Effects of Sanionia uncinata extracts in protecting against and inducing DNA cleavage by reactive oxygen species

Abstract

When mosses are exposed to increased quantities of ultraviolet (UV) radiation, they produce more secondary metabolites. Antarctica moss Sanionia uncinata (Hedw.) Loeske has presented high carotenoid contents in response to an increase in UVB radiation. This moss has been recommended as a potential source of antioxidants. In the present work, the protective and enhancing effects of aqueous (AE) and hydroalcoholic (HE) extracts of S. uncinata on the cleavage of supercoiled DNA were evaluated through topological modifications, quantified by densitometry after agarose gel electrophoresis. Total phenolic contents reached 5.89 mg/g. Our data demonstrated that the extract does not induce DNA cleavage. Furthermore, both extracts showed antioxidant activity that protected the DNA against cleavage induced by (i) O₂^??, 89% (AE) and 94% (HE) (P < 0.05), and (ii) .OH, 17% (AE) and 18% (HE). However, the extracts intensified cleavage induced by Fenton-like reactions: (i) Cu²⁺/H₂O₂, 94% (AE) and 100% (HE) (P < 0.05), and (ii) SnCl₂, 62% (AE) and 56% (HE). DNA damages seem to follow different ways: (i) in the presence of Fenton-like reactions could be via reactive oxygen species generation and (ii) with HE/Cu²⁺ could have also been triggered by other mechanisms.     

Protective mechanism of metallothionein against copper-1, 10-phenanthroline induced DNA cleavage

Metallothionein (MT) has been shown to protect DNA against cleavage induced by a variety of mutagenic agents. The mechanism has been attributed to its ability to either chelate transitional metals that participate in the Fenton reaction, or scavenge free radicals by means of the abundant cystenyl residues of the proteins. In the present study, the protective action of MT against DNA cleavage by the copper-1,10-phenanthroline [(OP)(2)Cu(+)] complex was studied in situ. At 0.1 microM, MT inhibited the (OP)(2)Cu(+) induced DNA cleavage by about 50% (IC(50) approximately 0.1 microM). At 2.5 microM, the cleavage activity was completely inhibited. Similar to MT, cysteine can protect against DNA cleavage by (OP)(2)Cu(+) (IC(50) of approximately 3 mM), however, its action was 1500-fold less efficient than MT. The combined action of MT and cysteine was additive. Reduced glutathione (1 and 10 mM) did not protect the (OP)(2)Cu(+) induced DNA cleavage. Sodium azide could inhibit the cleavage only at high concentrations (IC(40) approximately 25 mM). Spectrophotometric analysis showed that MT can inhibit the formation of the DNA[(OP)(2)Cu(+)] complex possibly by chelating Cu. It can also cause a dissociation of the complex after it was formed. In the later case, the mechanism through which MT protects against the DNA cleavage might occur when MT fitted in closely with the complex, competing with the hydroxyl groups of the nucleotides base for Cu, which, in turn, terminate the Fenton-like free radical reaction.     

DNA binding and nucleolytic properties of Cu(ii) complexes of salicylaldehyde semicarbazones

The copper(ii) complexes of two salicylaldehyde semicarbazones, HOC(6)H(4)CH[double bond, length as m-dash]N-NHCONR(2) [H(2)Bnz(2) (R = CH(2)Ph) and H(2)Bu(2) (R = Bu)], were evaluated for their DNA binding and cleavage properties by spectrophotometric DNA titration, ethidium bromide displacement assay and electrophoretic mobility shift assay. Results showed that the Cu(ii) complexes can bind to DNA via a partial intercalation mode with binding constants of 1.1 × 10(4) and 9.5 × 10(3) M(-1) for [Cu(HBnz(2))Cl] and [Cu(HBu(2))Cl], respectively. These complexes also cleave DNA in the presence of ascorbic acid, most likely through hydroxyl radicals that are generated via the reduction of a Cu(ii) to a Cu(i) species. The complexes show similar DNA cleavage activity, which is reflected in the similarity of their frontier molecular orbital energies calculated by density functional theory. These results are discussed in relation to the anticancer properties of the complexes.     

南极三洋藓(Sanionia uncinata)细菌群落组成分析

【背景】南极苔藓中蕴藏着丰富的极地微生物资源,然而目前对南极苔藓细菌群落组成的研究较少,限制了对这类极端生境微生物资源的开发和应用。【目的】在南极长城站不同地点随机取样,揭示南极三洋藓(Sanioniauncinata)的细菌群落结构组成。【方法】采用IlluminaHiSeq高通量测序技术对3个苔藓样品的16S rRNA基因V4区进行测序及生物信息学分析。【结果】3个苔藓样品中共获得细菌总优化序列273 367条。基于97%序列相似度,细菌优化序列聚类为9 579个OTU。分类地位明确的细菌可归为14门27纲50属。优势类群是变形菌门(Proteobacteria)(30.70%)、拟杆菌门(Bacteroidetes)(19.67%)、疣微菌门(Verrucomicrobia)(12.43%)、蓝细菌门(Cyanobacteria)(10.55%)及放线菌门(Actinobacteria)(9.36%)。在属水平上,56.73%属于未知细菌。【结论】南极三洋藓(Sanionia uncinata)中具有丰富多样的细菌,该研究为今后极地微生物研究奠定了基础。     

DNA breaks induction by mimosine

Mouse erythroleukemic F4 N cells were treated with mimosine, etoposide, Fe(II)-EDTA, and Cu(II) in the presence of ascorbate. DNA was isolated and subjected to agarose gel electrophoresis and the size and distribution of the DNA fragments produced by the agents were compared. With increasing concentration of Cu(II) the production of DNA fragments was increased without decrease of the average length of the fragments, and their sizes were similar to those produced by etoposide as expected for cleavage of DNA at the nuclear matrix attachments sites. In contrast, mimosine and Fe(II) produced fragments of random size and with the progression of the reaction the average length of the fragments decreased. These results indicate that mimosine cuts DNA in a random fashion, regardless of its higher order chromatin organization. A conclusion is drawn that the DNA fragments obtained after mimosine treatment are a result of mimosine-assisted, Fe(II) dependent Fenton-like reactions randomly cutting chromosomal DNA.     

Principles of the bark of Amphipterygium adstringens (Julianaceae) with anti-inflammatory activity.

Despite the fact that Amphipterygium adstringens (usually known as "cuachalalate") is used intensively in traditional medicine throughout México, there are, to our knowledge, no previous studies concerning the actual therapeutic, anti-inflammatory properties of this species. This lack of data prompted us to evaluate the aqueous (AE) and hexane (HE) extracts from A. adstringens in two models of acute inflammation: 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ear edema and carrageenan-induced paw edema. The results showed that HE possesses dose-dependent activity, while AE showed no anti-inflammatory effect on TPA-induced edema. Reverse effects were found in the carrageenan test, wherein AE showed a 73.5% of inhibition of edema, while HE showed only a 14.4% activity at 100 mg/kg body weight. These results could indicate that AE and HE possess different anti-inflammatory mechanisms of action. On the other hand, it is known that masticadienonic (1) and 3alpha-hydroxymasticadienonic (2) acids are the main constituents of the organic extract of A. adstringens bark. Because of this knowledge, we tested 1 and 2 in the same experimental models. The results showed that 2 possesses a dose-dependent effect, while 1 does not show a dose-dependent response in TPA-induced edema. In carrageenan-induced edema tests, both 1 and 2 showed almost the same activity (approximately 44% inhibition at 100 mg/kg body weight). In order to determine whether the anti-inflammatory activities of AE, HE, 1 and 2 are involved in the alteration of inducible nitric oxide synthase (iNOS) activity, we evaluated these substances by examining nitric oxide generation in lipopolysaccharide (LPS)-activated peritoneal macrophages. The results showed that 1 presented the highest activity (93.3%), followed by 2 (86.5%), while AE (57%) and HE (33.6%) showed the lowest. In the cytotoxic MTT assay, however only 1 and 2 showed any activity whatsoever.     

Different modes of DNA cleavage activity of dihydroxo-bridged dicopper(II) complexes having phenanthroline bases

Dihydroxo-bridged dicopper(II) complexes [(Cu(phen))(2)(mu-OH)(2)](ClO(4))(2) (1), [(Cu(dpq))(2)(mu-OH)(2)](ClO(4))(2) (2) and [(Cu(dppz)(DMF))(2)(mu-OH)(2)](PF(6))(2) (3), where phen, dpq and dppz are 1,10-phenanthroline, dipyridoquinoxaline and dipyridophenazine, respectively, are prepared and their DNA binding and cleavage properties studied. Complex 3 has been structurally characterized by X-ray crystallography. The complexes have a (Cu(2)(mu-OH)(2))(2+) core with an essentially planar arrangement of two CuN(2)O(2) basal planes. The complexes are avid binder to calf thymus DNA (K(app) value of 4.8 x 10(6) and 5.9 x 10(6) M(-1) for 2 and 3, respectively, from ethidium displacement assay) and exhibits significant cleavage of supercoiled (SC) pUC19 DNA in dark in presence of mercaptopropionic acid. Besides, the dpq and dppz complexes display photo-induced DNA cleavage on UV (312 nm) and red light (632.8 nm) irradiations in absence of any additives. Mechanistic investigations reveal minor groove binding for the phen and dpq complexes, and major groove preference for the dppz species. The oxidative DNA cleavage reactions in presence of mercaptopropionic acid as a reducing agent involve hydroxyl radicals. The photo-cleavage reactions at UV light involve singlet oxygen as the reactive species, while similar reactions on red light irradiation (632.8 nm) proceed through the formation of hydroxyl radical. The complexes show significant DNA hydrolase activity in absence of any additives under dark reaction conditions.     

Role of poly(A) polymerase in the cleavage and polyadenylation of mRNA precursor.

To determine the role of poly(A) polymerase in 3'-end processing of mRNA, the effect of purified poly(A) polymerase antibodies on endonucleolytic cleavage and polyadenylation was studied in HeLa nuclear extracts, using adenovirus L3 pre-mRNA as the substrate. Both Mg2+- and Mn2+-dependent reactions catalyzing addition of 200 to 250 and 400 to 800 adenylic acid residues, respectively, were inhibited by the antibodies, which suggested that the two reactions were catalyzed by the same enzyme. Anti-poly(A) polymerase antibodies also inhibited the cleavage reaction when the reaction was coupled or chemically uncoupled with polyadenylation. These antibodies also prevented formation of specific complexes between the RNA substrate and components of nuclear extracts during cleavage or polyadenylation, with the concurrent appearance of another, antibody-specific complex. These studies demonstrate that (i) previously characterized poly(A) polymerase is the enzyme responsible for addition of the poly(A) tract at the correct cleavage site and probably for the elongation of poly(A) chains and (ii) the coupling of these two 3'-end processing reactions appears to result from the potential requirement of poly(A) polymerase for the cleavage reaction. The results suggest that the specific endonuclease is associated with poly(A) polymerase in a functional complex.     

Photosensitized DNA damage induced by NADH: site specificity and mechanism

Increasing evidence reveals the carcinogenicity of UVA radiation. We demonstrated that UVA-irradiated NADH induced damage to (32)P-labeled DNA fragments obtained from the p53 gene in the presence of Cu(II). Formamidopyrimidine glycosylase (Fpg)-sensitive lesions were formed at guanine residues, whereas piperidine-labile lesions occurred frequently at thymine residues. Formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), upon UVA exposure in the presence of Cu(II), increased depending on NADH concentration. Catalase and bathocuproine, a Cu(I)-specific chelator, inhibited the DNA damage, suggesting the involvement of reactive species derived from H(2)O(2) and Cu(I). UVA-irradiated riboflavin induced DNA cleavage through electron transfer at 5' guanine of the 5'-GG-3' sequence with both Fpg and piperidine treatments; Fpg induced less cleavage at the guanine residues than piperidine. These results imply that NADH may participate as an endogenous photosensitizer in UVA carcinogenesis via H(2)O(2) generation, producing metal-mediated mutagenic lesions such as 8-oxodG.     

Oxidative damage of DNA induced by the cytochrome C and hydrogen peroxide system

To elaborate the peroxidase activity of cytochrome c in the generation of free radicals from H2O2, the mechanism of DNA cleavage mediated by the cytochrome c/H2O2 system was investigated. When plasmid DNA was incubated with cytochrome c and H2O2, the cleavage of DNA was proportional to the cytochrome c and H2O2 concentrations.Radical scavengers, such as azide, mannitol, and ethanol, significantly inhibited the cytochrome c/H2O2 system-mediated DNA cleavage. These results indicated that free radicals might participate in the DNA cleavage by the cytochrome c and H2O2 system. Incubation of cytochrome c with H2O2 resulted in a time-dependent release of iron ions from the cytochrome c molecule. During the incubation of deoxyribose with cytochrome c and H2O2, the damage to deoxyribose increased in a time-dependent manner, suggesting that the released iron ions may participate in a Fenton-like reaction to produce dOH radicals that may cause the DNA cleavage. Evidence that the iron-specific chelator, desferoxamine (DFX), prevented the DNA cleavage induced by the cytochrome c/H2O2 system supports this mechanism. Thus we suggest that DNA cleavage is mediated via the generation of dOH by a combination of the peroxidase reaction of cytochrome c and the Fenton-like reaction of free iron ions released from oxidatively damaged cytochrome c in the cytochrome c/H2O2 system.     

DNA cleavage mediated by copper superoxide dismutase via two pathways

The known action of Cu, Zn superoxide dismutase (Cu(2)Zn(2)SOD) that converts O(2)(-) to O(2) and H(2)O(2) plays a crucial role in protecting cells from toxicity of oxidative stress. However, the overproduction of Cu(2)Zn(2)SOD does not result in increased protection but rather creates a variety of unfavorable effects, suggesting that too much Cu(2)Zn(2)SOD may be injurious to the cells. The present study examined the DNA cleavage activity mediated by a Cu(n)SOD that contains 1-4 copper ions, in order to obtain an insight into the aberrant copper-mediated oxidative chemistry in the enzyme. A high SOD activity was observed upon metallation of the apo-form of Cu(2)Zn(2)SOD with Cu(II), indicating that nearly all of the Cu(II) in the Cu(n)SOD is as active as the Cu(II) in the copper site of fully active Cu(2)Zn(2)SOD. Using a supercoiled DNA as substrate, significant DNA cleavage was observed with the Cu(n)SOD in the presence of hydrogen peroxide or mercaptoethanol, whereas DNA cleavage with free Cu(II) ions can occur only <5% under the same conditions. Comparison with other proteins shows that the DNA cleavage activity is specific to some proteins including the Cu(n)SOD. The steady state study suggests that a cooperative action between the SOD protein and the Cu(II)may appear in the DNA cleavage activity, which is independent of the number of Cu(II) in the Cu(n)SOD. The kinetic study shows that a two-stage reaction was involved in DNA cleavage. The effects of various factors including EDTA, radical scavengers, bicarbonate anion, and carbon dioxide gas molecules on the Cu(n)SOD-mediated DNA cleavage activity were also investigated. It is proposed that DNA cleavage occurs via both hydroxyl radical oxidation and hydroxide ion hydrolysis pathways. This work implies that any form of the copper-containing SOD enzymes (including Cu(2)Zn(2)SOD and its mutants) might have the DNA cleavage activity.     

DNA Polymerase-Associated and Autonomous 3"→5" Exonucleases in Invertebrates, Unicellulars, and Bacteria

Recently we have revealed a high content of autonomous 3"5" exonucleases (AE), i.e., those not bound covalently with DNA polymerases, in cells of vertebrates, from fish to human [1]. In the present work, using gel filtration method, cell-free extracts were studied from 15 objects located at different positions on the phylogenetic tree, such as archaebacteria, eubacteria, fungi, infusorians, coelenterates, annelids, and arthropods. It is shown that enzymatic activity of AE accounts for from 30 to 88% of the total 3"5" exonuclease activity of the extracts. A part of AE is revealed in zone of high-molecular DNA polymerases and can be separated by change of the chromatography conditions. It indicates a probable formation of complexes of AE with DNA polymerases. The high AE activity in cells of different organisms, from archae- and eubacteria to human, allows suggesting these enzymes to play a significant role in correction of polymerase errors in the processes of DNA replication and reparation, as well as in postreplicative correction of heteroduplexes in DNA.     

Interactions of competent Streptococcus sanguis (Wicky) cells with native or denatured, homologous or heterologous deoxyribonucleic acids.

Competent cell-deoxyribonucleic acid (DNA) interactions were examined using tritium-labeled homologous or heterologous native or denatured DNAs and competent Streptococcus sanguis Wicky cells (strain WE4). The DNAs used were extracted from WE4 cells, Escherichia coli B cells, and E. coli bacteriophages T2, T4, T6, and T7. The reactions examined were: (i) total DNA binding, (ii) deoxyribonuclease-resistant DNA binding, and (iii) the production of acid-soluble products from the DNA. Optimal temperatures for the reactions were as follows: reaction (i), between 30 and 40 degrees C; reaction (ii), 30 degrees C; and reaction (iii), greater than 40 degrees C. The rates for the reactions (expressed as molecules of DNA that reacted per minute per colony-forming unit) did not vary greatly from one DNA source to another. With a constant competent cell concentration and differing DNA concentrations below a saturation level (from a given source), a different but constant fraction of the added DNA was cell bound, deoxyribonuclease resistant, and degraded to acid-soluble products. In experiments where the number of competent cells was varied and the DNA concentration was held constant, again essentially the same result was obtained. The extent of reactions (i), (ii), and (iii) depended upon the numbers as well as the source of DNA molecules applied to competent cells. Calcium ion essential for native DNA-cell reactions was also found essential for denatured DNA-cell reactions. Data obtained from competition experiments lead to the conclusion that competent WE4 cells contain specific sites for native as well as denatured DNAs.     

Acridine conjugates of 3-clip-phen: influence of the linker on the synthesis and the DNA cleavage activity of their copper complexes

To increase the DNA cleavage activity and the cell delivery of the bis(phenanthroline) DNA cleaver "3-Clip-Phen", conjugates between 3-Clip-Phen and the intercalators acridine and 6-chloro-2-methoxyacridine, through amino acid linkers of various length, were prepared. After complexation with CuCl(2), the ability of these conjugates to cleave phiX 174 DNA in the presence of a reductant and air was compared. The results indicated that (i) the coupling of 3-Clip-Phen to an acridine derivative increased the DNA cleavage efficiency of the copper complexes, (ii) the acridine derivatives were more active than 6-chloro-2-methoxyacridine derivatives, (iii) the linker length influenced cleavage efficiency, the highest DNA cleavage activity being obtained for an aminocaproic spacer.     

The bacteriophage lambda terminase. Partial purification and preliminary characterization of properties

The maturation of bacteriophage lambda DNA and its packaging into preformed heads to produce infectious phage is under the control of the two leftmost genes on the lambda chromosome, i.e., Nu1 and A. Based on its ability to complement lambda A- phage-infected cell extracts for packaging of lambda DNA in vitro, a single protein, designated terminase (ter) has been extensively purified using adsorption, ion exchange, and affinity column chromatography. The final preparation represents an approximately 60,000-fold purification over the activity found in crude extracts and is about 30 to 80% homogeneous as judged by visualizing the protein after electrophoresis in sodium dodecyl sulfate-polyacrylamide gel. In addition to packaging, terminase can also catalyze the endonucleolytic cleavage of lambda cohesive-end site DNA; both of these reactions require ATP. In some preparations, certain terminase fractions of extreme purity require protein factors present in extracts of uninfected Escherichia coli in order to catalyze the cohesive-end site cleavage reaction. On ion exchange columns purified terminase co-chromatographs with a DNA-dependent ATPase activity, hydrolyzing ATP to ADP and Pi in the presence of any of several types of DNA tested including those of non-lambda origin. The molecular weight of the native enzyme is 117,000 and appears to be a hetero-oligomer composed of 2 nonidentical subunits. The most likely composition of terminase is one gpA (gene product of A), Mr = 74,000 and two gpNu1, Mr = 21,000.     

Enhancing effect of melatonin on chemiluminescence accompanying decomposition of hydrogen peroxide in the presence of copper

The oxidation of melatonin (MEL) using the Cu(II) + H₂O₂ + HO⁻ (the Fenton-like reaction) system was investigated by chemiluminescence (CL), fluorescence, spectrophotometric, and EPR spin trapping techniques. The reaction exhibits CL in the 400–730 nm region. The light emission from the Fenton-like reaction was greatly enhanced in the presence of MEL and was strongly dependent on its concentration. The spectrum measured with cut-off filters revealed maxima at around 460, 500, 580–590, 640–650, and 690–700 nm. The band at 460 nm may be due to the excited cleavage product, N¹-acetyl-N²-formyl-5-methoxykynuramine, whereas the bands at 500, 580–590, 640–650, and 700 nm were similar to those observed for singlet molecular oxygen (¹O₂). The effect of reactive oxygen species (ROS) scavengers on the light emission was studied. The CL was strongly inhibited by the ¹O₂ scavengers in a dose-dependent manner; at concentration 1 mM the potency of ¹O₂ scavenging was 5,5-dimethylcyclohexandione-1,3 > methionine > histidine > hydroquinone. The potency of HO^• scavenging by thiourea, tryptophan, cysteine at concentration 5 mM was 79–94%, by 1 mM glutathione and trolox 75 and 94%, respectively, and by 10 mM cimetidine 18%. Specific acceptors of O₂^•− such as p-nitroblue tetrazolium chloride and 4,5-dihydroxy-1,3-benzene disulfonic acid (tiron) at concentration 5 mM decreased the CL by 51 and 95%, respectively, whereas superoxide dismutase (SOD) does not reduce the emission at concentration 2.8 U/ml. At higher concentration SOD substantially enhanced the light emission. Addition of 1360 U/ml catalase and 100 μM desferrioxamine strongly inhibited CL (96 and 90%, respectively). The increased generation of ¹O₂ from the Cu/H₂O₂ system in the presence of MEL was confirmed using the spectrophotometric method based on the bleaching of p-nitrosodimethylaniline and by trapping experiments with 2,2,6,6-tetramethylpiperidine (TEMP) and subsequent electron paramagnetic (EPR) spectroscopy. These findings suggest the increased production of reactive oxygen species (O₂^•−, HO^•, ¹O₂) from the Fenton-like reaction in the presence of MEL. This means that the hormone is not able to act as classical chain-breaking antioxidant even at low concentration, and may show clear prooxidant activity at higher concentrations. In addition, long-lived carbonyl product of the MEL transformation in the triplet state can also be toxic by transferring its energy to organelles and causing a photochemical process.     

陇中黄土高原典型草原生物土壤结皮发育对土壤养分的影响

为了解生物土壤结皮发育与土壤养分特征的关系,该研究选取处于不同发育阶段和不同盖度的藓结皮(M-crust)和地衣结皮(L-crust),对其结皮层生物体及结皮下层3 cm土壤进行养分分析。结果显示：(1)藓结皮生物体对土壤全氮(TN)、全磷(TP)、铜(Cu)和锌(Zn)的生物富集作用较强,其富集系数分别高达807.4%、175%、122.4%和244.5%,而地衣结皮生物体对TN、TP和钙(Ca)的生物富集作用较强,其富集系数分别高达950.8%、126.2%和208.6%;除Ca外,藓结皮生物体中TP、钠(Na)、钾(K)、Cu、铁(Fe)、镁(Mg)、锰(Mn)和Zn均显著高于地衣结皮。(2)土壤生物结皮盖度与其对应的土壤养分含量之间无显著线性相关关系,但发育有不同盖度藓结皮的土壤其平均TN、TP、Cu、Fe、K、Na和Ca含量均高于地衣结皮。研究表明,不同类型生物土壤结皮对养分吸收、富集效率不同,处于发育后期的藓结皮较发育中期的地衣结皮具有更强的养分累积效应,提高了土壤的养分贮存量,对黄土高原典型草原生态系统的恢复与重建具有潜在的重要影响。     

Effect of charge transfer bands on the photo-induced DNA cleavage activity of [1-(2-thiazolylazo)-2-naphtholato]copper(II) complexes

Ternary copper(II) complex [Cu(TAN)(O2CMe)] (1), where H-TAN is 1-(2-thiazolylazo)-2-naphthol, is prepared and structurally characterized by X-ray crystallography. The complex has a distorted square pyramidal (4+1) CuN2O3 coordination geometry with the acetate showing chelating axial-equatorial binding mode and TAN as a tridentate ligand bonded to the metal in the basal plane. Complex 1 is one-electron paramagnetic and displays ligand-to-metal charge transfer bands at 575 and 398 nm in dimethylformamide. The reactions of 1 with bases (B) like 1,10-phenanthroline (phen) and kanamycin-A (kan-A) afford ternary complexes of formulation [Cu(TAN)B]+ (B=phen, 2; kan-A, 3) under in situ reaction conditions. Complexes 2 and 3, prepared to explore their DNA binding and photo-induced DNA cleavage activity, display good binding propensity to calf thymus (CT) DNA giving a relative order: 2-3>1. The apparent binding constant (Kapp) for 1 is determined as 9.8 x 10(5)M(-1) from fluorescence quenching experiments using ethidium bromide. The quenching constants (K) values of 1-3, obtained from the Stern-Volmer plots, are 0.28, 0.52 and 0.49, respectively. All the complexes show photo-induced DNA cleavage activity when irradiated with a monochromatic UV light of 365 nm wavelength. A 200 microM complex 1 cleaves approximately 75% supercoiled (SC) DNA on 2h exposure time at 365 nm. A 50 microM solution of 1 in presence of 100 microM phen and kanamycin-A cleaves approximately 99% and approximately 60% SC DNA to its nicked circular form, respectively, for an exposure of 30 min. The complexes also exhibit significant cleavage of SC DNA on irradiation with visible light of wavelengths 532, 575 and 632.8 nm. Control experiments reveal the minor groove binding nature of the complexes. The cleavage reactions involve the formation reactive hydroxyl species as significant inhibition in the presence of dimethyl sulfoxide (DMSO) and catalase is observed. There is no apparent inhibition in cleavage in the presence of singlet oxygen quenchers like sodium azide. The cleavage activity has been found to be higher at the CT band position of 575 nm in comparison to those at 532 and 632.8 nm. The results indicate the involvement of the CT band in the photo-excitation process.     

Photosynthesis of two moss crusts from the Tengger Desert with contrasting sensitivity to supplementary UV-B radiation

Predicting the effects of increased ultraviolet-B (UV-B) radiation due to stratospheric ozone depletion on temperate desert ecosystems requires better knowledge of the ecophysiological response of common moss species. The aim of the current work was to determine whether elevated UV-B radiation affected photosynthetic performance and chloroplast ultrastructure of two moss crusts and whether response differences were observed between the crusts. In laboratory experiments, Bryum argenteum and Didymodon vinealis, which show microdistributions and are dominant in soil crusts at the Tengger Desert, Northern China, were subjected to four levels of UV-B radiation of 2.75 (control), 3.08, 3.25, and 3.41 W m^?2 for 10 days, simulating 0, 6, 9, and 12% of stratospheric ozone at the latitude of Shapotou, respectively. The results showed that chlorophyll a fluorescence parameters (i.e., the maximal quantum yield of PSII photochemistry, the effective quantum yield of PSII photochemistry, and photochemical quenching coefficient), pigment contents, soluble protein contents, and the ultrastructure were negatively influenced by elevated UV-B radiation and the degree of detrimental effects significantly increased with the intensity of UV-B radiation. Moreover, results indicated that B. argenteum was probably more sensitive to supplementary UV-B radiation than D. vinealis. Therefore, we propose the use of B. argenteum crusts as a bioindicator of responses to elevated UV-B radiation.     

Histone peptide AKRHRK enhances H(2)O(2)-induced DNA damage and alters its site specificity

Histone proteins are involved in compaction of DNA and the protection of cells from oxygen toxicity. However, several studies have demonstrated that the metal-binding histone reacts with H(2)O(2), leading to oxidative damage to a nucleobase. We investigated whether histone can accelerate oxidative DNA damage, using a minimal model for the N-terminal tail of histone H4, CH(3)CO-AKRHRK-CONH(2), which has a metal-binding site. This histone peptide enhanced DNA damage induced by H(2)O(2) and Cu(II), especially at cytosine residues, and induced additional DNA cleavage at the 5'-guanine of GGG sequences. The peptide also enhanced the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine and ESR spin-trapping signal from H(2)O(2) and Cu(II). Cyclic redox reactions involving histone-bound Cu(II) and H(2)O(2), may give rise to multiple production of radicals leading to multiple hits in DNA. It is noteworthy that the histone H4 peptide with specific sequence AKRHRK can cause DNA damage rather than protection under metal-overloaded condition.