DNA and RNA strand scission by copper, zinc and manganese superoxide dismutases

Copper/zinc (Cu/ZnSOD) and manganese (MnSOD) superoxide dismutases which catalyze the dismutation of toxic superoxide anion, O _inf2 ^sup– , to O₂ and H₂O₂, play a major role in protecting cells from toxicity of oxidative stress. However, cells overexpressing either form of the enzyme show signs of toxicity, suggesting that too much SOD may he injurious to the cell. To elucidate the possible mechanism of this cytotoxicity, the effect of SOD on DNA and RNA strand scission was studied. High purity preparations of Cu/ZnSOD and MnSOD were tested in an in vitro assay in which DNA cleavage was measured by conversion of phage X174 supercoiled double-stranded DNA to open circular and linear forms. Both types of SOD were able to induce DNA strand scission generating single- and double-strand breaks in a process that required oxygen and the presence of fully active enzyme. The DNA strand scission could be prevented by specific anti-SOD antibodies added directly or used for immunodepletion of SOD. Requirement for oxygen and the effect of Fe(II) and Fe(III) ions suggest that cleavage of DNA may be in part mediated by hydroxyl radicals formed in Fenton-type reactions where enzyme-bound transition metals serve as a catalyst by first being reduced by superoxide and then oxidized by H₂O₂. Another mechanism was probably operative in this system, since in the presence of magnesium DNA cleavage by SOD was oxygen independent and not affected by sodium cyanide. It is postulated that SOD, by having a similar structure to the active center of zinc-containing nucleases, is capable of exhibiting non-specific nuclease activity causing hydrolysis of the phosphodiester bonds of DNA and RNA. Both types of SOD were shown to effectively cleave RNA. These findings may help explain the origin of pathology of certain hereditary diseases genetically linked to Cu/ZnSOD gene.     

Is photocleavage of DNA by YOYO-1 using a synchrotron radiation light source sequence dependent?

The photocleavage of double-stranded and single-stranded DNA by the fluorescent dye YOYO-1 was investigated in real time by using the synchrotron radiation light source ASTRID (ISA, Denmark) both to initiate the reaction and to monitor its progress using Couette flow linear dichroism (LD) throughout the irradiation period. The dependence of LD signals on DNA sequences and on time in the intense light beam was explored and quantified for single-stranded poly(dA), poly[(dA-dT)₂], calf thymus DNA (ctDNA) and Micrococcus luteus DNA (mlDNA). The DNA and ligand regions of the spectrum showed different LD kinetic behaviors, and there was significant sequence dependence of the kinetics. However, in contrast to expectations from the literature, we found that poly(dA), mlDNA, low salt ctDNA and low salt poly[(dA-dT)₂] all had significant populations of groove-bound YOYO. It seems that this mode was predominantly responsible for the catalysis of DNA cleavage. In homopolymeric DNAs, intercalated YOYO was unable to cleave DNA. In mixed-sequence DNAs the data suggest that YOYO in some but not all intercalated binding sites can cause cleavage. It is also likely that cleavage occurs at transient single-stranded regions. The reaction rates for a 100 mA beam current of 0.5-μW power varied from 0.6 h⁻¹ for single-stranded poly(dA) to essentially zero for low salt poly[(dG-dC)₂] and high salt poly[(dA-dT)₂]. At the conclusion of the experiments with each kind of DNA, uncleaved DNA with intercalated YOYO remained.     

Possible accumulation of non-active molecules of catalase and superoxide dismutase in S. cerevisiae cells under hydrogen peroxide induced stress

The effect of hydrogen peroxide on the activities of catalase and superoxide dismutase (SOD) in S. cerevisiae has been studied under different experimental conditions: various H₂O₂ concentrations, time exposures, yeast cell densities and media for stress induction. The yeast treatment with 0.25–0.50 mM H₂O₂ led to an increase in catalase activity by 2–3-fold. At the same time, hydrogen peroxide caused an elevation by 1.6-fold or no increase in SOD activity dependently on conditions used. This effect was cancelled by cycloheximide, an inhibitor of protein synthesis in eukaryotes. Weak elevation of catalase and SOD activities in cells treated with 0.25–0.50 mM H₂O₂ found in this study does not correspond to high level of synthesis of the respective enzyme molecules observed earlier by others. It is well known that exposure of microorganisms to low sublethal concentrations of hydrogen peroxide leads to the acquisition of cellular resistance to a subsequent lethal oxidative stress. Hence, it makes possible to suggest that S. cerevisiae cells treated with low sublethal doses of hydrogen peroxide accumulate non-active stress-protectant molecules of catalase and SOD to survive further lethal oxidant concentrations.     

Kidney glomerular explants in serum-free media: Demonstration of intracellular antioxidant enzymes and active oxygen metabolites

Summary Guinea pig glomeruli were grown for 22 d in a serum-free medium composed of Waymouth's MB 752/1 supplemented with sodium pyruvate, nonessential amino acids, and antibiotics (the basic medium). Intracellular cellular activity of the antioxidant enzymes superoxide dismutase (SOD; both copper-zinc [Cu,Zn] and manganese [Mn] forms) and catalase, and intracellular active oxygen metabolites (hydrogen peroxide [H₂O₂] and superoxide [O_{2 −} ^· ]) were measured with time in culture. The results were compared to results obtained from glomeruli grown in different serum-free media, including the basic medium plus fibronectin (FN), the basic medium plus transferrin and FN, and a complex medium containing insulin, transferrin, selenium (Se), triiodothyronine, and FN (complete medium). In general, although the intracellular activity of antioxidant enzymes and active oxygen metabolites varied over time in culture in all media, there were only a few statistically significant differences among different media. Both CuZn SOD and Mn SOD activity were demonstrated, in isolated glomeruli. The CuZn SOD activity per DNA ratio decreased slightly with time in culture in all media tested except the complete medium, in which CuZn SOD activity per DNA ratio remained more constant. The Mn SOD activity per DNA ratio did not vary significantly over time in culture. Catalaselike activity was very low in isolated glomeruli and declined sharply with time in culture in all media except the complete medium. Both H₂O₂ and O_{2 −} ^· were detected intracellularly in glomerular culture. Our results indicate that intracellular antioxidant enzymes and active oxygen metabolites in glomeruli vary with time in culture and, in some instances, with culture conditions. Supported by grants to Dr. Terry Oberley from the University of Wisconsin Graduate School and by the Veterans Administration. Mr. Steinert was a predoctoral fellow supported by National Institutes of Health training grant 5-T32 ES0715.     

Excess copper induces accumulation of hydrogen peroxide and increases lipid peroxidation and total activity of copper–zinc superoxide dismutase in roots of Elsholtzia haichowensis

The effects of excess copper (Cu) on the accumulation of hydrogen peroxide (H₂O₂) and antioxidant enzyme activities in roots of the Cu accumulator Elsholtzia haichowensis Sun were investigated. Copper at 100 and 300 μM significantly increased the concentrations of malondialdehyde and H₂O₂, and the activities of catalase (E.C. 1.11.1.6), ascorbate peroxidase (E.C. 1.11.1.11), guaiacol peroxidase (GPOD, E.C. 1.11.1.7) and superoxide dismutase (SOD, E.C. 1.15.1.1). Isoenzyme pattern and inhibitor studies showed that, among SOD isoforms, only copper–zinc superoxide dismutase (CuZn–SOD) increased. Excess Cu greatly increased the accumulation of superoxide anion (O₂ ^·−) and H₂O₂ in E. haichowensis roots. This study also provides the first cytochemical evidence of an accumulation of H₂O₂ in the root cell walls as a consequence of Cu treatments. Experiments with diphenyleneiodonium as an inhibitor of NADPH oxidase, 1,2-dihydroxybenzene-3,5-disulphonic acid as an O₂ ^·− scavenger, and N-N-diethyldithiocarbamate as an inhibitor of SOD showed that the source of H₂O₂ in the cell walls could partially be NADPH oxidase. The enzyme can use cytosolic NADPH to produce O₂ ^·−, which rapidly dismutates to H₂O₂ by SOD. Apoplastic GPOD and CuZn–SOD activities were induced in roots of E. haichowensis with 100 μM Cu suggesting that these two antioxidant enzymes may be responsible for H₂O₂ accumulation in the root apoplast.     

Bacteriophytochrome-Dependent Regulation of Light-Harvesting Complexes in Rhodopseudomonas palustris Anaerobic Cultures

Bacteriophytochromes (Bphs) are photoreceptors that help bacteria sense changes in light wavelength and intensity. Bphs contain a linear tetrapyrrole chromophore that, upon absorption of red or far-red light, undergoes a cis–trans isomerization that leads to a conformational change in the holoprotein. The conformation and type of Bph affects the expression of genes. The linear tetrapyrrole bound by Bphs is thought to come from O₂-dependent cleavage of heme by a heme oxygenase. We have discovered that the absence of O₂ does not inhibit the normal function of two Bphs in the regulation of Rhodopseudomonas palustris light-harvesting complexes. These observations imply that: (i) a linear tetrapyrrole can be made anaerobically, either through anaerobic heme cleavage by a novel enzyme or directly from the heme precursor hydroxymethylbilane without ring cleavage; or (ii) that Bph-dependent signal transduction does not require a chromophore.     

Mechanism of bleomycin action: in vitro studies

The cytotoxic activity of bleomucin results from DNA cleavage, which is also accomplished $\text{in vitro}$ by reaction mixtures containing Fe(II), drug and O₂. Bleomycin forms a complex with Fe(II) and O₂ in the presence or absence of DNA. The species attacking DNA forms rapidly from this complex. The nature of the attacking species and of the primary lesion(s) to DNA are not yet known, but two major insults to DNA have been characterized. They are the release of free bases from their glycosidic linkages and, at other residues, the cleavage of the polymer backbone at the deoxyribose C3-C4 bond.     

Fermentative production of superoxide dismutase with <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis>

This work sought to develop a fermentative process for the microbial production of superoxide dismutase (SOD), to overcome extraction from animal tissues. Twenty-eight wild-type yeast strains were screened for SOD productivity. Kluyveromyces marxianus L3 showed the highest SOD activity (62 U mg⁻¹) and was used for process development. Oxidative stress conditions and parameters affecting oxygen transfer rate were exploited to improve production. The effects of dilution rate (0.067 vs 0.2 h⁻¹), aeration pressure (0.3 vs 1.2 bar) and H₂O₂ (0 vs 50 mM) were studied during chemostat experiments. Low dilution rate, high pressure and H₂O₂ resulted in an increase in CuZn–SOD up to 475 U mg⁻¹. When a regulation of oxygen saturation was applied during batch cultures, CuZn–SOD was progressively higher at 60, 80 and 90% dissolved oxygen tension (DOT) (250, 330 and 630 U mg⁻¹, respectively). Furthermore, the highest growth rate and biomass yield were achieved at 90% DOT, this being therefore the best DOT condition for high overall productivity. Growth and productivity on different carbon sources were compared. Specific activity was higher on glycerol than on lactose or glucose (496, 454 and 341 U mg⁻¹, respectively). The highest biomass yield was achieved on lactose. It may be therefore the best substrate for SOD production.     

Neutralization of radical toxicity by temperature-dependent modulation of extracellular SOD activity in coral bleaching pathogen Vibrio shiloi and its role as a virulence factor

Vibrio shiloi is the first and well-documented bacterium which causes coral bleaching, particularly, during summer, when seawater temperature is between 26 and 31°C. Coral bleaching is the disruption of the symbiotic association between coral hosts and their photosynthetic microalgae zooxanthellae. This is either due to lowered resistance in corals to infection or increased virulence of the bacterium at the higher sea surface temperature. The concentration of the oxygen and resulting oxygen radicals produced by the zooxanthellae during photosynthesis are highly toxic to bacteria, which also assist corals in resisting the infection. Hence, in this study we examined the effect of different temperatures on the activity of a novel extracellular SOD in V. shiloi. We also partially characterized the SOD and clearly confirmed that the extracellular SOD produced by V. shiloi is Mn–SOD type, as it was not inhibited by H₂O₂ or KCN. Performing chemical susceptibility killing assay, we confirmed that extracellular SOD may act as first line of defense for the bacteria against the reactive oxygen species. Since, increased activity of novel Mn–SOD at higher temperature, leads to the neutralization of radical toxicity and facilitates the survival of V. shiloi. Hence, the extracellular Mn–SOD may be considered as a virulence factor.     

Relationship between copper biosorption and microbial inhibition of hydroxyl radical formation in a copper(II)–hydrogen peroxide system

The microbial retardation of the spin adduct, DMPO-OH, formed in a copper(II)–hydrogen peroxide–DMPO (5,5-dimethyl-1-pyrroline N-oxide) solution was examined in relation to copper biosorption. A hydroxyl radical is formed in the solution through two steps, the reduction of Cu(II) to Cu(I) by H₂O₂ and the Fenton-type reaction of Cu(I) with H₂O₂. The resultant radical is trapped by DMPO to form DMPO-OH. Microbial cells retarded the DMPO-OH in the Cu(II)–H₂O₂–DMPO far more significantly than in the UV-irradiated H₂O₂–DMPO solution. Egg albumin showed a higher DMPO-OH retardation than microbial cells both in the Cu(II)–H₂O₂–DMPO and the UV-irradiated H₂O₂–DMPO solutions. These results indicated that the retardation effect is related to organic matter and not to microbial activity. Microorganisms having higher affinities for copper ion retarded DMPO-OH more significantly. The linear relationship between the amounts of copper biosorption and the inverse of the median inhibitory doses for DMPO-OH indicated that the microbial cells inhibited the reduction of Cu(II) to Cu(I) by H₂O₂, followed by the decrease of hydroxyl radical formation and the retardation of DMPO-OH. These results also suggest that the coupling between microbial cells and Cu(II) ion can be estimated from their ability to retard DMPO-OH.     

Antioxidant defense enzymes in cell vacuoles of red beet roots

The vacuolar fraction isolated from red beet (Beta vulgaris L.) taproots was shown to contain the cyanide-sensitive Cu,Zn-activated superoxide dismutase (SOD; EC 1.15.1.1). The enzyme was represented by three isoforms located in the aqueous phase (in the vacuolar sap) without association to the membrane. Effective operation of SOD in plant cells, especially of its H₂O₂-sensitive molecular forms, is known to depend on peroxide-utilizing enzymes; this study revealed the existence of phenol-dependent peroxidase (EC 1.11.1.7) in the plant vacuoles. It was shown that the vacuolar peroxidase of red beet roots has a high affinity to benzidines and exhibits optimal activity at low pH (pH range 4–6 depending on substrate species). This peroxidase was represented by numerous molecular forms of acidic and basic nature. The isoenzyme composition of peroxidase in storage roots was highly labile: it depended on the duration of dormant period and comprised from 10 to 17 isoforms. The peroxidase isoforms were located both in the aqueous phase (vacuolar sap) and in the membrane, being weakly associated with the tonoplast. The presence of SOD and peroxidase in the vacuolar sap indicates the existence in vacuoles of an antioxidant defense system that protects vacuolar molecular structures against the impact of superoxide radicals and excessive amounts of H₂O₂.     

A hydrogen peroxide biosensor based on the direct electrochemistry of hemoglobin modified with quantum dots

Direct electron transfer of hemoglobin modified with quantum dots (QDs) (CdS) has been performed at a normal graphite electrode. The response current is linearly dependent on the scan rate, indicating the direct electrochemistry of hemoglobin in that case is a surface-controlled electrode process. UV–vis spectra suggest that the conformation of hemoglobin modified with CdS is little different from that of hemoglobin alone, and the conformation changes reversibly in the pH range 3.0–10.0. The hemoglobin in a QD film can retain its bioactivity and the modified electrode can work as a hydrogen peroxide biosensor because of its peroxidase-like activity. This biosensor shows an excellent response to the reduction of H₂O₂ without the aid of an electron mediator. The catalytic current shows a linear dependence on the concentration of H₂O₂ in the range 5 × 10⁻⁷–3 × 10⁻⁴ M with a detection limit of 6 × 10⁻⁸ M. The response shows Michaelis–Menten behavior at higher H₂O₂ concentrations and the apparent Michaelis–Menten constant is estimated to be 112 μM.     

Structural characteristic,pH and thermal stabilities of apo and holo forms of caprine and bovine lactoferrins

Apo and holo forms of lactoferrin (LF) from caprine and bovine species have been characterized and compared with regard to the structural stability determined by thermal denaturation temperature values (T _m), at pH 2.0–8.0. The bovine lactoferrin (bLF) showed highest thermal stability with a T _m of 90 ± 1°C at pH 7.0 whereas caprine lactoferrin (cLF) showed a lower T _m value 68 ± 1°C. The holo form was much more stable than the apo form for the bLF as compared to cLF. When pH was gradually reduced to 3.0, the T _m values of both holo bLF and holo cLF were reduced showing T _m values of 49 ± 1 and 40 ± 1°C, respectively. Both apo and holo forms of cLF and bLF were found to be most stable at pH 7.0. A significant loss in the iron content of both holo and apo forms of the cLF and bLF was observed when pH was decreased from 7.0 to 2.0. At the same time a gradual unfolding of the apo and holo forms of both cLF and bLF was shown by maximum exposure of hydrophobic regions at pH 3.0. This was supported with a loss in α-helix structure together with an increase in the content of unordered (aperiodic) structure, while β structure seemed unchanged at all pH values. Since LF is used today as fortifier in many products, like infant formulas and exerts many biological functions in human, the structural changes, iron binding and release affected by pH and thermal denaturation temperature are important factors to be clarified for more than the bovine species.     

Iron–sulfur cluster biosynthesis: characterization of IscU–IscS complex formation and a structural model for sulfide delivery to the [2Fe–2S] assembly site

Recent work on the bacterial iron–sulfur cluster (isc) family of gene products, and eukaryotic homologs, has advanced the molecular understanding of cellular mechanisms of iron–sulfur cluster biosynthesis. Members of the IscS family are pyridoxyl-5′-phosophate dependent proteins that deliver inorganic sulfide during assembly of the [2Fe–2S] cluster on the IscU scaffold protein. Herein it is demonstrated through calorimetry, fluorescence, and protein stability measurements that Thermotoga maritima IscS forms a 1:1 complex with IscU in a concentration-dependent manner (K _D varying from 6 to 34 μM, over an IscS concentration range of approximately 2–50 μM). Docking simulations of representative IscU and IscS proteins reveal critical contact surfaces at the N-terminal helix of IscU and a C-terminal loop comprising a chaperone binding domain. Consistent with the isothermal titration calorimetry results described here, an overall dominant contribution of charged surfaces with a change in the molar heat capacity of binding, ΔC _p ~ 199.8 kcal K⁻¹ mol⁻¹, is observed that accounts for approximately 10% of the total accessible surface area at the binding interface. Both apo and holo IscUs and homologs were found to bind to IscS in an enthalpically driven reaction with comparable K _D values. Both helix and loop regions are highly conserved among phylogenetically diverse organisms from a pool of archael, bacterial, fungal, and mammalian representatives.     

Purification and biochemical characterization of a superoxide dismutase from the soluble fraction of the cyanobacterium, <Emphasis Type="Italic">Spirulina platensis</Emphasis>

A superoxide dismutase (SOD) was purified from Spirulina platensis sonicate. The SOD was purified to homogeneity (48-fold and 0.24% yield) through ammonium sulphate precipitation and DEAE-52 anion exchange chromatography. The SOD from S. platensis appeared to be a homodimer with a molecular weight of 30 kDa and a subunit MW of 15 kDa as determined by both native polyacrylamide gel electrophoresis and mass spectrometry. The enzyme activity was stable at pH 6.5–10.0 and 50 °C. Using group-specific chemical modifying reagents, the amino acids arginine, histidine, tryptophan, tyrosine and aspartic acid were identified to be essential for S. platensis SOD activity. The amino acid composition was found to lack methionine and cysteine. The inhibition of activity by H₂O₂ suggests that the enzyme may be an iron containing SOD.     

DNA electrochemical sensor based on an adduct of single-walled carbon nanotubes and ferrocene

A novel electrochemical sandwich-type gene sensing system was designed by using a DNA probe (DNA-probe1) immobilized on a gold electrode, the target DNA, and another DNA probe (DNA-probe2) conjugated on a single-walled carbon nanotubes/ferrocene (Fc–SWNT) adduct. In this sandwich-type gene-sensing electrode, the Fc–SWNT adduct could significantly amplify the electrochemical response of the reduction of H₂O₂. The target DNA could be detected selectively and sensitively based on the much enhanced electrochemical catalytic property of the Fc–SWNT adduct toward H₂O₂ reduction.     

Enhancement of superoxide dismutase activity in the leaves of white clover (Trifolium repens L.) in response to polyethylene glycol-induced water stress

Effects of polyethylene glycol (PEG)-induced water stress on the activities of total leaf superoxide dismutase (SOD) and chloroplast SOD (including thylakoid-bound SOD and stroma SOD) are described in white clover (Trifolium repens L.) grown in solution culture from rooted cuttings. Both leaf SOD and chloroplast SOD activities were markedly enhanced with increasing concentration of PEG stress, generating osmotic potentials around the roots 0, −0.5, −1.0, −1.5 MPa. The effects increased with time up to 72 h. Chloroplast Fe-containing SOD represented about 30% of the total leaf SOD activity in the control plants and a significant increase in chloroplast SOD activity was found during the stress period. This accounted for about 35.5–71.1% of the total leaf SOD activity. The proportion of chloroplast SOD in total leaf SOD not only increased with the decreasing of osmotic potential, but also increased with incubation time. Furthermore, the increase in thylakoid-bound SOD activity was much higher than that of stroma SOD in chloroplast of plants under water stress. The enhanced chloroplastic SOD activity, especially thylakoid-bound SOD activity, demonstrated in Trifolium repens suggests that Fe-SOD located in chloroplasts play a more important role than cytosolic Cu/Zn-containing SODs in scavenging O₂ ⁻.     

Superoxide dismutase (SOD) in boar spermatozoa: Purification,biochemical properties and changes in activity during semen storage (16 °C) in different extenders

The antioxidant system in semen is composed of enzymes, low-molecular weight antioxidants and seminal plasma proteins. Loss of enzymatic activity of superoxide dismutase (SOD) during semen preservation may cause insufficient antioxidant defense of boar spermatozoa. The aim of this study was to isolate and characterize SOD molecular forms from spermatozoa and to describe changes in SOD activity in boar sperm during preservation at 16 °C. Sperm extracts were prepared from fresh or diluted semen and used for SOD purification or activity measurement. Ion-exchange chromatography and gel filtration was used to purify SOD molecular forms. BTS, Dilu Cell, M III and Vitasem were used as diluents for 5-day storage of semen at +16 °C. The molecular form of SOD released from spermatozoa after cold shock and homogenization had a molecular weight of approximately 67 kDa. The activity of the SOD form was the highest at pH 10 within the temperature range between 20 and 45 °C. The enzymatic activity of form released after cold shock was inhibited by H₂O₂ and diethyldithiocarbamate (DDC; by 65 and 40%, respectively). The SOD form released by homogenization was inhibited by H₂O₂ and DDC (40%). The molecular form released after urea treatment was a 30 kDa protein with maximum activity at 20 °C and pH 10. Enzymatic activity of this form was inhibited by H₂O₂ by 35%, DDC by 80% and 2-mercaptoethanol by 15%. The antigenic determinants of SOD isolated from boar seminal plasma and spermatozoa were similar to each other. Susceptibility of spermatozoa to cold shock increased during storage, but the differences between extenders were statistically non-significant.     

Role of hydrogen peroxide and apoplastic peroxidase in tomato — Botrytis cinerea interaction

The aim of the research was to estimate the sensitivity of tomato tissue and spore from necrotrophic isolate of B. cinerea on H₂O₂. The influence of exogenic H₂O₂ and B. cinerea on plant tissue and on the activity of peroxidases (PO), catalase (CAT) and superoxide dismutase (SOD) in apoplastic tomato leaves fraction were investigated. It was proved that 40 mM H₂O₂ damaged the cells of a host, and inhibited in vitro germination of B.cinerea spores. Complete inhibition of germination was observed after the use 100 mM H₂O₂. In the presence of spores H₂O₂ was decomposed to H₂O and O₂. Trace activity of catalase was observed in a solution of spores used for inoculation. Necrosis which appeared on the leaves after 40 mM H₂O₂ treatment resembled hypersensitive response. On the leaves pretreated at this concentration the development of infection was observed. The H₂O₂ concentration harmful for the tissues, stimulated the PO activity measured with NADH — responsible for generation of ^·O ₂ ⁻ , as well as with syringaldazine (S) and ferulic acid (FA), substrates characteristics of forms lignifying and strengthening the cell wall. Clear increase in CAT activity, resulting from infection and early pretreatment with H₂O₂ was observed in apoplast. No effect on SOD activity was observed. A hypothesis may be put forward, that germinating spores produce enzymes which allow them to decompose H₂O₂ generated in apoplast, so there is little likelihood that B. cinerea can be directly inhibited by reactive oxygen forms (ROS) during initial stages of infection. Necrotic lesions resembling HR generated by exogenous H₂O₂ as well as induction of activity of apoplastic plant enzymes, particularly PO connected with strengthening and lignification of cell wall, were not sufficient factors to inhibit fungal expansion.     

2-(3,5-Dibromo-4-hydroxyphenyl)imidazo[4,5-<Emphasis Type="Italic">f</Emphasis>][1,10]phenanthrolinoruthenium(II) complexes: synthesis,characterization, cytotoxicity,apoptosis, DNA-binding and antioxidant activity

A new ligand DBHIP and its two ruthenium(II) complexes [Ru(dmb)₂(DBHIP)](ClO₄)₂ (1) and [Ru(dmp)₂(DBHIP)](ClO₄)₂ (2) have been synthesized and characterized. The cytotoxicity of DBHIP and complexes 1 and 2 has been assessed by MTT assay. The apoptosis studies were carried out with acridine orange/ethidium bromide (AO/EB) staining methods. The binding behaviors of these complexes to calf thymus DNA (CT-DNA) were studied by absorption titration, viscosity measurements, thermal denaturation and photoactivated cleavage. The DNA-binding constants of complexes 1 and 2 were determined to be 8.64 ± 0.16 × 10⁴ (s = 1.34) and 2.79 ± 0.21 × 10⁴ (s = 2.17) M⁻¹. The results suggest that these complexes interact with DNA through intercalative mode. The studies on the mechanism of photocleavage demonstrate that superoxide anion radical (O₂ ^•–) and singlet oxygen (¹O₂) may play an important role in the DNA cleavage. The experiments on antioxidant activity show that these compounds also exhibit good antioxidant activity against hydroxyl radical (OH^•).