Uranium(VI) reduction by nanoscale zero-valent iron in anoxic batch systems: the role of Fe(II) and Fe(III).

The role of Fe(II) and Fe(III) in U(VI) reduction by nanoscale zerovalent iron (nanoFe0) was investigated using two iron chelators 1,10-phenanthroline and triethanolamine (TEA) under a CO2-free anoxic condition. The results showed that U(VI) reduction was strongly inhibited by 1,10-phenanthroline and TEA in a pH range from 6.9 to 9.0. For instance, at pH 6.9 the observed U(VI) reduction rates decreased by 81% and 82% in the presence of 1,10-phenanthroline and TEA, respectively. The inhibition was attributed to the formation of stable complexes between 1,10-phenanthroline and Fe(II) or TEA and Fe(III). In the absence of iron chelators, U(VI) reduction can be enhanced by surface-bound Fe(II) on nanoFe0. Our results suggested that Fe(III) and Fe(II) possibly acted as an electron shuttle to ferry the electrons from nanoFe0 to U(VI), therefore a combined system with Fe(II), Fe(III) and nanoFe0 could facilitate U(VI) reductive immobilization in the contaminated groundwater.

Identification of metabolites in WZS-miniature pig urine after oral administration of Danshen decoction by HPLC coupled with diode array detection with electrospray ionization tandem ion trap and time-of-flight mass spectrometry.

Danshen (DS) is a widely used traditional Chinese medicine for treating cardiovascular and cerebrovascular diseases. A simple, rapid and sensitive method was developed for identification of the in vivo metabolites in urine of WZS-miniature pigs after oral administration of DS decoction by HPLC coupled with diode array detection with electrospray ionization tandem ion trap and time-of-flight mass spectrometry. This method has been successfully applied to simultaneous identification of 50 compounds (including 11 new ones) in pig urine. In addition, one new compound, (3-hydroxyphenyl) crylic acid glycine methyl ester (C1), along with eight known ones were first isolated by column chromatography and identified by spectroscopic means, including 1D/2DNMR and mass spectrometry, as reference substances. Ten phenolic compounds (protocatechuic aldehyde, protocatechuic acid, caffeic acid, danshensu, ferulic acid, isoferulic acid, rosmarinic acid and salvianolic acid A/B/D) were found to be the main absorbed original constituents of DS decoction, which underwent the metabolic reactions of glucuronidation, sulfation, methylation, hydrogenation and glycine conjugation in vivo. In conclusion, the developed method is applicable to the analysis and identification of constituents in biological matrices after administration of DS decoction.

Synthesis, characterization, nucleic acid binding, and cytotoxic activity of a Ru(II) polypyridyl complex.

The binding properties of [Ru(bpy)(2)(H(2)IIP)](2+) (1) {bpy=2,2'-bipyridine, H(2)IIP=2-(indole-3-yl)-imidazolo[4,5-f][1,10]phenanthroline} with calf thymus DNA (CT-DNA) and yeast tRNA have been investigated comparatively by different spectroscopic and viscosity measurements. The results suggest that the affinity of complex 1 binding with yeast tRNA is stronger than that of complex 1 binding with CT-DNA, and complex 1 is a better enantioselective binder to yeast tRNA than to CT-DNA. The toxicity of complex 1 was concentration dependent, and HL-60 cells are more sensitive to complex 1 than Hep-G2 cells; complex 1 could induce Hep-G2 cell apoptosis.

Direct vasorelaxation by a novel phytoestrogen tanshinone IIA is mediated by nongenomic action of estrogen receptor through endothelial nitric oxide synthase activation and calcium mobilization.

Salvia miltiorrhiza (Danshen) has been widely used in China and other Asian countries for treating various cardiovascular diseases resulting from its ability to improve coronary microcirculation and increase coronary blood flow. Tanshinone IIA (Tan IIA), the major active lipophilic ingredient responsible for the beneficial actions of Salvia miltiorrhiza, has been shown to induce vasodilation in coronary arteries. Because our recent study identified Tan IIA as a new member of the phytoestrogens, we hypothesized that its action might be mediated by estrogen receptor (ER) in vascular endothelial cells. The aim of the present study was to assess whether cardiovascular protection exerted by Tan IIA is mediated by the ER signal pathway and whether the genomic or nongenomic action of ER is involved within arteries and vascular endothelial cells. The effect of Tan IIA on blood vessels was investigated by vascular ring assay using endothelium-intact and endothelium-denuded rat aortas. Similar to estrogen, Tan IIA caused an nitric oxide- and endothelium-dependent relaxation, which was blocked by ER antagonist ICI 182,780. Primary cardiac microvascular endothelial cells were used as a model to study the cellular and molecular mechanisms of Tan IIA-induced vasorelaxation. We demonstrate that Tan IIA is capable of activating the estrogen receptor signal pathway, leading to increased endothelial nitric oxide synthase gene expression, nitric oxide production, ERK1/2 phosphorylation, and Ca mobilization. Collectively, these effects contribute to Tan IIA's vasodilative activity effects of y ER antagonist Cnt of cardiovascular diseases. Our findings support a continued effort in discovering and developing novel phytoestrogens as an alternative hormone replacement therapy for safer and more effective treatment of cardiovascular diseases.

Dinitrosyl iron complexes with thiol-containing ligands and apoptosis: studies with HeLa cell cultures.

No pro-apoptotic effect of dinitrosyl iron complexes (DNIC) with glutathione, cysteine or thiosulfate was established after incubation of HeLa cells in Eagle's medium. However, DNIC with thiosulfate manifested pro-apoptotic activity during incubation of HeLa cells in Versene's solution supplemented with ethylene diamine tetraacetate (EDTA) known to induce the decomposition of these DNIC. The water-soluble о-phenanthroline derivative bathophenanthroline disulfonate (BPDS) had a similar effect on DNIC with glutathione during incubation of HeLa cells in Eagle's medium. It was assumed that EDTA- or BPDS-induced pro-apoptotic effect of DNIC with thiosulfate or glutathione is coupled with the ability of decomposing DNIC to initiate S-nitrosylation of proteins localized on the surface of HeLa cells. Presumably, the pro-apoptotic effect of S-nitrosoglutathione (GS-NO) on HeLa cells preincubated in Eagle's medium is mediated by the same mechanism, although the pro-apoptotic effect based on the ability of GS-NO to initiate the release of significant amounts of NO and its oxidation to cytotoxic peroxynitrite in a reaction with superoxide should not be ruled out either. No apoptotic activity was found in the presence of bivalent iron and glutathione favoring the conversion of GS-NO into DNIC with glutathione. It is suggested that interaction of HeLa cells with intact DNIC with glutathione or thiosulfate results in the formation of DNIC bound to cell surface proteins.

Interaction study of two diterpenes, cryptotanshinone and dihydrotanshinone, to human acetylcholinesterase and butyrylcholinesterase by molecular docking and kinetic analysis.

Alzhemier's disease (AD) is a common form of dementia in the ageing population which is characterized by depositions of amyloids and a cholinergic neurotransmission deficit in the brain. Current therapeutic intervention for AD is primarily based on the inhibition of brain acetylcholinesterase (AChE) to restore the brain acetylcholine level. Cryptotanshinone (CT) and dihydrotanshinone (DT) were diterpenoids extracted from Salvia miltiorrhiza Bge. having anti-cholinesterase activity. Here we characterized the inhibition property of these two diterpenoids towards human AChE and butyrylcholinesterase (BChE). Both CT and DT were found to be mixed non-competitive inhibitors for human AChE and an uncompetitive inhibitor for human BChE. The docking analyses of CT and DT into the active sites of both cholinesterases indicate that they interact with the allosteric site inside the active-site gorge mainly by hydrophobic interactions.

Structural elucidation of metabolites of tanshinone I and its analogue dihydrotanshinone I in rats by HPLC-ESI-MSn.

Tanshinone I and its analogue dihydrotanshinone I are the major active components isolated from Salvia miltiorrhiza Bunge and Salvia Przewalskii Maxim. These compounds have been found to possess significant antibacterial, anti-dermatophytic, antioxidant, anti-inflammatory and anticancer activities. Fifteen phase I metabolites and two phase II metabolites of tanshinone I and dihydrotanshinone I in rat bile were elucidated and identified by a sensitive HPLC-ESI-MS(n) method. The molecular structures of the metabolites are presented on the basis of the characteristics of their precursor ions, product ions and chromatographic retention times. The results indicate that the phase I metabolites are biotransformed through four main pathways: dehydrogenation, hydroxylation, furan ring cleavage and oxidation metabolism. Phase II metabolites were mainly identified as the sulfated conjugates which showed a characteristic neutral loss of 80 Da. The biotransformed pathways of tanshinone I and dihydrotanshinone I were proposed on the basis of the investigation.

Microsphere resin chromatography combined with microbial biotransformation for the separation and purification of salvianolic acid B in aqueous extract of roots of Salvia multiorrihza Bunge.

Salvianolic acid B was separated and purified from Salvia miltiorrhiza Bunge (danshen) by microbial transformation together with chromatography of microsphere resin. The aqueous extract of danshen was transformed by Fusarium graminearum in a bioreactor containing phosphate buffer (PBS), in which rosmarinic acid was transformed into danshensu and caffeic acid and the yield of salvianolic acid B was higher than 85%. After biotransformation, salvianolic acid B was purified by microsphere resin. A parallel test for making a comparison of microsphere resin chromatography between elution by methanol water solution and water was done. The purity of salvianolic acid B was up to 95% at the yield of 62% when impurities and salvianolic acid B were eluted by 45% and 55% methanol solution respectively. The purity of salvianolic acid B was up to 99% at the yield of 90% when distilled water was used to elute the impurities and salvianolic acid B. The total yield of salvianolic acid B was up to 75% at the purity over 99% while biotransformation combined with microsphere resin chromatography by water elution. Microbial biotransformation together with water elution of microsphere resin supplied an efficient method to eliminate the micromolecular impurities and a possible method to purify water-soluble compounds in traditional Chinese medicine.

DNA cleavage in red light promoted by copper(II) complexes of alpha-amino acids and photoactive phenanthroline bases.

Ternary copper(II) complexes [Cu(L-trp)(B)(H(2)O)](NO(3)) (1-3) and [Cu(L-phe)(B)(H(2)O)](NO(3)) (4-6) of L-tryptophan (L-trp) and L-phenylalanine (L-phe) having phenanthroline bases (B), viz. 1,10-phenanthroline (phen, 1 and 4), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq, 2 and 5) and dipyrido[3,2-a:2',3'-c]phenazine (dppz, 3 and 6), were prepared and characterized by physico-chemical techniques. Complexes 3 and 6 were structurally characterized by X-ray crystallography and show the presence of a square pyramidal (4 + 1) CuN(3)O(2) coordination geometry in which the N,O-donor amino acid (L-trp or L-phe) and N,N-donor phenanthroline base bind at the equatorial plane with an aqua ligand coordinated at the elongated axial site. Complex 3 shows significant distortion from the square pyramidal geometry and a strong intramolecular pi-pi stacking interaction between the pendant indole ring of L-trp and the planar dppz aromatic moiety. All the complexes display good binding propensity to the calf thymus DNA giving an order: 3,6 (dppz) > 2,5 (dpq) > 1,4 (phen). The binding constant (K(b)) values are in the range of 2.1 x 10(4)-1.1 x 10(6) mol(-1) with the binding site size (s) values of 0.17-0.63. The phen and dpq complexes are minor groove binders while the dppz analogues bind at the DNA major groove. Theoretical DNA docking studies on 2 and 3 show the close proximity of two photosensitizers, viz. the indole moiety of L-trp and the quinoxaline/phenazine of the dpq/dppz bases, to the complementary DNA strands. Complexes 2 and 3 show oxidative DNA double strand breaks (dsb) of supercoiled (SC) DNA forming a significant quantity of linear DNA along with the nicked circular (NC) form on photoexposure to UV-A light of 365 nm and red light of 647.1 nm (Ar-Kr laser). Complexes 1,5 and 6 show only single strand breaks (ssb) forming NC DNA. The red light induced DNA cleavage involves metal-assisted photosensitization of L-trp and dpq/dppz base resulting in the formation of a reactive singlet oxygen ((1)O(2)) species.

Plant polyphenols mobilize nuclear copper in human peripheral lymphocytes leading to oxidatively generated DNA breakage: implications for an anticancer mechanism.

It was earlier proposed that an important anti-cancer mechanism of plant polyphenols may involve mobilization of endogenous copper ions, possibly chromatin-bound copper and the consequent pro-oxidant action. This paper shows that plant polyphenols are able to mobilize nuclear copper in human lymphocytes, leading to degradation of cellular DNA. A cellular system of lymphocytes isolated from human peripheral blood and comet assay was used for this purpose. Incubation of lymphocytes with neocuproine (a cell membrane permeable copper chelator) inhibited DNA degradation in intact lymphocytes. Bathocuproine, which is unable to permeate through the cell membrane, did not cause such inhibition. This study has further shown that polyphenols are able to degrade DNA in cell nuclei and that such DNA degradation is inhibited by neocuproine as well as bathocuproine (both of which are able to permeate the nuclear pore complex), suggesting that nuclear copper is mobilized in this reaction. Pre-incubation of lymphocyte nuclei with polyphenols indicates that it is capable of traversing the nuclear membrane. This study has also shown that polyphenols generate oxidative stress in lymphocyte nuclei which is inhibited by scavengers of reactive oxygen species (ROS) and neocuproine. These results indicate that the generation of ROS occurs through mobilization of nuclear copper resulting in oxidatively generated DNA breakage.

Plant polyphenols mobilize endogenous copper in human peripheral lymphocytes leading to oxidative DNA breakage: a putative mechanism for anticancer properties.

Plant polyphenols are important components of human diet and a number of them are considered to possess chemopreventive and therapeutic properties against cancer. They are recognized as naturally occurring antioxidants but also act as prooxidants catalyzing DNA degradation in the presence of transition metal ions such as copper. Using human peripheral lymphocytes and Comet assay we have previously confirmed that resveratrol-Cu(II) is indeed capable of causing DNA degradation in cells. In this paper we show that the polyphenols alone (in the absence of added copper) are also capable of causing DNA breakage in cells. Incubation of lymphocytes with neocuproine inhibited the DNA degradation confirming that Cu(I) is an intermediate in the DNA cleavage reaction. Further, we have also shown that polyphenols generate oxidative stress in lymphocytes which is inhibited by scavengers of reactive oxygen species and neocuproine. These results are in further support of our hypothesis that anticancer mechanism of plant polyphenols involves mobilization of endogenous copper, possibly chromatin bound copper, and the consequent prooxidant action.

Phenolics as potential antioxidant therapeutic agents: mechanism and actions.

Accumulating chemical, biochemical, clinical and epidemiological evidence supports the chemoprotective effects of phenolic antioxidants against oxidative stress-mediated disorders. The pharmacological actions of phenolic antioxidants stem mainly from their free radical scavenging and metal chelating properties as well as their effects on cell signaling pathways and on gene expression. The antioxidant capacities of phenolic compounds that are widely distributed in plant-based diets were assessed by the Trolox equivalent antioxidant capacity (TEAC), the ferric reducing antioxidant power (FRAP), the hypochlorite scavenging capacity, the deoxyribose method and the copper-phenanthroline-dependent DNA oxidation assays. Based on the TEAC, FRAP and hypochlorite scavenging data, the observed activity order was: procyanidin dimer>flavanol>flavonol>hydroxycinnamic acids>simple phenolic acids. Among the flavonol aglycones, the antioxidant propensities decrease in the order quercetin, myricetin and kaempferol. Gallic acid and rosmarinic acid were the most potent antioxidants among the simple phenolic and hydroxycinnamic acids, respectively. Ferulic acid displayed the highest inhibitory activity against deoxyribose degradation but no structure-activity relationship could be established for the activities of the phenolic compounds in the deoxyribose assay. The efficacies of the phenolic compounds differ depending on the mechanism of antioxidant action in the respective assay used, with procyanidin dimers and flavan-3-ols showing very potent activities in most of the systems tested. Compared to the physiologically active (glutathione, alpha-tocopherol, ergothioneine) and synthetic (Trolox, BHA, BHT) antioxidants, these compounds exhibited much higher efficacy. Plant-derived phenolics represents good sources of natural antioxidants, however, further investigation on the molecular mechanism of action of these phytochemicals is crucial to the evaluation of their potential as prophylactic agents.

Impaired protein binding of Chinese medicine DanShen in uremic sera and sera with hyperbilirubinemia: rapid assessment of total and free DanShen concentrations using the fluorescence polarization immunoassay for digoxin.

DanShen is a Chinese medicine that is used to treat cardiovascular disorders. DanShen is moderately to strongly protein bound, mainly to albumin. Because impaired protein binding of albumin-bound drugs in uremia has been reported, we studied protein binding of DanShen by measuring the digoxin-like immunoreactive component of this Chinese medicine. We observed a significantly higher percentage of free fraction of DanShen in uremic sera in vitro. Impaired protein binding of DanShen was also observed in sera from patients with liver disease, who had elevated concentrations of bilirubin. Treating uremic sera with activated charcoal significantly improved the protein binding of DanShen, indicating that uremic compounds are responsible for the impaired protein binding of DanShen. On the other hand, when various amounts of bilirubin were added to aliquots of the normal pool supplemented with DanShen, we observed only a modest displacement of DanShen from the protein-binding sites by bilirubin, indicating that hypoalbuminemia may play a major role in impaired protein binding of DanShen in sera with elevated bilirubin concentrations. We conclude that protein binding of DanShen is lower in uremic sera and in sera with elevated bilirubin concentrations.

A novel 1,10-phenanthroline-sensitive membrane sensor for potentiometric determination of Hg(II) and Cu(II) cations.

Preparation, characterization, and applications of a 1,10-phenanthrolinium cation (phenH(+))-sensitive potentiometric sensor are described. The sensor incorporates a liquid polymeric membrane consisting of phenH-tetraphenylborate, nitrophenyloctyl ether, and poly(vinyl chloride) as ion exchanger, plasticizer, and polymeric support, respectively. The sensor exhibits a fast and Nernstian response to phenH(+) over the concentration range of 6 x 10(-6)-2 x 10(-4) M with a monovalent cationic slope of 58.0+/-0.5 mV/log[phenH(+)] in acetate buffer of pH 4.2. The sensor is successfully applied to the monitoring of the potentiometric titration of Hg(II) and Cu(II) ions with phen solution in the presence of citrate and acetate buffers of pH 4.2, respectively. Sharp inflection breaks (90-180 mV) at 1:1 (metal:phen reaction) are obtained in the presence of chloride and thiocyanate background. This stoichiometry is explained by the formation of insoluble [HgCl(2)(phen)], [Hg(SCN)(2)(phen)], and [Cu(SCN)(2)(phen)] complexes. Optimization of each titration and the effect of foreign ions are evaluated. The method offers the advantages of adequate sensitivity, accuracy, and selectivity for the determination of mercury and copper in pharmaceutical, rock, and tea samples. The results are in good agreement with those obtained using the standard atomic absorption spectrometric and United States Pharmacopeial methods.

Activated charcoal is more effective than equilibrium dialysis in removing Chinese medicines Chan Su and Dan Shen from serum and activated charcoal also prevents further absorption of these agents from G.I. tract in mice: monitoring the effect in clinical laboratory by measuring digoxin activity in serum.

BACKGROUND: Chinese medicines are freely available without prescription and are widely used by the general population. Chan Su and Dan Shen are both indicated for the treatment of cardiac diseases. Severe toxicity from Chan Su has been reported. We studied the possibility of removing Chan Su and Dan Shen from human sera using activated charcoal and equilibrium dialysis, and also examined the potential benefit of preventing absorption of these agents from the G.I. tract in the mouse model. METHODS: For in vitro studies, drug-free serum pools were supplemented with Chan Su or Dan Shen and then either treated with activated charcoal (10 and 25 mg/ml), or passed through a column packed with activated charcoal. Serum pools supplemented with Chan Su or Dan Shen were also subjected to equilibrium dialysis against phosphate buffer (pH 7.4) using dialysis membrane with molecular cut-off of 25,000 Da. Removal of Chan Su or Dan Shen from the serum was monitored by measuring the apparent digoxin concentration using the fluorescence polarization immunoassay (FPIA) for digoxin (Abbott Laboratories). RESULTS: We observed the fast and effective removal of both Chan Su and Dan Shen from the serum by activated charcoal. We also observed significant removal of both Chan Su and Dan Shen when the serum pools containing these Chinese medicines were passed through columns packed with activated charcoal. Although equilibrium dialysis was also effective in removing these Chinese medicines from the serum, 24 h was required for complete removal of Dan Shen activity, and for Chan Su, complete removal was not achieved even after 24 h. In our in vivo model, we observed significantly less digoxin activity in the group of mice that received activated charcoal compared to the control group. CONCLUSIONS: Activated charcoal is effective in preventing absorption of these Chinese medicines from the G.I. tract and can also remove these agents from the serum.

Chelation and intercalation: complementary properties in a compound for the treatment of Alzheimer's disease.

Selective application of metal chelators to homogenates of human Alzheimer's disease (AD) brain has led us to propose that the architecture of aggregated beta-amyloid peptide, whether in the form of plaques or soluble oligomers, is determined at least in part by high-affinity binding of transition metals, especially copper and zinc. Of the two metals, copper is implicated in reactive oxygen species generating reactions, while zinc appears to be associated with conformational and antioxidant activity. We tested the copper chelators trientine, penicillamine, and bathophenanthroline for their ability to mobilize brain Abeta as measured against our benchmark compound bathocuproine (BC). All of these agents were effective in solubilizing brain Abeta, although BC was the most consistent across the range of AD brain tissue samples tested. Similarly, all of the copper chelators depleted copper in the high-speed supernatants. BC alone had no significant effect upon zinc levels in the soluble fraction. BC extraction of brain tissue from C100 transgenic mice (which express human Abeta but do not develop amyloid) revealed SDS-resistant dimers as Abeta was mobilized from the sedimentable to the soluble fraction. NMR analysis showed that, in addition to its copper chelating properties, BC interacts with Abeta to form a complex independent of the presence of copper. Such hybrid copper chelating and "chain breaking" properties may form the basis of a rational design for a therapy for Alzheimer's disease.

Optimizing the targeted chemical nuclease activity of 1,10-phenanthroline-copper by ligand modification.

Our interest in improving the efficiency of targeted scission reagents has prompted us to study the influence of ring substituents on the nuclease activity of 1,10-phenanthroline-copper conjugated to oligonucleotides and DNA-binding proteins. Since methyl substitution at all but the 2 and 9 positions enhances the copper-dependent chemical nuclease activity of 1,10-phenanthroline, we have compared the activity of conjugates prepared from 5-(aminomethyl)-1,10-phenanthroline (MOP) to those of conjugates prepared from 5-amino-1,10-phenanthroline (amino-OP). Tethering MOP derivatives to the Escherichia coli Fis protein enhances DNA scission several-fold at the weaker cleavage sites initially observed with conjugates prepared from amino-OP. However, scission efficiency is not increased at the stronger cleavage sites, or when scission is targeted to single-stranded DNA by a complementary oligonucleotide. These results are consistent with a change in the rate-determining step for cleavage associated with the differential accessibility of the DNA-bound coordination complex to solvent and reductant. Although the free bis cuprous complex of 2,9-dimethyl-1,10-phenanthroline (neocuproine) is redox-inactive, an oligonucleotide tethered to neocuproine through C5 of the phenanthroline ring efficiently cleaves a complementary DNA sequence. These results establish that the nucleolytic species in targeted scission is the 1:1 cuprous complex and suggest that the oxidative reaction proceeds through a copper-oxo intermediate rather than a metal-coordinated peroxy species. However, substituents at the 2 and 9 positions of the ligand will often hinder close approach of the phenanthroline-copper moiety to the oxidatively sensitive ribose as shown by the preference of the oligonucleotide-targeted chimera for cleavage of single-stranded regions and the failure of neocuproine-DNA-binding protein chimeras and a C2-tethered chimera to cleave DNA.

Recognition and cleavage of hairpin structures in nucleic acids by oligodeoxynucleotides.

The possibility of designing antisense oligodeoxynucleotides complementary to non-adjacent single-stranded sequences containing hairpin structures was studied using a DNA model system. The structure and stability of complexes formed by a 17mer oligonucleotide with DNA fragments containing hairpin structures was investigated by spectroscopic measurements (melting curves) and chemical reactions (osmium tetroxide reaction, copper-phenanthroline cleavage). A three-way junction was formed when the oligonucleotide was bound to both sides of the hairpin structure. When the complementary sequences of the two parts of the oligonucleotide were separated by a sequence which could not form a hairpin, the oligonucleotide exhibited a slightly weaker binding than to the hairpin-containing target. An oligodeoxynucleotide-phenanthroline conjugate was designed to form Watson-Crick base pairs with two single-stranded regions flanking a hairpin structure in a DNA fragment. In the presence of Cu2+ ions and a reducing agent, two main cleavage sites were observed at the end of the duplex structure formed by the oligonucleotide-phenanthroline conjugate with its target sequence. Competition experiments showed that both parts of the oligonucleotide must be bound in order to observe sequence-specific cleavage. Cleavage was still observed with target sequences which could not form a hairpin, provided the reaction was carried out at lower temperatures. These results show that sequence-specific recognition and modification (cleavage) can be achieved with antisense oligonucleotides which bind to non-adjacent sequences in a single-stranded nucleic acid.

Sequence-specific scission of DNA by RNAs linked to a chemical nuclease.

RNAs linked to the chemical nuclease 1,10-phenanthroline-copper cut double-stranded DNA of complementary sequence. This cleavage reaction is applicable to all sequences and can be used to measure the distance between marker genes in base pairs, map the size of a transcription unit and define positions of chromosomal breakpoints.

Interaction of hepatic microsomal epoxide hydrolase derived from a recombinant baculovirus expression system with an azarene oxide and an aziridine substrate analogue.

A recombinant baculovirus (vEHX) encoding rat hepatic microsomal epoxide hydrolase has been constructed. Infection of Spodoptera frugiperda (Sf9) cells with the recombinant virus results in the expression of the enzyme at a level estimated to be between 5% and 10% of the cellular protein. The enzyme, which can be purified in 15% yield by a simple three-step procedure involving detergent extraction, DEAE-cellulose chromatography, and removal of the detergent on hydroxylapatite, has physical and kinetic properties very close to those of the enzyme obtained from rat liver microsomes. The interaction of the enzyme with two nitrogen-containing analogues of the substrate phenanthrene 9,10-oxide (1) was investigated in order to delineate the contributions of the oxirane group and the hydrophobic surface of the substrate to substrate recognition. The enzyme exhibits altered kinetic properties toward 1,10-phenanthroline 5,6-oxide (2) in which the biphenyl group of 1 is replaced with a bipyridyl group, suggesting that hydrophobic interaction between the complementary surfaces of the substrate and active site has an influence on catalysis. The conjugate acid of the aziridine analogue of 1, phenanthrene 9,10-imine (3), in which the oxirane oxygen is replaced with NH, has a pKa of 6.1, which allows the characterization of both the neutral and protonated aziridine (3H+) as substrate analogues for the enzyme. The pH dependence of the solvolysis reveals that 3H+ rearranges to a 65/35 mixture of 9-aminophenanthrene and 9-amino-10-hydroxy-9,10-dihydrophenanthrene 10(3)-fold faster than does 3. The neutral aziridine is a competitive inhibitor (Ki = 26 microM) of the enzyme at pH 8.(ABSTRACT TRUNCATED AT 250 WORDS)