10-fold. These results suggest a novel role for Apo A-IV in the regulation of intracellular glutathione redox balance and the modulation of redox-dependent apoptosis via stimulation of G6PD activity. [ABSTRACT FROM AUTHOR]

Abstract: A novel class of anthranilic diamides has been discovered with exceptional insecticidal activity on a range of Lepidoptera. These compounds have been found to exhibit their action by release of intracellular Ca2+ stores mediated by the ryanodine receptor. The discovery, synthesis, structure–activity, and biological results are presented. [Copyright &y& Elsevier]

Abstract: The synthesis of four diamide derivatives of the p-tert-butylcalix[4]arenes from the reaction of 5,11,17,23-tetra-tert-butyl-25,27-diethoxycarbonylmethoxy-26,28-dihydroxycalix[4]arene 2 with various primary amines were reported. The 1H and 13C NMR, data showed that the synthesized compounds exist in the cone conformation. The complexing properties of these compounds toward Cr2O7 2−/HCr2O7 − anions are also studied. It has been observed that receptors 5 and 6 are better extractant than the compounds 3 and 4 . The protonated alkyl ammonium form of 5 and 6 is an effective extractant for transferring HCr2O7 −/Cr2O7 2− anions from an aqueous phase into a dichloromethane layer. [Copyright &y& Elsevier]

Abstract: In rat heart mitochondria, auranofin, arsenite, diamide, and BCNU increase H2O2 formation, further stimulated by antimycin. However, in submitochondrial particles, H2O2 formation and oxygen uptake are not affected, indicating that these substances do not alter respiration. Mitochondria are also able to rapidly metabolize added H2O2 in a process partially prevented by BCNU or auranofin. Calcium does not modify the production of H2O2 and the mitochondrial thioredoxin system is not affected by calcium ions. Auranofin, arsenite, and diamide determine a large mitochondrial permeability transition, while BCNU and acetoacetate are ineffective. Thiols and glutathione are modified only by BCNU and diamide. However, all the compounds tested cause the release of cytochrome c that occurs also in the absence of mitochondrial swelling. In conclusion, the compounds utilized share the common feature of shifting the mitochondrial thiol-linked redox balance towards a more oxidized condition that is responsible of the observed effects. [Copyright &y& Elsevier]

Abstract: The cellular response of mycobacteria to thiol specific oxidative stress was studied in Mycobacterium bovis BCG cultures. Two-dimensional gel electrophoresis revealed that upon diamide treatment at least 60 proteins were upregulated. Fourteen of these proteins were identified by MALDI-MS; four proteins, AhpC, Tpx, GroEL2, and GroEL1 are functionally related to oxidative stress response; eight proteins, LeuC, LeuD, Rv0224c, Rv3029c, AsnB, Rv2971, PheA and HisH are classified as part of the bacterial intermediary metabolism and respiration pathways; protein EchA14 belong to lipid metabolism, and NrdE, belongs to the mycobacterial information pathway category. Reverse transcription followed by quantitative real time PCR in response to diamide stress demonstrated that protein expression is directly proportional to the corresponding gene transcription. [Copyright &y& Elsevier]

Abstract: The sigH gene of Corynebacterium glutamicum encodes ECF sigma factor σH. The gene apparently plays an important role in other stress responses as well as heat stress response. In this study, we found that deleting the sigH gene made C. glutamicum cells sensitive to the thiol-specific oxidant diamide. In the sigH mutant strain, the activity of thioredoxin reductase markedly decreased, suggesting that the trxB gene encoding thioredoxin reductase is probably under the control of σH. The expression of sigH was stimulated in the stationary growth phase and modulated by diamide. In addition, the SigH protein was required for the expression of its own gene. These data indicate that the sigH gene of C. glutamicum stimulates and regulates its own expression in the stationary growth phase in response to environmental stimuli, and participates in the expression of other genes which are important for survival following heat and oxidative stress response. [Copyright &y& Elsevier]

An ERK MAP kinase-mediated contractile mechanism previously reported to be activated by peroxide and stretch in bovine coronary arteries is shown in this study to be present in endothelium-denuded bovine pulmonary arteries and subject to regulation by modulation of cytosolic NAD(H) redox through the lactate dehydrogenase reaction. Although our previous work identified an acute Po2-dependent per- oxide-mediated relaxation of bovine pulmonary arteries on exposure to lactate, a 30-mm treatment with 10 mM lactate enhanced ERK phosphorylation and increased force generation to 30 mM KCl. Hypoxia inhibited these responses to lactate. Increases in ERK phosphorylation and the enhancement of force generation by lactate and stretch are attenuated in the presence of inhibitors of Nox oxidase (0.1 mM apocynin) or ERK activation (10 µM PD-98059) and by 0.1 mM ebselen. Additionally, incubation of pulmonary arteries with 10 mM pyruvate lowered basal levels of ERK phosphorylation, and it inhibited both the ERK phosphorylation and the enhancement in force generation to 30 mM KCl caused by stretch. Treatment of pulmonary arteries with the thiol oxidant diamide (1 µM) elicited what appears to be a peroxide-independent increase in force and ERK phosphorylation that were both attenuated by PD-9 8059. Thus pulmonary arteries possess a peroxide-elicited contractile mechanism involving ERK MAP kinase, which is stimulated by stretch and regulated through the control of Nox oxidase activity by the availability of cytosolic NADH. [ABSTRACT FROM AUTHOR]

Abstract: Protein–glutathione mixed disulfide formation was investigated in vitro by exposure of human platelets to the thiol-specific oxidant azodicarboxylic acid-bis-dimethylamide (diamide). We found that diamide causes a decrease in the reduced form of glutathione (GSH), paralleled by an increase in protein–GSH mixed disulfides (S-glutathionylated proteins), which was not accompanied by any significant increase in the basal level of glutathione disulfide (GSSG). The increase in the appearance of S-glutathionylated proteins was inversely correlated with ADP-induced platelet aggregation. Platelet cytoskeleton was analyzed by SDS–PAGE followed by Western immunoblotting with anti-GSH antibody. The main S-glutathionylated cytoskeletal protein proved to be actin, which accounts for 35% of the platelet total protein content. Our results suggest that neither GSSG formation nor a consequent thiol–disulfide exchange mechanism is involved in actin S-glutathionylation of human platelets exposed to diamide. Instead, a mechanism involving the initial oxidative activation of actin thiol groups, which then react with GSH to the protein–GSH mixed disulfides, makes it likely that platelet actin is S-glutathionylated without any significant increase in the GSSG content. [Copyright &y& Elsevier]

Abstract: Bach1 is a transcriptional repressor of heme oxygenase-1, one of the most inducible phase 2 proteins. Bach1 binds in conjunction with a small Maf protein to tandem repeats of the antioxidant response element (ARE) and quenches the target gene expression. On the other hand, the transactivator Nrf2 binds and up-regulates the ARE-governed gene expression. By using a sulfhydryl oxidizing agent, diamide, here we provide evidence which indicates that the Bach1 function is regulated by the redox state. Diamide showed restricted Nrf2 nuclear translocation and ARE-driven reporter activity but reversed the ARE transcriptional activity suppressed by ectopically expressed Bach1. Substitution of the conserved cysteine residue in the DNA binding domain of Bach1 to serine (C574S mutant) caused a refractory response to the diamide-mediated reactivation of the Bach1-suppressed reporter activity. Moreover, diamide induced cytoplasmic translocation of the GFP–Bach1 fusion protein but failed to translocate the fusion protein consisting of the C574S mutant. These data suggest that redox regulation of Bach1 is an alternative mechanism to induce multiple ARE-governed genes. [Copyright &y& Elsevier]

Abstract: The extraction of 75 elements by N,N,N′,N′-tetraoctyl-3-oxapentanediamide (TODGA) from nitric acid to n-dodecane was investigated and discussed concerning distribution ratios (D) of metal ions and their ionic radii. The extractability for elements depends strongly on their oxidation state. Monovalent and pentavalent ions were not extractable. In divalent metal extraction, Ca(II) (ionic radius: 100pm) showed the highest D-value and the D decreased with increase or decrease of the ionic radius. Trivalent and tetravalent ions with ionic radii of 87–113 and 83–94pm, respectively, showed the D over 1000, and the extracted chemical forms were determined to be M(TODGA)3(NO3)n or M(TODGA)4(NO3)n (n=3 or 4). The oxonium ions with higher oxidation states than tetravalent one forms the complicated metal-complex with TODGA, and the number of TODGA associated in those extraction reactions was confirmed to be two at most. [Copyright &y& Elsevier]

UDP-galactose 4-epimerase from Kluyveromyces fragilis is a stable homodimer of 75 kDa/subunit with non-covalently bound NAD acting as cofactor. Partial proteolysis with trypsin in the presence of 5′-UMP, a strong competitive inhibitor, led to a degraded product which was purified. Results from SDS–PAGE, size-exclusion (SE)-HPLC and ultracentrifugation indicated its monomeric status and size between 43 and 45 kDa. `Two-step assay'' with UDP-glucose dehydrogenase as coupling enzyme in the presence of NAD ensured epimerase activity of the monomer. The possibility of transient dimerization of monomeric epimerase during catalysis was excluded by SE-HPLC in the presence of excess substrate and NAD. This truncated enzyme retained catalytic site related properties like Km for UDP-galactose, `NADH-like coenzyme fluorescence'' and `reductive inhibition'' similar to its dimeric counterpart. Reversible reactivation of the monomer was achieved up to 95% within 3 min from 8 M urea induced unfolded state, indicating that the catalytic site could form independent of its quaternary structure. Equilibrium unfolding between 0 and 8 M urea indicated that the monomer was less stable compared to the dimer. Chemical modification of amino acids and reconstitution with etheno-NAD suggested that the architecture around the catalytic site of the monomer was conserved. Specific modification reagents further confirmed that the cysteine residues required for catalysis and coenzyme fluorophore reside exclusively on a single subunit negating a `subunit sharing model'' of its catalytic site. [Copyright &y& Elsevier]

A chiral diamide stationary phase (CSP) with different length of spacers has been synthesized by chemically bonding the N-(3,5-dimethylbenzoyl)-d-phenylglycine on the aminopropylsilica gel or silica gel bonded with different carbon chain length of spacers. These CSPs have successfully been used for the chiral resolutions of the benzoyl-dl-octylamine and benzoyl-dl-amino acid isopropyl esters in normal-phase liquid chromatography (NP-LC). The spacer length and the introduction of a secondary amine group in the CSPs have been investigated. It has been found that a long spacer of CSPs generally benefits on the enantioselectivity. The introduction of secondary amine group in the spacer leads to the decline of separation factors. Further, the effect of organic modifiers has been examined. Proper use of organic modifiers resulted in an interesting ‘masking effect’ on the residing silanol groups of the CSPs. The addition of suitable amount of organic modifiers improves the enantioselectivity, and enantioselectivity depends on the shape of organic modifiers in the mobile phases. [Copyright &y& Elsevier]

Cellular thiol groups modulate various aspects of cellular function, including cell death. In this study, we found that a thiol oxidant, diamide, induced morphological changes such as cell swelling, membrane blebbing, and chromatin condensation in Aplysia cultured sensory neurons. Furthermore, diamide induced biphasic changes in the membrane potential, where hyperpolarization was followed by depolarization. Moreover, these diamide-induced cytotoxic effects were completely blocked by the equimolar addition of the disulfide reducing agent dithiothreitol (DTT). We also found that during H2O2-induced cell death, DTT attenuated cell swelling and membrane blebbing, but not DNA breakage, whereas the vitamin E analogue trolox attenuated DNA breakage, but not cell swelling and membrane blebbing. These results demonstrate that during H2O2-induced cell death, apoptotic features such as DNA breakage are mediated in part by free radical generation, whereas necrotic features such as cell swelling and membrane blebbing are primarily mediated by the oxidation of cellular thiol groups. [Copyright &y& Elsevier]

The extraction properties of the trivalent lanthanides (Ln(III)) with the bidentate N,N′-dimethyl-N,N′-diphenyl-malonamide (MA) and the tetradentate N,N′-dimethyl-N,N′-diphenyl-3,6-dioxaoctanediamide (DOODA) were investigated. These diamides formed by coupling two amide groups with methylene and/or ether groups are bidentate for the MA and tetradentate for the DOODA. By adding a previous data regarding the tridentate N,N′-dimethyl-N,N′-diphenyl-diglycolamide (DGA), these extraction results enabled us systematically study an effect of number of oxygen donor on its extraction behavior of Ln(III). The change in the distribution ratios (Ds) of Lu(III) with an increase in the HNO3 concentration is greater than that of La(III) in both the MA and DOODA systems. Therefore, the relationship between the D and atomic number, i.e., the lanthanide pattern, changes with the HNO3 concentration: the Ds decrease with an increasing atomic number at lower HNO3 concentrations. The Ds of the lighter Ln(III) are similar to the Ds of the heavier Ln(III) at higher HNO3 concentrations. The number of the extractant in the extracted species for La(III) and Lu(III) obtained from slope analysis at 4 M HNO3 in the MA system are about 3, while those in the DOODA system are quite different, i.e., 2 for La(III) and 1.5–3 for Lu(III). The comparison of the extractability of Ln(III) by MA, DOODA, and DGA shows that the magnitude of the Ds is in the sequence of MA < DOODA ≪ DGA. This suggests the introduction of one ether oxygen atom to the principal chain in the diamides leads to a good extractability for the Ln(III) from HNO3 solution. [ABSTRACT FROM AUTHOR]

Photo-oxidation of large malonamides N,N′-dimethyl, N,N′-dibutyl, tetradecyl malonamide (DMDBTDMA) and N,N′-dimethyl, N,N′-dibutyl, dodecylethoxy malonamide (DMDBDDEMA)—potential extractants for minor actinides in nuclear fuel reprocessing—leads mainly to the corresponding monoamides: N-butyl N-methyl hexadecanamide (BMHDA) and N-butyl N-methyl dodecyl oxy butanamide (BMDDOBA), respectively. The evolution of these main photoproducts was monitored during the degradation of malonamide solutions, and also pure monoamide solutions. Irradiation, for 1 h, with a low mercury pressure lamp can destroy BMHDA when diluted to 0.075 mol l−1 in n-dodecane. The two monoamides only differ by an ether bond and the weakness introduced by such a function in an alkyl chain is shown by slightly faster degradation. The relative weakness of BMDDOBA is independent of oxidant addition. Global degradation can be monitored by GC/FID or UV detection at 200 nm. [Copyright &y& Elsevier]

An overview of the work carried out on the development of amides/diamides as alternate extractants in the back end of fuel cycle has been given. Whereas a straight chain amide, viz. N,N-dihexyl octanamide (DHOA) is a promising alternate to tri-n-butyl phosphate (TBP) in the reprocessing of irradiated uranium-based fuels, a branched chain amide, viz. N,N-di(2-ethylhexyl) isobutyramide (D2EHIBA) has been found suitable for the selective recovery of 233U from irradiated Th. DHOA has been found to extract Pu more efficiently than TBP under uranium loading conditions. On the other hand, significant improvement in the separation of 233U from Th and fission products has been achieved by employing 0.5 M D2EHIBA in n-dodecane as compared to that of 5% TBP (the THOREX solvent). Stripping behaviour of U for DHOA and D2EHIBA is better than that of TBP. N,N′,N,N′-dimethyl dibutyl tetradecyl malonamide (DMDBTDMA) has been evaluated for the partitioning of minor actinides (MA) from high-level nuclear waste with large uranium content (∼20 g/l). Designed amide ligands, viz. D2EHIBA showed improved separation of the desired actinide constituents like Pu (mainly 239Pu) and 233U over the fission products and the major matrix elements like U and Th. [Copyright &y& Elsevier]

The dithiol-reducing thioredoxin/thioredoxin reductase system normally maintains the reduced state of key enzymes responsible for the cell’s anti-oxidant defences. We therefore addressed the question of whether AW 464—a novel thioredoxin inhibitor—as well as broad spectrum dithiol ligands diamide and phenylarsine oxide are able to induce and execute a regular apoptotic sequence of events without overwhelming the cell’s ability to detoxify reactive oxygen species. All three agents were found to target the thioredoxin system in a cell-free assay. In HL-60 leukaemia cells, they were also found to induce Bak activation, cytochrome c release from mitochondria, ↓ΔΨm, chromatin condensation, phosphatidyl serine exposure and Tdt-sensitive DNA nicks. At the onset of apoptosis there was no evidence of increases in oxygen free radicals or peroxide in cells treated with AW 464 or diamide. Phenylarsine oxide induced both free radicals and hydrogen peroxide, but this did not appear to interfere with apoptosis. We conclude that pharmacological targeting of thioredoxin can induce a well-orchestrated apoptotic programme. [Copyright &y& Elsevier]

A series of new polymerizable non-ionic and ionic surfactants (surfmers) with amides groups on both sides of the C=C double bonds have been prepared upon reaction of maleic isoimide carrying a long alkyl chain (or a benzyl group) with a hydrophilic amine derivative. Their critical micellar concentration (CMC) was measured with a surface tensiometer. They have been engaged in batch emulsion polymerization of styrene, and semi-batch seeded copolymerization of styrene and butyl acrylate, giving stable latexes during the polymerization process, and upon extraction with ethanol, showing a high rate of incorporation at the particle surface. However these surfmers do not confer good steric stabilization properties, which may be expected from the use of non-ionic surfactants. To cite this article: I. Klimenkovs et al., C. R. Chimie 6 (2003). [Copyright &y& Elsevier]

The red cell deformation under the conditions of oscillating centrifugal fields was studied. Experiments were carried out with a modified Cell-Elastometer operating in oscillating mode (0.02 to 0.30 Hz). Gravitational acceleration was sinusoidally modulated between 620g and 2250g. At low frequencies (below 0.08 Hz), native red cells followed the applied stress without delay. At 0.09 Hz and up, the cellular deformation was still periodical and included an additional perturbation due to intracellular movements. This perturbation was analysed and quantified. The influence of alterations on the erythrocyte membrane by diamide was analysed to verify the sensitivity of this method. On increasing the membrane stiffness with low concentrations of diamide, the response to oscillatory centrifugal stress was impaired characteristically in terms of amplitude deformation. Based on tangential and centrifugal accelerations, a physical model was developed that describes the basic observable changes on varying the oscillation frequency. From the data it can be concluded that viscoelastic properties of red cells can be analysed and quantified using oscillatory centrifugal accelerations. The described method can become a valid tool to differentiate between membrane alterations or intracellular viscous modifications. [ABSTRACT FROM AUTHOR]

In order to study whether the endothelial cells (ECs) with lipid peroxidation induced by diamide can express and secrete macrophage inflammatory protein 1α (MIP-1α), the expression of MIP-1α protein in the cells was detected by cell enzyme-linked immunosorbent assay (ELISA) and that of MIP-1α mRNA was determined by cell in situ hybridization and nuclease S1 protection assay after the ECs were exposed to different concentrations of diamide for 4 h. The chemotactic activity of MIP-1α was tested by micropore filter method using modified Boyden chambers. Cell ELISA showed that the expression of MIP-1α protein in endothelial cells exposed to 1 μmol/L, 5 μmol/L and 10 μmol/L diamide was 1.9-fold, 2.3-fold and 1.7-fold respectively as much as that in the control cells, which was statistically significant by analysis of variance. In situ hybridization revealed that the mRNA expression of ECs treated with 1 μmol/L, 5 μmol/L and 10 μmol/L diamide was 1.3-fold, 3.0-fold and 1.7-fold as much as that in the control group, which had statistical significance ( F=188. 93, P<0.01). The mRNA expression in 5 μmol/L dimide treated ECs, measured by nuclease S1 protection assay, was 3.4-fold as much as that in the control group ( t=8.70, P<0.05). Chemotactic response (99.50±4.31 μm) to the culture medium conditioned by 5 μmol/L diamide treated ECs, which was stronger than that (66.47±3.25 μm) conditioned by the ECs ( F=404.31, P<0.05), was significantly decreased ( F=192.25, P<0.05) after adding MIP-1α antibody. It suggests that diamide, a lipid peroxidation inducer, could stimulate ECs to produce high level of MIP-1α, and might play an important role in atherogenesis by promoting the migration of peripheral blood monocytes into arterial intima. [ABSTRACT FROM AUTHOR]

In this work, a new diamide-type chiral stationary phase was developed by chemically bonding the N-stearoyl-L-leucine on the aminopropylsilica gel. It has been successfully used for the chiral resolution of the benzoyl-, dinitrobenzoyl-, and trifluoroacethyl amino acids in the modes of normal phase, as well as for the underivatized amino acids in the mode of reversed phase. It was found that the hydrogen bonding makes significant contribution to the chiral resolution. It has been demonstrated that the chiral resolution, by the use of diamide-type chiral stationary phase (CSP), can be achieved without the π–π interaction between the CSP and analytes. [ABSTRACT FROM AUTHOR]

In order to investigate the relationship between the pore diameter and the rate of uptake of U(VI) ions and Ce(III) ions, N,N,N′,N′-tetramethylmalonamide (TMMA) chelating resins with different pore diameters were synthesized, using different pore producing solvents. The batch technique was used to study the kinetics of the adsorption U(VI) and Ce(III) ions from 3 M HNO3, acid, and 3 M NaNO3+0.05 M HNO3, salt, media. The process was found to be controlled by the particle diffusion model. The calculated values for the diffusion coefficient were found to be directly proportional to the pore diameter, i.e. the resin with the largest pore diameter showed the fastest rate of uptake. [Copyright &y& Elsevier]

The photo-oxidation of two potential extractants, the N,N′-dimethyl, N,N′-dibutyl, tetradecyl malonamide (DMDBTDMA) and the N,N′-dimethyl, N,N′-dibutyl, dodecylethoxy malonamide (DMDBDDEMA) for minor actinides in nuclear fuel reprocessing was followed. The main photoproducts, identified by coupled techniques (GC/FTIR and GC/MS) are malonamides, monoamides and carbonylate derivatives. An irradiation, for 1 h, with a low mercury pressure lamp can destroy initial diamides diluted at 0.075 mol l−1 in n-dodecane. With pure solutions of diamides (2.05 mol l−1), destruction needs several hours to be complete. Both diamides only differ by an ether bond, their reactivity under different conditions of photo-oxidation is discussed. [Copyright &y& Elsevier]

: ObjectiveTo investigate the influence of human sperm nuclear chromatin on fertilization.: DesignProspective study.: SettingAssisted reproductive technology unit at a university teaching hospital.: Patient(s)Fifty men starting an IVF-ET program.: Intervention(s)Epifluorescent microscopic observation of human-ejaculated sperm nuclei stained with diamide–acridine orange. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis of extracted sperm nucleoproteins.: Main outcome measure(s)Usefulness of diamide–acridine orange in analysis of human sperm nuclear chromatin and fertilization ability.: Result(s)There was no correlation between the semen parameters and the diamide–acridine orange observation. A positive correlation was observed between the fertilization rate after conventional IVF and the green-type increase ratio (percentage of green-pattern sperm after diamide–acridine orange staining/percentage of green-pattern sperm after acridine orange staining). Furthermore, it was suggested by SDS-PAGE that structural differences were noticed between the fertile men and the men with sperm immaturity diagnosed after diamide–acridine orange staining.: Conclusion(s)Diamide–acridine orange staining was a more precise method for detecting chromatin abnormalities in human-ejaculated sperm and evaluating fertilization ability than acridine orange staining alone. This method can be used as a diagnostic tool to assess the fertilization ability of human-ejaculated spermatozoa before IVF procedures. [Copyright &y& Elsevier]

In order to separate U(VI) ions and lanthanide ions from acidic media, N,N,N′,N′-tetramethylmalonamide, (TMMA), chelating resin was synthesized by chemically bonding the functional group to the main chain of chloromethylstyrene–divinylbenzene copolymer. Characterization of the resin in terms of IR, solid-NMR, porosity and elemental analysis suggested that TMMA was uniformly linked to the polymer particles following the expected synthetic scheme and that the introduction ratio was ca. 75%. The uptake behavior of Ce(III) ions and of U(VI) ions from different acidic media were investigated using the batch technique. The kinetic and equilibrium studies showed that the obtained resin is promising for the proposed separation. [Copyright &y& Elsevier]

We have reported reversible oxidoreductive regulation of DNA-binding activity of Pax-8: oxidation inhibits its binding and subsequent reduction restores the binding. Here, we show that Cys-45 and Cys-57 in the paired domain of rat Pax-8, which are conserved in all Pax members, are responsible for the redox regulation of its binding. Electrophoretic mobility shift assay using deletion mutants and mutants with substitution of cysteine with serine revealed that oxidation by diamide of either Cys-45 or Cys-57 loses the DNA binding of Pax-8. An intracellular oxidoreductive enzyme redox factor-1 (Ref-1) could reduce the oxidized Cys-45 or Cys-57 and restored the binding. Furthermore, reporter gene assay showed that transcriptional activity of wild-type Pax-8 was enhanced by co-expression of Ref-1. When the mutant with double substitutions of Cys-45 and Cys-57, which was insensitive to oxidation, was transfected, the basal transactivation level was much higher than that of wild-type Pax-8, while it was not enhanced by Ref-1. These results demonstrated that oxidoreductive modification of Cys-45 and Cys-57 via Ref-1 plays a role in redox regulation of Pax-8 in living cells. [Copyright &y& Elsevier]