Effect of three different modes of alcohol administration on the activity of the rat hypothalamic-pituitary-adrenal axis

The review presents specific interactions that occur in complexes of Cu(II) ions with peptides composed only of amino acids with nonco-ordinating side chains. Three classes of such peptides are discussed. The first type (NSFRY analogues) is characterised by the presence of a specific combination of bulky and aromatic residues, leading to a formation of multiple weak interactions around Cu(II) that result in an extremely high stability of complexes. The second class is composed of complexes of vasopressins and oxytocins, achieving superstability through a pre-conformation in the peptide molecule. The third group are oligopeptides containing one or two proline residues. These peptides form exotic macrochelate loops with Cu(II) in a result of the break-point effect of Pro residues. Particular emphasis in the review was given to stability constants of complexes, compared to oligoglycine or oligoalanine peptides.     

DNA strand break induction and enhanced cytotoxicity of propyl gallate in the presence of copper(II)

The antioxidant propyl gallate (PG) induced single strand breaks in PM2 DNA at concentrations higher than 0.25 microM when it was combined with copper concentrations at 5 microM and above. In combination with 100 microM CuCl2, extensive double strand breakage was also observed. Neither PG alone nor CuCl2 showed any strand breaking properties. DNA strand breakage was inhibited by addition of catalase or the Cu(I) chelator neocuproine, indicating the involvement of H2O2 and a Cu(II)/Cu(I) redox cycle in the DNA damage. DNA damage of PG/Cu(II) was also observed in human fibroblasts. Using the alkaline elution technique concentrations of 0.15-0.5 mM PG induced DNA strand breaks in combination with 2.5 mM CuCl2, while the single substances did not show any effect. At these concentrations cell viability measured by the MTT assay was not reduced by more than 10%; however, cell growth was inhibited by PG in combination with Cu(II). This growth inhibition was apparently due to the DNA damage incurred by PG/Cu(II). The synergistic interaction between PG and Cu(II) is probably caused by a redox reaction between both compounds, whereby reactive species such as ROS are formed, which are responsible for the observed genotoxic and cytotoxic effects. Our results demonstrate that the antioxidative and cytoprotective properties of propyl gallate may change to prooxidative, cytotoxic and genotoxic properties in the presence of Cu(II).     

Induction and mechanism of DNA single- and double-strand breaks by tetracycline/Cu(II) in the absence of light

In the absence of light, tetracycline (TC) induced single- and double-strand breaks in PM2 DNA at micromolar concentrations in combination with CuCl2, whereas TC or CuCl2 alone had no effect. Strand break formation was completely suppressed by catalase and the specific Cu(I) scavenger neocuproine. The extent of strand break formation depended on the ratio of Cu(II):TC. At a ratio of > or = 2 most DNA damage was observed. The influence of the kind of Cu(II)/TC complexation on DNA strand break formation is discussed. The DNA damage in PM2 DNA provoked by TC/CuCl2 was indirectly detected also in human fibroblasts by the induction of DNA repair. The results are discussed with regard to human risk from TC/Cu(II).     

DNA strand scission by polycyclic aromatic hydrocarbon o-quinones: role of reactive oxygen species, Cu(II)/Cu(I) redox cycling, and o-semiquinone anion radicals,

In previous studies, benzo[a]pyrene-7,8-dione (BPQ), a polycyclic aromatic hydrocarbon (PAH) o-quinone, was found to be 200-fold more potent as a nuclease than (+/-)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9, 10-tetrahydrobenzo[a]pyrene, a suspect human carcinogen. The mechanism of strand scission mediated by naphthalene-1,2-dione (NPQ) and BPQ was further characterized using either phiX174 DNA or poly(dG).poly(dC) as the target DNA. Strand scission was extensive, dependent on the concentration of o-quinone (0-10 microM), and required the presence of NADPH (1 mM) and CuCl2 (10 microM). The production of reactive species, i.e., superoxide anion radical, o-semiquinone anion (SQ) radical, hydrogen peroxide (H2O2), hydroxyl radical (OH.), and Cu(I), was measured in the incubation mixtures. The formation of SQ radicals was measured by EPR spectroscopy under anaerobic conditions in the presence of NADPH. A Cu(II)/Cu(I) redox cycle was found to be critical for DNA cleavage. No strand scission occurred in the absence of Cu(II) or when Cu(I) was substituted, yet Cu(I) was required for OH* production. Both DNA strand scisson and OH. formation were decreased to an equal extent, albeit not completely, by the inclusion of OH. scavengers (mannitol, soduim benzoate, and formic acid) or Cu(I) chelators (bathocuproine and neocuproine). In contrast, although the SQ radical signals of NPQ and BPQ were quenched by DNA, no strand scission was observed. When calf thymus DNA was treated with PAH o-quinones, malondialdehyde (MDA) was released by acid hydrolysis. The formation of MDA was inhibited by OH. scavengers suggesting that OH* cleaved the 2'-deoxyribose moiety in the DNA to produce base propenals. These studies indicate that for PAH o-quinones to act as nucleases, NADPH, Cu(II), Cu(I), H2O2, and OH*, were necessary and that the primary species responsible for DNA fragmentation was OH., generated by a Cu(I)-catalyzed Fenton reaction. The genotoxicity of PAH o-quinones may play a role in the carcinogenicity and mutagenicity of the parent hydrocarbons.     

The treatment of flexion contracture of the knee in myelomeningocele

Sequential modification of two amino acid residues (a histidyl and a cysteinyl residue), both essential for the enzymatic function of bacterial luciferase from Beneckea harveyi, has been conducted to determine if the inactivation arising from the chemical modification of either of these residues is due to a conformational change. This experimental approach has shown that modification of the histidyl or cysteinyl residue did not affect the reactivity of the remaining 'essential' residue, suggesting that chemical modification had not caused a change in conformation. Furthermore, since substrates protect luciferase against inactivation due to modification of either of these residues, it was possible to determine if the initial modification of the histidyl or cysteinyl residue prevented substrate binding by conducting the modification of the remaining residue (i.e., the cysteinyl or histidyl residue, respectively) in the presence of substrates. The results have shown that after modification of the histidyl residue substrates no longer protected the cysteinyl residue against modification, whereas after modification of the cysteinyl residue substrates still protected the histidyl residue against modification. These results have provided evidence that the histidyl residue and not the cysteinyl residue of luciferase is essential for the binding of substrates in the bacterial bioluminescent reaction.     

Adrenoleucodystrophy: dermatological findings and skin surface lipid study

Co(II), Ni(II), Cu(II) and Zn(II) complexes of levamisole (LMS) were prepared and characterized by elemental analyses, IR spectroscopy, 1H and 13C NMR and mass spectrometry. The following general formula was derived: M(LMS)2Cl2, where M = Co, Ni, Cu, Zn. It was established that LMS behaved as a monodentate ligand and the coordination was accomplished through the N-7 atom. The toxicity and the immunomodulating activity of the complexes on mice and rats in comparison with uncomplexed LMS was assayed. The metals in the complexes exerted different changes in the toxicity of LMS. The complex containing Zn(II) was less toxic and manifested higher immunomodulating activity than LMS.     

Localization of critical histidyl residues required for vinblastine-induced tubulin polymerization and for microtubule assembly

Vinblastine-induced tubulin polymerization is electrostatically regulated and shows pH dependence with a pI approximately 7.0 suggesting the involvement of histidyl residues. Modification of histidyl residues of tubulin with diethylpyrocarbonate (DEPC) at a mole ratio of 0.74 (DEPC/total His residues) for 3 min at 25 degreesC completely inhibited vinblastine-induced polymerization with little effect on microtubule assembly. Under these conditions DEPC reacts only with histidyl residues. For complete inhibition two histidyl residues have to be modified. Demodification of the carboxyethyl histidyl derivatives by hydroxylamine led to nearly complete recovery of polymerization competence. Labeling with [14C]DEPC localized both of these histidyl residues on beta-tubulin at beta227 and beta264. Similarly, tubulin modification with DEPC for longer times (8 min) resulted in complete inhibition of microtubule assembly, at which time approximately 4 histidyl residues had been modified. This inhibition by DEPC was also reversed by hydroxylamine. The third histidyl residue was found on alpha-tubulin at alpha88. Thus, two charged histidyl residues are obligatorily involved in vinblastine-induced polymerization, whereas a different histidyl residue on a different tubulin monomer is involved in microtubule assembly.     

AP site structural determinants for Fpg specific recognition

The binding of Escherichia coli and Lactococcus lactis Fapy-DNA glyosylase (Fpg) proteins to DNA containing either cyclic or non-cyclic abasic (AP) site analogs was investigated by electrophoretic mobility shift assay (EMSA) and by footprinting experiments. We showed that the reduced AP site is the best substrate analog for the E.coli and L.lactis enzymes ( K Dapp = 0.26 and 0.5 nM, respectively) as compared with the other analogs tested in this study ( K Dapp >2.8 nM). The 1,3-propanediol (Pr) residue-containing DNA seems to be the minimal AP site structure allowing a Fpg specific DNA binding, since the ethyleneglycol residue is not specifically bound by these enzymes. The newly described cyclopentanol residue is better recognized than tetrahydrofuran (for the E.coli Fpg, K Dapp = 2.9 and 25 nM, respectively). These results suggest that the hemiacetal form of the AP site is negatively discriminated by the Fpg protein suggesting a hydrogen bond between the C4'-hydroxyl group of the sugar and a Fpg residue. High-resolution hydroxyl radical footprinting using a duplex containing Pr shows that Fpg binds to six nucleotides on the strand containing the AP site and only the base opposite the lesion on the undamaged complementary strand. This comparative study provides new information about the molecular mechanism involved in the Fpg AP lyase activity.     

Copper-binding amyloid precursor protein undergoes a site-specific fragmentation in the reduction of hydrogen peroxide

The extracellular domain of transmembrane Abeta amyloid precursor protein (APP) has a Cu(II) reducing activity upon Cu(II) binding associated with the formation of a new disulfide bridge. The complete assignment of the disulfide bond revealed the involvement of cysteines 144 and 158 around copper-binding histidine residues. The vulnerability of APP-Cu(I) complexes to reactive oxygen species was elaborated as a site-specific and random fragmentation of APP in a time-dependent manner and at low concentrations of H2O2. Analysis of the specific reaction revealed the generation of C-terminal polypeptides, containing the Abeta domain. APP catalyzed the reduction of H2O2 and oxidation of Cu(I) to Cu(II) in a "peroxidative" reaction in vitro. The resulting bound copper-hydroxyl radical intermediate [APP-Cu(II)(.OH)] then likely participated in a Fenton type of reaction with radical formation as a prerequisite for protein degradation. Evidence from two observations suggests that the reaction takes place in two phases. Bathocuproine, a trapping agent for Cu(I), abolished the initial fragmentation, and chelation of Cu(II) by DTPA (diethylenetriaminepentaacetic acid) interrupted the reaction cascade induced by H2O2 at later stages. Consequently, the results suggest that a cytotoxic gain-of-function of APP-Cu(I) complexes might result in a perturbation of free radical homeostasis. What significance such a perturbation may have for the pathogenesis of Alzheimer's disease remains to be determined.     

Differences in weight gain in relation to race, gender, age and education in young adults: the CARDIA Study. Coronary Artery Risk Development in Young Adults

Copper/zinc (Cu/ZnSOD) and manganese (MnSOD) superoxide dismutases which catalyze the dismutation of toxic superoxide anion, O(2-)-, 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 be 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 phi 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.     

Mutational analysis of Chlorella virus DNA ligase: catalytic roles of domain I and motif VI

A conserved catalytic core of the ATP-dependent DNA ligases is composed of an N-terminal domain (domain 1, containing nucleotidyl transferase motifs I, III, IIIa and IV) and a C-terminal domain (domain 2, containing motif VI) with an intervening cleft. Motif V links the two structural domains. Deletion analysis of the 298 amino acid Chlorella virus DNA ligase indicates that motif VI plays a critical role in the reaction of ligase with ATP to form ligase-adenylate, but is dispensable for the two subsequent steps in the ligation pathway; DNA-adenylate formation and strand closure. We find that formation of a phosphodiester at a pre-adenylated nick is subject to a rate limiting step that does not apply during the sealing of nicked DNA by ligase-adenylate. This step, presumably conformational, is accelerated or circumvented by deleting five amino acids of motif VI. The motif I lysine nucleophile (Lys27) is not required for strand closure by wild-type ligase, but this residue enhances the closure rate by a factor of 16 when motif VI is truncated. We find that a more extensively truncated ligase consisting of only N-terminal domain 1 and motif V is inert in ligase--adenylate formation, but competent to catalyze strand closure at a pre-adenylated nick. These results suggest that different enzymic catalysts facilitate the three steps of the DNA ligase reaction.     

Site-specific DNA damage induced by NADH in the presence of copper(II): role of active oxygen species

Oxidative DNA damage by NAD(P)H in the presence of metal ions has been characterized by using 32P 5' end-labeled DNA fragments obtained from human p53 tumor suppressor gene and c-Ha-ras-1 protooncogene. NADH, as well as other endogenous reductants, induced DNA damage in the presence of Cu(II). The order of inducing effect on Cu(II)-dependent DNA damage was ascorbate > reduced glutathione (GSH) > NADH > NADPH. Although NADH caused no or little DNA damage in the presence of Fe(III)-EDTA, the addition of H2O2 induced the DNA damage. The Cu(II)-mediated DNA damage induced by NADH was inhibited by catalase and bathocuproine, a Cu(I)-specific chelator; but not by scavengers of hydroxyl free radical (.OH), suggesting the involvement of active species derived from hydrogen peroxide (H2O2) and Cu(I) rather than .OH. The predominant cleavage sites were thymine residues located 5' and/or 3' to guanine. The cleavage pattern was similar to that induced by Cu(II) plus GSH, Cu(II) plus ascorbate, or Cu(I) plus H2O2. Formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine by NADH increased with its concentration in the presence of Cu(II). UV-visible spectroscopy indicated the facilitation of reduction of Cu(II) by NADH under some conditions. ESR spin-trapping experiments and mass spectrometry showed that the carbon-centered radical was formed during the reaction of NADH with Cu(II). These results suggest that optimal molar ratios of DNA/metal ion yield copper with a high redox potential which catalyzes NADH autoxidation to NAD. being further oxidized to NAD+ with generation of superoxide radical and that H2O2 reacts with Cu(I) to form active oxygen species such as copper(I)-peroxide complex causing DNA damage.     

Competitive solvation and complexation of Cu(I), Cu(II), Pb(II), Zn(II), and Ag(I) in aqueous ethanol,acetonitrile, and dimethylsulfoxide solutions containing chloride ion with applications to hydrometallurgy

The changes in reduction potential and single ion activity of Cu(I), Cu(II), Pb(II), Zn(II), and Ag(I) have been measured in a range of aqueous ethanol (EtOH), acetonitrile (AN), and dimethylsulfoxide (DMSO) compositions containing excess chloride ion. The results are compared with changes in such solutions in the absence of chloride ion and with the changes in strong brines and rationalized in terms of the various competitive ion-solvent and ion-chloride interactions. Organic solvents are shown to generally enhance chloride ion activity and promote complex ion formation. But AN is a stronger ligand for Cu(I) and DMSO is a stronger ligand for Cu(II) and Zn(II) than is Cl⁻ or the other solvents. The decrease in metal ion activity in mixed aqueous solvents containing Cl⁻ is greater than that in concentrated aqueous chloride salt solutions, according to the strength of the chloro- or solvo-complex. These fundamental changes lead to applications in the extraction of metal ion complexes and promote the dissolution of AgCl, PbCl₂, and CuCl in aqueous DMSO containing Cl⁻. Formerly Doctoral Student, Murdoch University     

The role of protonation and metal chelation preferences in defining the properties of mercury-binding coiled coils

To define the delicate interplay between metal chelation, protein folding and function in metalloproteins, a family of de novo-designed peptides was synthesized that self-assemble in aqueous solution to form two and three-stranded alpha-helical coiled coils. Each peptide contains a single Cys residue at an a or d position of the heptad repeat. Peptide association thus produces a Cys-rich coordination environment that has been used to bind Hg(II) ions. These peptides display a pH-dependent association, with trimers observed above the pKa of Glu side-chains and dimers below this value. Finite-difference Poisson-Boltzmann calculations suggest that the dimeric state decreases the unfavorable electrostatic interactions between positively charged Lys side-chains (relative to the trimer). The Cys-containing peptides bind Hg(II) in a position-dependent fashion. Cys at a positions form three-coordinate Hg complexes at high pH where the trimeric aggregation state predominates, and two-coordinate complexes at lower pH. A d position Cys, however, is only able to generate the two-coordinate complex, illustrating the difference in coordination geometry between the two positions in the coiled coil. The binding of Hg(II) was also shown to substantially increase the stability of the helical aggregates.     

Dimeric beta-cyclodextrin-based supramolecular ligands and their copper(II) complexes as metalloenzyme models

New supramolecular ligands possessing linear 13- and 15-membered pyridine diamidetriamine chelators between the primary sides of two beta-cyclodextrin cavities were synthesized, and characterized by MALDI-MS, NMR, IR and UV-Visible spectroscopy. Fluorescence and pH-metric titration were carried out in order to ascertain their behavior as bifunctional hosts for fluorescent guests and Cu(II) ion. The pKa value for the Cu(II) promoted deprotonation of amide ligands was determined to be 6.2 from pH-absorbance profile. Above pH 8.0, two deprotonated amides and three amino groups chelated Cu(II) ion, and yielded penta-coordinated Cu(II) complexes. The Cu(II) complexes catalyzed the hydrolysis of p-nitrophenyl acetate, adamantate and amino acids. Especially, the complex containing 13-membered chelator is an artificial metalloesterase with catalytic rate constant kcat = 3.8 x 10(-3) min-1 and Michaelis constant K(m) = 3.5 x 10(-4) M for the hydrolysis of p-nitrophenyl adamantate via metal-hydroxide mechanism.     

Purification of tryptophan containing synthetic peptides by selective binding of the alpha-amino group to immobilised metal ions

Immobilised metal ion affinity chromatography (IMAC) based on selective binding via the alpha-amino group to Cu2+ and Ni2+ ions has been used to purify tryptophan containing synthetic peptides. A free alpha-amino group, serving as an affinity handle, is present only in the target peptide when the peptides are synthesised by the solid-phase method and remaining amino groups after each coupling step are blocked by acetylation. A free alpha-amino group is necessary to retain the peptide on the column. The tryptophan residue may contribute to the binding only if the peptide is simultaneously anchored via the alpha-amino group.     

Architecture of a complex between the sigma70 subunit of Escherichia coli RNA polymerase and the nontemplate strand oligonucleotide. Luminescence resonance energy transfer study

We used luminescence energy transfer measurements to determine the localization of 5'- and 3'-ends of a 12-nucleotide nontemplate strand oligonucleotide bound to sigma70 holoenzyme. Five single reactive cysteine mutants of sigma70 (cysteine residues at positions 1, 59, 366, 442, and 596) were labeled with a europium chelate fluorochrome (donor). The oligonucleotide was modified at the 5'- or at the 3'-end with Cy5 fluorochrome (acceptor). The energy transfer was observed upon complex formation between the donor-labeled sigma70 holoenzyme and the acceptor-labeled nontemplate strand oligonucleotide, whereas no interaction was observed with the template strand oligonucleotide. The oligonucleotide was bound in one preferred orientation. This observation together with the sequence specificity of single-stranded oligonucleotide interaction suggests that two mechanisms of discrimination between the template and nontemplate strand are used by sigma70: sequence specificity and strand polarity specificity. The bound oligonucleotide was found to be close to residue 442, confirming that the single-stranded DNA binding site of sigma70 is located in an alpha-helix containing residue 442. The 5'-end of the oligonucleotide was oriented toward the COOH terminus of the helix.     

Anti-inflammatory activity of (cresoxyacetate)copper complexes]

In groups of mononuclear aqua(cresoxyacetato)copper(II) complexes of the composition [Cu(ROCH2COO)2(H2O)n] (R= 2- and 3-methylphenyl, n = 2; 4-methylphenyl, n = 3) and binu- clear phenazone(o-, m-, p-cresoxyacetato)copper(II) complexes of the composition [Cu2(ROCH2COO)4(phz)2] the anti-inflammatory activity was assayed in rat paw carrageenan-induced edema. The effects of the complexes were compared with each other and with those of the free acids. Salicylic acid and its cupric salt (tetrahydrate) were used as standards of comparison. All copounds were applied i.p. in a single dose of 50 mumol/kg body weight (calculated for the RCOO-fragment). In general, all Cu(II) complexes tested were clearly more effective (71-88%) than the corresponding free isomeric cresoxyacetic acids (47, 37, 45%), with the exception of diaquabis (o-cresoxyacetato)copper(II) complex (43%). Copper(II) salicylate tetrahydrate (57%) and salicylic acid (41%) were less active, too. The observed activities of complexes are discussed in relation to their structures.     

Preliminary screening of non-platinum complexes of Schiff bases as antitumour agents using fluorimetry

Based on the consistency of the in vivo and in vitro interactions of drugs with DNA, a fluorimetric method has been developed as a new in vitro method for preliminary screening of antitumour agents. This method was tested using Schiff bases synthesized from salicylaldehyde with 1-alanine, 1-asparagine and 1-histidine, and complexes of these Schiff bases with Cu(II), Zn(II), Ni(II) and Sn(IV) as potential antitumour agents. The study of the interaction of the complexes with DNA by a fluorescence probe ethidium bromide (EthBr)-DNA system indicated the parallelism between the binding constants and antineoplastic ratios. The relationship between structure and antitumour activity was investigated.