Architectures based on the use of gold nanoparticles and ruthenium complexes as a new route to improve genosensor sensitivity

The preparation of DNA-sensing architectures based on gold nanoparticles (Au-NPs) in conjunction with an "in situ" prepared ruthenium complex as a new route to improve the analytical properties of genosensors is described. In the development of these architectures several strategies to obtain Au-NPs modified gold electrodes (Au-NP/Au) have been essayed, in particular covalent binding and electrochemical deposition from a solution containing Au-NPs previously synthesized. UV-vis absorption measurements in conjunction with transmission electron microscope (TEM) images reveal that the synthesized Au-NPs are stable for at least 4 weeks and have a narrow size distribution. Atomic force microscopy (AFM) was employed to characterize the morphology and to estimate the Au-NPs surface coverage of the modified gold electrodes obtained following the different modification strategies. In order to assess the utility of these architectures as DNA-sensing devices, a thiolated capture probe sequence from Helicobacter pylori was immobilized onto the as-prepared surface. This sequence was chosen as a case of study within the framework of developing approaches of wide applicability. The hybridization event is detected using a water-soluble pentaamin ruthenium [3-(2-phenanthren-9-yl-vinyl)-pyridine] complex (Ru(NH(3))(5)L) prepared "in situ". This complex, due to its intercalative character, is able to bind to double stranded DNA more efficiently than to single stranded DNA. In addition, the metal provides with a redox center that can be used as an electrochemical indicator. On the basis of this strategy, complementary target sequences of H. pylori have been detected over the range of 40-800pmol with a detection limit of 25+/-2pmol.     

Detection of protein-DNA interaction with a DNA probe: distinction between single-strand and double-strand DNA-protein interaction

A simple, direct method for the detection of DNA-protein interaction was developed with electrochemical methods. Single-stranded DNA (ss-DNA) probes were prepared through the chemical bonding of an oligonucleotide to a polymer film bearing carboxylic acid groups, and double-stranded DNA (ds-DNA) probes were prepared through hybridization of the complementary sequence DNA on the ss-DNA probe. Impedance spectroscopy and differential pulse voltammetry (DPV) distinguished the interaction between the DNA probes with mouse Purbeta (mPurbeta), an ss-DNA binding protein, and with Escherichia coli MutH, a ds-DNA binding protein. Impedance spectra obtained before and after the interaction of DNA probes with these proteins clearly showed the sequence-specific ss-DNA preference of mPurbeta and the sequence-specific ds-DNA preference of MutH. The concentration dependence of proteins on the response of the DNA probes was also investigated, and the detection limits of MutH and mPurbeta were 25 and 3 microg/ml, respectively. To confirm the impedance results, the variation of the current oxidation peak of adenine of the DNA probe was monitored with DPV. The formation constants of the complexes formed between the probe DNA and the proteins were estimated based on the DPV results.     

Immobilization of homooligonucleotide probe layers onto Si/SiO(2) substrates: characterization by electrochemical impedance measurements and radiolabelling

Radiolabelling and electrochemical impedance measurements were used to characterize the immobilization of single stranded homooligonucleotides onto silica surfaces and their subsequent hybridization with complementary strands. The immobilization procedure consists of grafting an epoxysilane onto microelectronic grade Si/SiO(2) substrates, and coupling oligonucleotides bearing a hexylamine linker onto the epoxy moiety. Radiolabelling was used as a reference method to quantify the amount of immobilized and hybridized oligonucleotides. These results show that the Si/SiO(2) substrates modified with an epoxysilane yield a surface concentration of approximately 10(11) strands/cm(2) for the immobilized oligonucleotides, after vigorous washings, and that approximately 36% of these undergo hybridization with complementary strands. The impedance measurements, which provide a direct means of detecting variations in electrical charge accumulation across the semiconductor/oxide/electrolyte structure when the oxide surface is chemically modified, show that the semiconductor's flat band potential undergoes reproducible shifts of -150 and -100 mV following the immobilization and the hybridization step, respectively. These results demonstrate that electrochemical impedance measurements using chemically modified semiconductor/oxide/electrolyte structures of this type offer a viable alternative for the direct detection of complementary DNA strands upon hybridization.     

Detection of protein–DNA interaction with a DNA probe: distinction between single-strand and double-strand DNA–protein interaction

A simple, direct method for the detection of DNA–protein interaction was developed with electrochemical methods. Single-stranded DNA (ss-DNA) probes were prepared through the chemical bonding of an oligonucleotide to a polymer film bearing carboxylic acid groups, and double-stranded DNA (ds-DNA) probes were prepared through hybridization of the complementary sequence DNA on the ss-DNA probe. Impedance spectroscopy and differential pulse voltammetry (DPV) distinguished the interaction between the DNA probes with mouse Purβ (mPurβ), an ss-DNA binding protein, and with Escherichia coli MutH, a ds-DNA binding protein. Impedance spectra obtained before and after the interaction of DNA probes with these proteins clearly showed the sequence-specific ss-DNA preference of mPurβ and the sequence-specific ds-DNA preference of MutH. The concentration dependence of proteins on the response of the DNA probes was also investigated, and the detection limits of MutH and mPurβ were 25 and 3 μg/ml, respectively. To confirm the impedance results, the variation of the current oxidation peak of adenine of the DNA probe was monitored with DPV. The formation constants of the complexes formed between the probe DNA and the proteins were estimated based on the DPV results.     

Electrochemical spectroscopic investigations on the interaction of an ytterbium complex with DNA and their analytical applications such as biosensor

Metal ion-DNA interactions are important in nature, often changing the genetic material's structure and function. A new Yb complex of YbCl₃ (tris(8-hydroxyquinoline-5-sulfonic acid) ytterbium) was synthesized and utilized as an electrochemical indicator for the detection of DNA oligonucleotide based on its interaction with Yb(QS)₃. Cyclic voltammetry (CV) and fluorescence spectroscopy were used to investigate the interaction of Yb(QS)₃ with ds-DNA. It was revealed that Yb(QS)₃ presented an excellent electrochemical activity on glassy carbon electrode (GCE) and could intercalate into the double helix of double-stranded DNA (ds-DNA). The binding mechanism of interaction was elucidated on glassy carbon electrode dipped in DNA solution and DNA modified carbon paste electrode by using differential pulse voltammetry and cyclic voltammetry. The binding ratio between this complex and ds-DNA was calculated to be 1:1. The extent of hybridization was evaluated on the basis of the difference between signals of Yb(QS)₃ with probe DNA before and after hybridization with complementary DNA. With this approach, this DNA could be quantified over the range from 1 × 10⁻⁸ to 1.1 × 10⁻⁷ M. The interaction mode between Yb(QS)₃ and DNA was found to be mainly intercalative interaction. These results were confirmed with fluorescence experiments.     

Introduction of hematoxylin as an electroactive label for DNA biosensors and its employment in detection of target DNA sequence and single-base mismatch in human papilloma virus corresponding to oligonucleotide

For the detection of DNA hybridization, a new electrochemical biosensor was developed on the basis of the interaction of hematoxylin with 20-mer deoxyoligonucleotides (from human papilloma virus, HPV). The study was performed based on the interaction of hematoxylin with an alkanethiol DNA probe self-assembled gold electrode (ss-DNA/AuE) and its hybridization form (ds-DNA/AuE). The optimum conditions were found for the immobilization of HPV probe on the gold electrode (AuE) surface and its hybridization with the target DNA. Electrochemical detection of the self-assembled DNA and the hybridization process were performed by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) over the potential range where the accumulated hematoxylin at the modified electrode was electroactive. Observing a remarkable difference between the voltammetric signals of the hematoxylin obtained from different hybridization samples (non-complementary, mismatch and complementary DNAs), we confirmed the potential of the developed biosensor in detecting and discriminating the target complementary DNA from non-complementary and mismatch oligonucleotides. Under optimum conditions, the electrochemical signal had a linear relationship with the concentration of the target DNA ranging from 12.5 nM to 350.0 nM, and the detection limit was 3.8 nM.     

A DFT method for the study of the antioxidant action mechanism of resveratrol derivatives

Quantum-chemical calculations using DFT, have been performed to explain the molecular structure antioxidant activity relationship of resveratrol (RSV) (1) analogues: 3,4-dihydroxy-trans-stilbene (3,4-DHS) (2); 4,4′-dihydroxy-trans-stilbene (4,4′-DHS) (3); 4-hydroxy-trans-stilbene (4-HS) (4); 3,5-dihydroxy-trans-stilbene (3,5-DHS) (5); 3,3′-dimethoxy-4,4′-dihydroxy-trans-stilbene (3,3′-DM-4,4′-DHS) (6); 2,4-dihydroxy-trans-stilbene (2,4-DHS) (7) and 2,4,4′-trihydroxy-trans-stilbene (2,4,4′-THS) (8). It was found that all compounds studied were effective antioxidants with the exception of 3, 5-DHS. The high antioxidant activity of both 3, 3′-DM-4, 4′-DHS and 3, 4-DHS may be due to the abstraction of the two hydrogen atoms of the para and ortho-position hydroxyls respectively, to form a quinone structure. Our results revealed that the antioxidant pharmacophore of 2,4-DHS and 2,4,4′-THS, exhibiting higher antioxidant activity than resveratrol, is the 2-hydroxystilbene, rather than 4-hydroxystilbene. Experimental observations were satisfactorily explained and commented.     

Electrochemical aptasensor using the tripropylamine oxidation to probe intramolecular displacement between target and complementary nucleotide for protein array

Tripropylamine (TPA) has different oxidation efficiency at double stranded (ds)-and single stranded (ss)-DNA-modified electrodes. Using this property, a simple but sensitive biosensor using TPA oxidation to probe the intramolecular displacement was constructed with the analysis of lysozyme as model for the first time. After the complementary ss-DNA strand of anti-lysozyme aptamer was immobilized onto gold electrode via gold-thiol bond, the incubation with the aptamer resulted in the formation of ds-DNA. Lysozyme (in 10 μL sample) binding with aptamer displaced the complementary strand because of the high affinity of lysozyme and its aptamer, corresponding to the dissociation of the ds-DNA. The modified electrode was swept in 20mM TPA solution from 0.2 to 0.95 V. The difference in oxidation current was used to quantify the content of lysozyme with a linear range from 1.0 pM to 1.1 nM. That means 10 amol or 6.0 × 10(6) lysozyme molecules can be detected. Because the signal is produced from the preconcentrated TPA at the electrode surface, the high sensitivity is achieved over the single site labelling strategy. The proposed method is simple, stable, specific, and time-saving while the complicated sample pre-treatment and the labelling to the DNA strand are avoided. The biosensor was validated by the analysis of the diluted egg white sample directly. The recovery and reproducibility were 93.3-100% and 1.4-4.2%, respectively.     

DNA damage induced by resveratrol and its synthetic analogues in the presence of Cu (II) ions: mechanism and structure-activity relationship

The prooxidant effect of resveratrol (3,5,4′-trihydroxy-trans-stibene) and its synthetic analogues (ArOH), that is, 3,4,4′-trihydroxy-trans-stibene (3,4,4′-THS), 3,4,5-trihydroxy-trans-stibene (3,4,5-THS), 3,4-dihydroxy-trans-stibene (3,4-DHS), 4,4′-dihydroxy-trans-stibene (4,4′-DHS), 2,4-dihydroxy-trans-stilbene (2,4-DHS), 3,5-dihydroxy-trans-stilbene (3,5-DHS) and 3,5,4′-trimethoxy-trans-stibene (3,5,4′-TMS), on supercoiled pBR322 plasmid DNA strand breakage and calf thymus DNA damage in the presence of Cu (II) ions has been studied. It was found that the compounds bearing ortho-dihydroxyl groups (3,4-DHS, 3,4,4′-THS, and 3,4,5-THS) or bearing 4-hydroxyl groups (2,4-DHS, 4,4′-DHS, and resveratrol) exhibit remarkably higher activity in the DNA damage than the ones bearing no such functionalities. Kinetic analysis by UV-visible spectra demonstrates that the formation of ArOH-Cu (II) complexes, the stabilization of oxidative intermediate derived from ArOH and Cu (II)/Cu (I) redox cycles, might be responsible for the DNA damage. This study also reveals a good correlation between antioxidant and prooxidant activity, as well as cytotoxicity against human leukemia (HL-60 and Jurkat) cell lines. The mechanisms and implications of these observations are discussed.     

DNA damage induced by resveratrol and its synthetic analogues in the presence of Cu (II) ions: Mechanism and structure-activity relationship

The prooxidant effect of resveratrol (3,5,4′-trihydroxy-trans-stibene) and its synthetic analogues (ArOH), that is, 3,4,4′-trihydroxy-trans-stibene (3,4,4′-THS), 3,4,5-trihydroxy-trans-stibene (3,4,5-THS), 3,4-dihydroxy-trans-stibene (3,4-DHS), 4,4′-dihydroxy-trans-stibene (4,4′-DHS), 2,4-dihydroxy-trans-stilbene (2,4-DHS), 3,5-dihydroxy-trans-stilbene (3,5-DHS) and 3,5,4′-trimethoxy-trans-stibene (3,5,4′-TMS), on supercoiled pBR322 plasmid DNA strand breakage and calf thymus DNA damage in the presence of Cu (II) ions has been studied. It was found that the compounds bearing ortho-dihydroxyl groups (3,4-DHS, 3,4,4′-THS, and 3,4,5-THS) or bearing 4-hydroxyl groups (2,4-DHS, 4,4′-DHS, and resveratrol) exhibit remarkably higher activity in the DNA damage than the ones bearing no such functionalities. Kinetic analysis by UV-visible spectra demonstrates that the formation of ArOH-Cu (II) complexes, the stabilization of oxidative intermediate derived from ArOH and Cu (II)/Cu (I) redox cycles, might be responsible for the DNA damage. This study also reveals a good correlation between antioxidant and prooxidant activity, as well as cytotoxicity against human leukemia (HL-60 and Jurkat) cell lines. The mechanisms and implications of these observations are discussed.     

The involvement of primary and secondary metabolism in the covalent binding of 1,2- and 1,4-dichlorobenzenes.

The microsomal oxidation of 1,2-[14C]- and 1,4-[14C]dichlorobenzene (DICB) was investigated with special attention for possible differences in biotransformation that might contribute to the isomer-specific hepatotoxicity. Major metabolites of both isomers were dichlorophenols (2,5-DICP for 1,4-DICB and 2,3- and 3,4-DICP for 1,2-DICB, respectively) and dichlorohydroquinones. The formation of polar dihydrodiols appeared to be a major route for 1,2-DICB but not 1,4-DICB. Both the hepatotoxic 1,2-DICB and the non-hepatotoxic 1,4-DICB were oxidized to metabolites that covalently interacted with protein and only to a small extent with DNA. Protein binding could be inhibited by the addition of the reducing agent ascorbic acid with a concomitant increase in the formation of hydroquinones and catechols, indicating the involvement of reactive benzoquinone metabolites in protein binding. However, in the presence of ascorbic acid, a substantial amount of protein-bound metabolites of 1,2-DICB was still observed, in contrast to 1,4-DICB where binding was nearly completely inhibited. This latter effect was ascribed to the direct formation of reactive benzoquinone metabolites in a single P450-mediated oxidation of para-substituted dichlorophenols (such as 3,4-DICP) in the case of 1,2-DICB. In contrast, the major phenol isomer derived from 1,4-DICB (i.e. 2,5-DICP) is oxidized to its hydroquinone derivative, which needs prior oxidation in order to generate the reactive benzoquinone species. Residual protein binding in the presence of ascorbic acid could also indicate the involvement of reactive arene oxides in the protein binding of 1,2-DICB, but not of 1,4-DICB. However, MO computer calculations did not provide indications for differences in chemical reactivity and/or stability of the various arene oxide/oxepin tautomers that can be formed from either 1,2-DICB or 1,4-DICB. In conclusion, reactive intermediates in the secondary metabolism of 1,2-DICB lead to more covalent binding than those derived from 1,4-DICB, which correlates very well with their reported hepatotoxic potency.     

Electrochemical detection of DNA hybridization using a change in flexibility

A novel electrochemical method of detecting DNA hybridization is presented based on the change in flexibility between the single and double stranded DNA. A recognition surface based on gold nanoparticles (GNPs) is firstly modified via mixing self-assembled monolayer of thiolated probe DNA and 1,6-hexanedithiol. The hybridization and electrochemical detection are performed on the surface of probe-modified GNPs and electrode, respectively. Here in our method the charge transfer resistance (R(ct)) signal is enhanced by blocking the surface of electrode with DNA covered GNPs. The GNPs will be able to adsorb on the gold electrode when covered with flexible single stranded DNA (ssDNA). On the contrary, it will be repelled from the electrode, when covered with stiff double stranded DNA (dsDNA). Therefore, different R(ct) signals are observed before and after hybridization. The hybridization events are monitored by electrochemical impedance spectroscopy (EIS) measurement based on the R(ct) signals without any external labels. This method provides an alternative route for expanding the range of detection methods available for DNA hybridization.     

Formation of DNA adducts by microsomal and peroxidase activation of p-cresol: role of quinone methide in DNA adduct formation.

We have investigated the activation of p-cresol to form DNA adducts using horseradish peroxidase, rat liver microsomes and MnO(2). In vitro activation of p-cresol with horseradish peroxidase produced six DNA adducts with a relative adduct level of 8.03+/-0.43 x 10(-7). The formation of DNA adducts by oxidation of p-cresol with horseradish peroxidase was inhibited 65 and 95% by the addition of either 250 or 500 microM ascorbic acid to the incubation. Activation of p-cresol with phenobarbital-induced rat liver microsomes with NADPH as the cofactor; resulted in the formation of a single DNA adduct with a relative adduct level of 0.28+/-0.08 x 10(-7). Similar incubations of p-cresol with microsomes and cumene hydroperoxide yielded three DNA adducts with a relative adduct level of 0.35+/-0.03 x 10(-7). p-Cresol was oxidized with MnO(2) to a quinone methide. Reaction of p-cresol (QM) with DNA produced five major adducts and a relative adduct level of 20.38+/-1.16 x 10(-7). DNA adducts 1,2 and 3 formed by activation of p-cresol with either horseradish peroxidase or microsomes, are the same as that produced by p-cresol (QM). This observation suggests that p-cresol is activated to a quinone methide intermediate by these activation systems. Incubation of deoxyguanosine-3'-phosphate with p-cresol (QM) resulted in a adduct pattern similar to that observed with DNA; suggesting that guanine is the principal site for modification. Taken together these results demonstrate that oxidation of p-cresol to the quinone methide intermediate results in the formation of DNA adducts. We propose that the DNA adducts formed by p-cresol may be used as molecular biomarkers of occupational exposure to toluene.     

The study of interactions between DNA and PcrA DNA helicase by using targeted molecular dynamic simulations

DNA helicases are important enzymes involved in all aspects of nucleic acid metabolism, ranging from DNA replication and repair to recombination, rescue of stalled replication and translation. DNA helicases are molecular motors. Through conformational changes caused by ATP hydrolysis and binding, they move along the template double helix, break the hydrogen bonds between the two strands and separate the template chains, so that the genetic information can be accessed. In this paper, targeted molecular dynamic simulations were performed to study the important interactions between DNA and PcrA DNA helicase, which can not be observed from the crystal structures. The key residues on PcrA DNA helicase that have strong interactions with both double stranded DNA (ds-DNA) and single stranded DNA (ss-DNA) have been identified, and it was found that such interactions mostly exist between the protein and DNA backbone, which indicates that the translocation of PcrA is independent of the DNA sequence. The simulations indicate that the ds-DNA is separated upon ATP rebinding, rather than ATP hydrolysis, which suggests that the two strokes in the mechanism have two different major roles. Firstly, in the power stroke (ATP hydrolysis), most of the translocations of the bases from one pocket to the next occur. In the relaxation stroke (ATP binding), most of the ‘work’ is being done to ‘melt’ the DNA at the separation fork. Therefore, we propose a mechanism whereby the translocation of the ss-DNA is powered by ATP hydrolysis and the separation of the ds-DNA is powered by ATP binding.     

DNA microdevice for electrochemical detection of Escherichia coli 0157:H7 molecular markers

An electrochemical DNA sensor based on the hybridization recognition of a single-stranded DNA (ssDNA) probe immobilized onto a gold electrode to its complementary ssDNA is presented. The DNA probe is bound on gold surface electrode by using self-assembled monolayer (SAM) technology. An optimized mixed SAM with a blocking molecule preventing the nonspecific adsorption on the electrode surface has been prepared. In this paper, a DNA biosensor is designed by means of the immobilization of a single stranded DNA probe on an electrochemical transducer surface to recognize specifically Escherichia coli (E. coli) 0157:H7 complementary target DNA sequence via cyclic voltammetry experiments. The 21 mer DNA probe including a C6 alkanethiol group at the 5' phosphate end has been synthesized to form the SAM onto the gold surface through the gold sulfur bond. The goal of this paper has been to design, characterise and optimise an electrochemical DNA sensor. In order to investigate the oligonucleotide probe immobilization and the hybridization detection, experiments with different concentration of DNA and mismatch sequences have been performed. This microdevice has demonstrated the suitability of oligonucleotide Self-assembled monolayers (SAMs) on gold as immobilization method. The DNA probes deposited on gold surface have been functional and able to detect changes in bases sequence in a 21-mer oligonucleotide.     

Electrochemical DNA biosensor for the detection of interaction between di[azino-di(5,6-azafluorene)-kappa2-NN'] dibromicoppers and DNA

A new Cu(II) complex CuL(2)Br(2) (L = azino-di(5,6-azafluorene)-kappa(2)-NN') was synthesized, and a new method of electrochemical probe has been proposed for the determination of hepatitis B virus (HBV) based on its interaction with [CuL(2)](2+). This ligand, containing functional groups, as well as planar aromatic domains, is capable of binding to double-stranded DNA (dsDNA) more efficiently than to single-stranded DNA (ssDNA). Emphasis has been placed on the elucidation of the nature of the interaction by electrochemical techniques. The electroactive [CuL(2)](2+) could be employed as an electrochemical indicator to detect hybridization events in DNA biosensors. These biosensors have been constructed by immobilization of a probe DNA sequence from HBV onto glassy carbon electrode (GCE). After hybridization with the complementary target sequence, [CuL(2)](2+) was accumulated within the dsDNA layer. Electrochemical detection was performed by differential pulse voltammetry over the potential range. Using this approach, complementary target sequences of HBV can be quantified over the range of 1.74 x 10(-9) to 3.45 x 10(-7) M, with a detection limit of 8.32 x 10(-10) M and a linear correlation coefficient of 0.9936.In addition, this approach is capable of detecting hybridization of complementary sequences containing one or three mismatched bases.     

Inhibition of free radical-induced peroxidation of rat liver microsomes by resveratrol and its analogues

Resveratrol (3,5,4'-trans-trihydroxystilbene) is a natural phytoalexin present in grapes and red wine, which possesses a variety of biological activities including antioxidative activity. To find more efficient antioxidants by structural modification, resveratrol analogues, that is, 3,4-dihydroxy-trans-stilbene (3,4-DHS), 4,4'-dihydroxy-trans-stilbene (4,4'-DHS), 4-hydroxy-trans-stilbene (4-HS) and 3,5-dihydroxy-trans-stilbene (3,5-DHS), were synthesized and their antioxidant activity studied for the free radical-induced peroxidation of rat liver microsomes in vitro. The peroxidation was initiated by either a water-soluble azo compound 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH) or Fe(2+)/ascorbate, and monitored by oxygen uptake and formation of thiobarbituric acid reactive substances (TBARS). It was found that all of these trans-stilbene derivatives are effective antioxidants against both AAPH- and iron-induced peroxidation of rat liver microsomes with an activity sequence of 3,4-DHS>4,4'-DHS>resveratrol>4-HS>3,5-DHS. The remarkably higher antioxidant activity of 3,4-DHS is discussed.     

Effects of stilbene derivatives on arachidonate metabolism in leukocytes

The effects of various alpha-phenylcinnamic acid derivatives (i.e., alpha-(3,4-dihydroxyphenyl)cinnamic acid, alpha-(3,4-dihydroxyphenyl)-3-hydroxycinnamic acid, alpha-(3,4-dihydroxyphenyl)-4-hydroxycinnamic acid and alpha-(3,4-dihydroxyphenyl)-3, 4-dihydroxycinnamic acid) synthesized from 3,4-dihydroxyphenyl acetic acid and hydroxy-benzaldehyde, and 3,3',4-trihydroxystilbene obtained by decarboxylation of alpha-(3,4-dihydroxyphenyl)-3-hydroxycinnamic acid on rat peritoneal polymorphonuclear leukocyte lipoxygenase and cyclooxygenase activities were studied. 3,3',4-Trihydroxystilbene was found to inhibit the 5-lipoxygenase product, 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HETE), and cyclooxygenase products, 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and thromboxane B2; its concentrations for 50% inhibition (IC50) were 0.885 +/- 0.016 microM for the leukocyte lipoxygenase product, 5-HETE, 7.70 +/- 0.104 microM for the formations of HHT and 7.96 +/- 0.143 microM for the formation of thromboxane B2. Alpha-(3,4-Dihydroxyphenyl)cinnamic acid, alpha-(3,4-dihydroxyphenyl)-3-hydroxycinnamic acid and alpha-(3,4-dihydroxyphenyl)-3,4-dihydroxycinnamic acid also inhibited the formations of 5-HETE, HHT and thromboxane B2, although less strongly. Their IC50 values were, respectively, 91.3 +/- 3.62 microM, 947.5 +/- 28.7 microM, 453.3 +/- 229.3 microM and 148.8 +/- 50.6 microM for the formation of 5-HETE, 894.0 +/- 5.57 microM, 792.5 +/- 15.9 microM, greater than 1000 microM and 925.0 +/- 7.64 microM for the formation of HHT and 941.0 +/- 18.0 microM, 825 +/- 14.4 microM, greater than 1000 microM and 932.7 +/- 3.93 microM for the formation of thromboxane B2.     

Detection of Z DNA binding proteins in tissue culture cells.

A gel electrophoresis DNA binding assay to detect Z DNA binding proteins has been developed utilising [32P] labelled poly [d(G-C)] which was converted to the Z form by incubation in 100 microM Co(NH3)6Cl3. The parameters of the assay were established using a Z DNA antibody as a model system and then applied to extracts of Hela and BHK21 cells. Using an anti-Z DNA antibody conditions were established which allowed resolution of antibody-DNA complexes and free DNA in the presence of 100 microM Co(NH3)6Cl3. The inclusion of unlabelled complementary homopolymers eliminated non-specific binding to the labelled Z-DNA probe. Competition experiments demonstrated that the assay was highly specific for double stranded non-B DNA. Application of the technique to extracts of mammalian cells demonstrated that human and hamster cells contain Z-DNA binding proteins; further characterisation by a blotting technique indicated that a 56,000 molecular weight cell protein preferentially binds Z-DNA.