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Superoxide dismutase from breef brain and rat liver was assayed in an enzymatic system, using xanthine oxidase, and a non-enzymatic system, based on aerobic reduction of nitro-blue tetrazolium in presence of phenazine methosulphate. The non-enzymatic assay is rapid and simple and permits simulatneous analysis of many samples. Similar results are found by the two methods of assay of superoxide dismutase.
In the unicellular Anacystis nidulans, the expression of both the H2-uptake (with phenazine methosulfate or methylene blue as the electron acceptor) and H2-evolution (with methyl viologen reduced by Na2S2O4) was dependent on Ni in the culture medium. In extracts from Anacystis and Anabaena 7119, H2-evolution and uptake activities were strongly inhibited by Cu2+, p-chloromercuribenzoate and HgCl2 suggesting that at least one functional SH-group is involved in catalysis by hydrogenase. Extracts from the N2-fixing Anabaena 7119 contained two different hydrogenase fractions which could be separated by chromatography on DE-52 cellulose using a linear NaCl concentration gradient. The fraction eluting with 0.13 M NaCl from the column catalyzed only the uptake of H2 with methylene blue as the electron acceptor but virtually not the evolution of H2 ("uptake" hydrogenase fraction). The fraction eluting at a NaCl strength of 0.195 M catalyzed both H2-uptake with methylene blue and H2-evolution with reduced methyl viologen ("reversible" hydrogenase fraction). Growth under anaerobic conditions drastically enhanced the activity levels of the "reversible" but not of the "uptake" hydrogenase fraction. The "uptake" hydrogenase but not the "reversible" protein was activated by reduced thioredoxin. It is suggested that thioredoxin activates the H2-uptake by the membrane-bound "uptake" hydrogenase also in intact cells. The occurrence of the number of hydrogenases in cyanobacteria will be reevaluated.
Analysis of a 32.8-kb segment of DNA from the rapamycin (Rp) producer, Streptomyces hygroscopicus ATCC 29253, revealed a new type-I polyketide synthase (PKS) cluster consisting of four open reading frames (ORF 1-4), each encoding a single PKS module. The four ORFs are transcribed in the same direction and are flanked by several smaller ORFs (ORF 5-9), which may be related to the PKS cluster. The first PKS-containing ORF has a ligase domain at the N-terminus of the polypeptide. This domain has 55% aa identity to the CoA ligase domain of the Rp PKS (Schwecke et al., 1995. Proc. Natl. Acad. Sci. 92, 7839-7843) which is also encoded in this strain (Lowden et al., 1996. Angew. Chem. Int. Ed. Engl. 35, 2249-2251). ORF5 (340 aa) and ORF6 (924 aa) were found to be homologous to RapK (41% aa identity) and RapH (35% aa identity), which are hypothesized to be a pteridine-dependent dioxygenase and a regulatory protein, respectively (Molnar et al., 1996. Gene 169, 1-7). In addition, ORF7 (391 aa) was found to have up to 42% aa identity to a number of plant 3-deoxy-D-arabino-heptulosonate-7-phosphate synthases (DAHPS) and 47% aa identity to PhzF, a bacterial DAHPS involved in phenazine antibiotic synthesis. The proximity of the DAHPS-encoding gene to the PKS cluster containing a Rp-like ligase domain suggests that a derivative of shikimate may be used as the PKS starter. ORF8 (283 aa) was found to have homology (32% aa identity) to a Synechocystis sp. gene of unknown function. The N-terminal portion of ORF9 was found to be similar to a tetracycline 6-hydroxylase (34% aa identity) from Streptomyces aureofaciens.
(1) The light-induced phosphorylation in spinach chloroplast is coupled with proton transfer. (2) We investigated the effect of volatile anesthetics (halothane, enflurane, isoflurane) on the cyclic and/or non-cyclic light-induced phosphorylation. (3) We used potassium ferricyanide as a Hill oxidant for non-cyclic phosphorylation and phenazin-methosulfate as an oxidation-reduction indicator for cyclic phosphorylation. (4) These three anesthetics inhibit the Hill reaction in light induced phosphorylation. The inhibition rate of this reaction generates concave curves with minimum values at 303 K in each of the anesthetics.
We have previously shown gentamicin to form a redox-active iron chelate. This study investigates whether other aminoglycosides can likewise stimulate the generation of reactive oxygen species (free radicals). Kanamycin, neomycin and streptomycin were compared to gentamicin in intact cells and in cell-free in vitro assays using luminescence detection with lucigenin or luminol. Neutrophils and Epstein-Barr virus-transformed lymphoblastoid cells served as cell models in which a respiratory burst of superoxide was induced by phorbol ester. The addition of millimolar amounts of any of the aminoglycosides increased the luminescence significantly. The drugs also increased the formation of free radicals in an enzymatic (hypoxanthine-xanthine oxidase) and a non-enzymatic (phenazine methosulfate-NADH) superoxide-generating system. Half-maximal stimulation was reached with (0.4 mM gentamicin, and there was an absolute requirement for an electron donor, arachidonic acid. In both intact cells and cell-free systems, gentamicin-enhanced luminosity was suppressed by iron chelators. These results demonstrate that different aminoglycoside antibiotics can stimulate the formation of free radicals in biological and in cell-free systems. Luminescence detection is a convenient assay method to investigate the redox properties of these drugs.
Reactive oxygen species (ROS) have emerged as important signaling molecules in the regulation of various cellular processes. In our study, we investigated the effect of a wide range of ROS on Chinese hamster lung fibroblast (V79) cell proliferation. Treatment with H2O2 (100 microM), superoxide anion (generated by 1 mM xanthine and 1 mU/ml xanthine oxidase), menadione, and phenazine methosulfate increased the cell proliferation by approximately 50%. Moreover, a similar result was observed after partial inhibition of superoxide dismutase (SOD) and glutathione peroxidase. This upregulation of cell proliferation was suppressed by pretreatment with hydroxyl radical scavengers and iron chelating agents. In addition to ROS, treatment with exogenous catalase and SOD mimic (MnTMPyP) suppressed the normal cell proliferation. Short-term exposure of the cells to 100 microM H2O2 was sufficient to induce proliferation, which indicated that activation of the signaling pathway is important as an early event. Accordingly, we assessed the ability of H2O2 to activate mitogen-activated protein kinases (MAPK). Jun-N-terminal kinase (JNK) and p38 MAPK were both rapidly and transiently activated by 100 microM H2O2, with maximal activation 30 min after treatment. However, the activity of extracellular signal-regulated kinase (ERK) was not changed. Pretreatment with SB203580 and SB202190, specific inhibitors of p38 MAPK, reduced the cell proliferation induced by H2O2. The activation of both JNK and p38 MAPK was also suppressed by pretreatment with hydroxyl radical scavenger and iron chelating agents. Our results suggest that the trace metal-driven Fenton reaction is a central mechanism that underlies cell proliferation and MAPK activation.
The human red blood cell (RBC) is known to be susceptible to oxidant damage, with both structural and functional properties altered consequent to oxidant attack. Such oxidant-related alterations may lead to changes of RBC rheologic behavior (i.e., deformability, aggregability). Two different models of oxidant stress were used in this study to generate superoxide anions either internal or external to the RBC. Our results indicate that generation of superoxide within the RBC by phenazine methosulfate decreases RBC deformability without effects on cell aggregation. Conversely, superoxide generated externally by the xanthine oxidase-hypoxanthine system primarily affects RBC aggregability: the shear rate necessary to disaggregate RBC was markedly increased while the extent of aggregation decreased slightly. Increased disaggregation shear rate (i.e., greater aggregate strength) as a result of superoxide radical damage may adversely affect the dynamics of blood flow in low-shear portions of the circulation, and may also play a role in the no-reflow phenomena encountered after ischemia-reperfusion.
An amperometric dimethyl sulfoxide (DMSO) sensor was constructed based on DMSO reductase (DMSO-R). DMSO-R from Rhodobacter sphaeroides f. sp. denitrificans was immobilized by BSA-glutaraldehyde cross-linking at the surface of a glassy carbon electrode. Mediators were added to the sample solution in a free form. Several mediators (methyl viologen (MV), benzyl viologen (BV), neutral red (NR), safranin T (ST), FMN, phenazine methosulfate (PMS)), which can donate electrons to DMSO-R, were examined with the DMSO-R immobilized electrode. Among them MV was selected as a model mediator because of its wide linear response range and fast response time. The response current was effected by the measurement temperature but hardly effected by the pH of the sample solution. The response current was increased with the measurement temperature up to 50 degrees C. A response current was observed at 1 microM DMSO and the response time was 20 s under the optimum conditions. The response was observed for approximately 2 weeks. By the reduction of Schiff base in the cross-linking layer the response range became narrower but most of the response current was retained at 300 microM of DMSO for more than 5 weeks.
Symmetrical dimers of lipophilic intercalating chromophores linked by cation-containing chains have recently been shown to have broad-spectrum in vivo anticancer activity. We report the preparation and evaluation of a series of both symmetric and unsymmetric dimers of a variety of intercalating chromophores of varied DNA binding strength, including naphthalimides, acridines, phenazines, oxanthrenes and 2-phenylquinolines. The unsymmetrical dimers were prepared by sequential coupling of the chromophores to linkers with selectively protected primary terminal amines to ensure high yields and unequivocal product. Protection of the internal (secondary) amines as BOC derivatives was used to ensure complete structural specificity, and was also an aid to the purification of these very polar compounds. The growth inhibitory abilities (as IC(50) values) of the compounds in a range of cell lines showed that the nature of the linker chain was important, and independent of the nature of the chromophore, with compounds containing the dicationic linker [-(CH2)2NH(CH2)2NH(CH2)2-] being on average 30-fold more potent than the corresponding compounds containing the monocationic linker [-(CH2)3NMe(CH2)3-]. However, the chromophores also play a role in determining biological activity, with the cytotoxicities of symmetric and unsymmetric dicationic dimers correlating with the overall DNA binding abilities of the chromophores.
Strain IC203, deficient in OxyR, and its oxyR(+) parent WP2 uvrA/pKM101 (denoted IC188) are the basis of a new bacterial reversion assay, the WP2 Mutoxitest, which has been used in the evaluation of 80 chemicals for oxidative mutagenicity. The following 31 oxidative mutagens were recognized by their greater mutagenic response in IC203 than in IC188: (1) peroxides: hydrogen peroxide (HP), t-butyl hydroperoxide (BOOH) and cumene hydroperoxide (COOH); (2) benzoquinones (BQ): 2-methyl-1,4-BQ, 2,6-dimethyl-1,4-BQ and 2,3, 5,6-tetramethyl-1,4-BQ; (3) naphthoquinones (NQ): 1,4-NQ, 2-methyl-1, 4-NQ and 2-hydroxy-1,4-NQ; (4) phenol derivatives: catechol, hydroquinone, pyrogallol, 1,2,4-benzenetriol, t-butylhydroquinone, gallic acid and 4-aminophenol; (5) catecholamines: DL- and L-dopa, DL- and L-epinephrine, dopamine and L-norepinephrine; (6) thiols: L-cysteine methyl ester, L-cysteine ethyl ester, L-penicillamine and dithiothreitol; (7) diverse: 3,4-dihydroxyphenylacetic acid, hypoxanthine and xanthine, both in the presence of xanthine oxidase, L-ascorbic acid plus copper (II) and phenazine methosulfate. Among these oxidative mutagens, 25 were found to be uniquely positive in IC203. With the exception of BOOH and COOH, mutagenesis by all oxidative mutagens was inhibited by catalase present in rat liver S9, indicating that it is mediated by HP generation, probably in autoxidation reactions. These catalase-sensitive oxidative mutagens were poor inducers of mutations derived from 8-oxoguanine lesions, whereas such mutations were efficiently induced by organic hydroperoxides. The results support the usefulness of incorporating IC203 in the bacterial battery for testing of chemicals. The well-characterized oxidative mutagens available with the use of the WP2 Mutoxitest may serve as a reference in studies on the genotoxicity of oxidative stress.
8-Hydroxyguanine (8-OH-G) DNA glycosylase is an enzyme involved in repair of oxidative DNA damage, e.g., 8-OH-G in DNA. In order to assess the roles of 8-OH-G in spontaneous and chemically-induced mutagenesis, the mutMST gene encoding 8-OH-G DNA glycosylase of Salmonella typhimurium was disrupted in several Ames tester strains, i.e., S. typhimurium TA1535 (hisG46, uvrB-, rfa), TA1975 (hisG46, uvr+, rfa) and TA102 (hisG428, uvr+, rfa). The spontaneous mutation frequencies were increased 2.4 and 1.6 times, respectively, by the mutMST deletions in strains TA1535 and TA1975, which are spontaneously reverted to His+ by mutations mainly at G:C base pairs. The resulting strains YG3001 (TA1535 delta mutMST) and YG3002 (TA1975 delta mutMST) were 2 to 8 times more sensitive to the mutagenicities of methylene blue plus visible light, neutral red plus visible light and 2-nitrofluorene than the parent strains. The strain YG3002 but not YG3001 was about 30 times more sensitive to the mutagenicity of 4-nitroquinoline N-oxide than the parent strain TA1975. Neither hydrogen peroxide nor phenazine methosulfate was mutagenic in the mutMST-deletion strains as well as in the parent strains. In contrast, the mutMST deletion did not affect the spontaneous mutation frequency of strain TA102, which has an A:T base pair at the critical site for reversion. The sensitivities of strain TA102 to the chemicals were not enhanced by the mutMST deletion except for hydrogen peroxide. These results suggest that 8-OH-G in DNA plays important roles in spontaneous mutagenesis occurring at G:C base pairs in S. typhimurium, and some nitro aromatics such as 4-nitroquinoline N-oxide or 2-nitrofluorene as well as the photosensitizers plus visible light can produce 8-OH-G in DNA, thereby inducing mutations. In the case of 4-nitroquinoline N-oxide, 8-OH-G rather than DNA adducts seems to play major roles in mutagenesis in uvr+ background. The new strains could be useful for the evaluation of the roles of 8-OH-G in mutagenesis in S. typhimurium and permit the efficient detection of some oxidative mutagens in the environment.
We previously reported the development of mutation-specific Escherichia coli B tester strains WP3101 to WP3106 from strain WP2uvrA. In this study we constructed their pKM101-containing derivatives WP3101P to WP3106P, and further isolated their rfa derivatives WP4101-WP4106 and WP4101P-WP4106P. The six kinds of F' plasmids (lacI-, lacZ-, proAB+), each of which carries a different lacZ allele, contained in the above strains were originally derived from E. coli K-12 strains CC101-CC106. All the tester strains show Lac- and Trp- phenotype. Assays for transitions and transversions are based upon Lac+ reversion of a specific mutation located within the lacZ gene on an F' plasmid. The trpE65(ochre) allele in the same strains enables them to be used for Trp+ reversion assays as well. In the present paper, we evaluated the sensitivity, specificity, and usefulness of the newly developed tester strains. Strains WP3101P-WP3106P were highly sensitive to determine mutational profile of heterocyclic amines with S9 mix-mediated metabolic activation and most of the oxidative mutagens and free radical generators tested. Every type of base-pair substitutions induced by 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) or 5-diazouracil were detected in strains WP3101P-WP3106P, while A:T-->C:G and G:C-->A:T mutations induced by MeIQ, and A:T-->C:G, G:C-->A:T, and G:C-->C:G by 5-diazouracil were not detected in pKM101-free tester strains. In pKM101-carrying strains, cumene hydroperoxide induced all types of base substitutions, while formaldehyde preferentially induced G:C-->T:A transversions. Phenazine methosulfate induced predominantly G:C-->A:T transitions and G:C-->T:A transversions, while H2O2 induced predominantly G:C-->T:A and A:T-->T:A transversions. Introduction of the rfa mutation considerably enhanced sensitivity to bulky mutagens such as polycyclic aromatic compounds. All six possible base substitutions induced by 9, 10-dimethyl-1,2-benzanthracene (DMBA) were detected in tester strains WP4101P-WP4106P. In conclusion, our tester strains WP3101P-WP3106P and WP4101P-WP4106P permitted rapid and simple detection of specific mutations induced by variety of mutagens.
The interaction of Ru(bipy)2(dppx)2+ (bipy = 2.2'-bipyridine,dppx = 7,8-dimethyldipyrido phenazine) with the calfthymus DNA has been studied with fluorescence and ultraviolet visible absorption spectroscopy. The results of fluorescence quenching and salt effect show that Ru(bipy)2(dppx)2+ intercalate into the double helix of DNA. The ultraviolet visible absorption spectrum of Ru(bipy)2(dppx)2+, calfthymus DNA, and their interaction indicate that Ru(bipy)2(dppx)2+ intercalate into the double helix of DNA via the ligand dppx.
A number of compounds were isolated from the medicinal plant Aster tataricus including shionone, epifriedelinol, quercetin, kaempferol, scopoletin, emodin, aurantiamide acetate and 1,7-dihydroxy-6-methyl-anthraquinone. The compounds were compared with regard to their ability in inhibiting hemolysis of rat erythrocytes induced by 2'-2' azobis (2-amidinopropane) dihydrochloride, lipid peroxidation using the FeSO(4)-ascorbic acid system, and generation of superoxide radicals using a phenazine methosulfate-nicotinamide adenine dinucleotide system. The effects on the Fe-bleomycin-induced DNA damage reflected pro-oxidant activity. Quercetin and kaempferol were most potent in inhibiting hemolysis, lipid peroxidation and superoxide radical generation. Scopoletin and emodin were similar to quercetin and kaempferol in inhibiting superoxide radical generation and second to them in inhibiting lipid peroxidation. Aurantiamide acetate exhibited some inhibitory activity toward superoxide radical generation. 1,7-dihydroxy-6-methyl-anthraquinone exerted an inhibitory activity only on superoxide radical generation. Shionone and epifriedelinol did not display any antioxidant activity. Quercetin and kaempferol, but not the remaining compounds, exhibited some pro-oxidant activity.
In an attempt to uncover electric field interactions between PS I and PS II during their functioning, fluorescence induction curves were measured on hydroxylamine-treated thylakoids of Chenopodium album under conditions ensuring low and high levels of photogenerated membrane potentials. In parallel experiments with Peperomia metallica chloroplasts, the photocurrents were measured with patch-clamp electrodes and served as indicator of electrogenic activity of thylakoid membranes in continuous light. Inhibition of linear electron flow at PS II donor side by hydroxylamine (0.1 mM) eliminated a slow rise of chlorophyll fluorescence to a peak level and suppressed photoelectrogenesis. Activation of PS I-dependent electron transport using cofactors of either cyclic (phenazine methosulfate) or noncyclic electron transport (reduced TMPD or DCPIP in combination with methyl viologen) restored photoelectrogenesis in hydroxylamine-treated chloroplasts and led to reappearance of slow components in the fluorescence induction curve. Exposure of thylakoids to valinomycin reduced the peak fluorescence in the presence of KCl but not in the absence of KCl. Combined application of valinomycin and nigericin in the presence of KCl exerted stronger suppression of fluorescence than valinomycin alone but was ineffective in the absence of KCl. In samples treated with hydroxylamine and PS I cofactors (DCPIP/ascorbate and methyl viologen), preillumination with a single-turnover flash or a multiturnover pulse shifted the induction curves of both membrane potential and chlorophyll fluorescence to shorter times, which confirms the supposed influence of PS I-generated electrical field on PS II fluorescence. A model is presented that describes modulating effect of the membrane potential on chlorophyll fluorescence and roughly simulates the fluorescence induction curves measured at low and high membrane potentials.
The electrocatalytic oxidation of DNA by a heterodimer, [(bpy)2Ru(tpphz)Os(bpy)2]4+ (tpphz: tetrapyrido[3,2-alpha: 2',3'-c:3'',2''-h:2''',3'''-j]phenazine) (1), was studied using cyclic voltammetry with digital simulation. This dimer was chosen because the Ru(III/II) couple (E1/2 = 1.09 V vs Ag/AgCl) is capable of catalyzing guanine oxidation while the Os(III/II) couple (E1/2 = 0.63 V) provides a convenient reporter on the binding and mass transport of the complex, which can then be determined in the same voltammetric sweep as the electrocatalysis. Proper description of the electrochemical response required careful measurement of the binding constant of 1 to herring testes (HT) DNA, which was (2.0 +/- 0.1) x 10(4) M(-1) by both absorption titration and normal pulse voltammetry. Thermal denaturation experiments were consistent with a nonintercalative binding mode and gave a deltaTm of only (2.4 +/- 0.5) degrees C. The minor groove binder distamycin did not displace 1 from HT DNA, suggesting that the complex binds in the major groove. As expected, acquisition of the cyclic voltammogram of 1 in the presence of DNA produced catalytic current for the Ru(III/II) couple, while a suppression of current was observed for the Os(III/II) couple. Although the catalytic current for the Ru(III/II) couple initially appeared as a current enhancement, higher concentrations suppressed the catalytic wave as a result of the slower mass transport of the DNA-bound complex. The binding studies were used to create a model for digital simulation that reproduced the behavior of 1 with DNA and gave rate constants that were independent of DNA concentration. The apparent second-order rate constant at 25 mV/s for oxidation of guanine in HT DNA (av 1000 bp, 25% guanine) by 1 was 3 +/- 1 x 10(4) M(-1) s(-1); similar values were obtained for a 200-bp fragment (7 +/- 3 x 10(4) M(-1) s(-1)) and a 435-bp fragment (8 +/- 2 x 10(4) M(-1) s(-1)). As observed in previous studies of these reactions, biphasic kinetics in the catalytic reaction led to a dependence of the rate constant determined by simulation on the sweep rate. Increasing the sweep rate led to a systematic increase in the simulated rate constant, consistent with a fast phase of the homogeneous catalytic reaction.
Selective detection of double-stranded DNA (ds-DNA) in solution was achieved by photoelectrochemistry using a high-affinity DNA intercalator, Ru(bpy)2dppz (bpy = 2,2'-bipyridine, dppz = dipyrido[3,2-a:2',3'-c]phenazine) as the signal indicator and tin oxide nanoparticle as electrode material. When Ru(bpy)2dppz alone was irradiated with 470-nm light, anodic photocurrent was detected on the semiconductor electrode due to electron injection from its excited state into the conduction band of the electrode. The current was sustained in the presence of oxalate in solution, which acted as a sacrificial electron donor to regenerate the ground-state metal complex. After addition of double-stranded calf thymus DNA into the solution, photocurrent dropped substantially. The drop was attributed to the intercalation of Ru(bpy)2dppz into DNA and, consequently, the reduced mass diffusion of the indicator to the electrode, as well as electrostatic repulsion between oxalate anion and negative charges on DNA. The degree of signal reduction was a function of the DNA concentration, thus forming the basis for real-time DNA detection. The signal reduction was selective for ds-DNA, as no such effect was observed for single-stranded polynucleotides such as poly-G, poly-C, poly-A, and poly-U. The detection limit of calf thymus ds-DNA reached 1.8 x 10(-10) M in solution.
Protein folding has been studied extensively with an aim to better understanding of the relationship between protein sequence, structure, and function. A large variety of techniques have been developed and utilized to probe protein conformation and folding/unfolding transition. In this report, electrochemical monitoring of urea-induced unfolding of a large cofactor-free protein, bovine serum albumin (BSA), is described. Enhanced electrochemical oxidation of tyrosine and tryptophan in free amino acids and in BSA was achieved on an indium tin oxide electrode by using an electron mediator, Os(bpy)2dppz (bpy = 2,2'-bipyridine, dppz = dipyrido[3,2-a:2',3'-c]phenazine). The oxidation current was used as a signal reporter in the monitoring of urea-induced BSA denaturation. At high urea concentrations, the electrochemical signal increased by 3-fold relative to the native protein. The increase is attributed to the closer contact between the oxidizable residues in the unfolded BSA and Os(bpy)2dppz. The degree of unfolding assessed by electrochemistry correlates well with the established fluorescence technique in the range of 0-10 M urea. The method can be used to investigate the unfolding process of other cofactor-free proteins.