The interaction of copper(II) and glycyl-L-histidyl-L-lysine, a growth-modulating tripeptide from plasma.

The interaction between Cu(II) and the growth-modulating tripeptide glycyl-L-histidyl-L-lysine in the presence and absence of L-histidine was investigated by potentiometric titration and visible-absorption spectrophotometry at 25 degrees C in 0.15 M-NaCl. Analyses of the results in the pH range 3.5--10.6 indicated the presence of multiple species in solution in the binary system and extensive amounts of the ternary complexes in the ternary system. The species distribution and the stability constants, as well as the visible-absorption spectra of the species, were evaluated. The combined results were used to propose the structure of some of the complexes. The influence of the epsilon-amino group of the peptide in the enhancement of the stability constants was reflected prominently when compared with those complexes formed by either glycyl-L-histidine or glycyl-L-histidylglycine. The results obtained from the equilibrium-dialysis experiments showed that this tripeptide was able to compete with albumin for Cu(II) at pH 7.5 and 6 degrees C. At equimolar concentrations of albumin and the peptide, about 42% of the Cu(II) was bound to the peptide. At the physiologically relevant concentrations of Cu(II), albumin, L-histidine and this peptide, about 6% of the Cu(II) was associated with the low-molecular-weight components. This distribution could be due to the binary as well as the ternary complexes. The possible physiological role of these complexes in the transportation of Cu(II) from blood to tissues is discussed.     

The interaction of Fe(III), adriamycin and daunomycin with nucleotides and DNA and their effects on cell growth of fibroblasts (NIH-3T3)

The interactions of the iron complexes of the anthracycline antitumour drugs daunomycin (DN) and adriamycin (ADM) with the mononucleotide AMP, herring sperm DNA, plasmic pBR322 and immortalized 3T3 fibroblasts were studied. By means of Mössbauer spectroscopy it was demonstrated that DNA is a powerful ferric iron chelator as compared with AMP, which is not able to compete with DN or acetohydroxamic acid for ferric iron. The difference between AMP and DNA is postulated to be based on the chelate effect. The Mössbauer spectra of the ternary Fe-anthracycline-DNA systems differ from Fe-anthracycline binary complexes, indicating rearrangement reactions. Dialysis experiments clearly disclose the formation of a ternary Fe-ADM-pBR322 complex, the topology of which differs substantially from intercalating ADM. The effect of Fe-ADM complexes (3:1) on the growth of immortalized mouse embryonal fibroblasts (NIH-3T3) was studied in comparison with ADM alone. No significant difference on the inhibition of cell growth was noticed, suggesting comparable cytotoxicity for the compounds. In contrast to literature data, no evidence was found for DNA cleavage by ferric ADM at molar ratios as high as 1/100 (ADM/base pair), even if the ternary systems were prepared in the light and in the presence of reducing or oxidizing agents. Based on our observations it seems that the cytotoxicity of both ADM and Fe-ADM oligomer is not based primarily on intercalation or direct interaction with DNA.     

Characterization of the ternary complexes formed in the reaction of cis-diamminedichloroplatinum (II), ethidium bromide and nucleic acids.

The purpose of this study was to characterize the ternary complexes formed in the reaction of cis-diamminedichloroplatinum (II) (cis-DDP) and nucleic acids, in the presence of the intercalating compound ethidium bromide (EtBr). In these ternary complexes, some EtBr is tightly bound to the nucleic acids. Tight binding is defined by resistance to extraction with butanol, assayed by filtration at acid pH or thin layer chromatography at basic pH. These ternary complexes are formed with double stranded but not with single stranded nucleic acids. They are not formed if cis-DDP is replaced by transdiamminedichloroplatinum(II). The amount of tightly bound EtBr depends upon the sequence of the nucleic acid, being larger with poly (dG-dC).poly(dG-dC) than with poly(dG).poly(dC). Spectroscopic results support the hypothesis that the tight binding of the dye is due to the formation of a bidentate adduct (guanine-EtBr)cis-platin. The visible spectrum of the ternary complexes is blue-shifted as compared to that of EtBr intercalated between the base pairs of unplatinated DNA and it depends upon the conformation of the ternary complex. The fluorescence quantum yield of the ternary complexes is lower than that of free EtBr in water. Tightly bound EtBr stabilizes strongly the B form versus the Z form of the ternary complex poly(dG-dC)-Pt-EtBr and slows down the transition from the B form towards the Z form. The sequence specificity of cis-DDP binding to a DNA restriction fragment in the absence or presence of EtBr is mapped by means of the 3'----5' exonuclease activity of T4 DNA polymerase. In the absence of the dye, all the d(GpG) sites and all the d(ApG) sites but one in the sequence d(TpGpApGpC) are platinated. The d(GpA) sites are not platinated. In the presence of EtBr, some new sites are detected. These results might help to explain the synergism for drugs used in combination with cis-DDP and in the design of new chemotherapeutic agents.     

Ternary complex consisting of DNA, polycation, and a natural polysaccharide of schizophyllan to induce cellular uptake by antigen presenting cells

A natural polysaccharide called schizophyllan (SPG) can form a complex with polynucleotides, and the complex has been shown to deliver biofunctional short DNAs such as antisense DNAs and CpG-DNAs. Although it is a novel and efficient method, there is a drawback: attachment of homo-polynucleotide tails [for example, poly(dA) or poly(C)] to the end of DNA is necessary to stabilize the complex, because DNA heterosequences cannot bind to SPG. The aim of this paper is to present an alternative method in which SPG/DNA complexes can be made without using the tails. The basic strategy is as follows: since SPG can form hydrophobic domains in aqueous solutions, hydrophobic objects should be encapsulated by this domain. DNA alone is highly hydrophilic; however, once DNA/polycation complexes are made, they should be included by the SPG hydrophobic domain. The aim of this paper is to prove the formation of the polycation/DNA/SPG ternary complex. Gel electrophoresis showed that presence of SPG influenced the migration pattern of polycation+DNA mixtures. With increasing the SPG ratio, the zeta potential (zeta) of the polycation+DNA+SPG mixture decreased drastically to reach almost zeta = 0 and the particle size distributions were altered due to the ternary complex formation. Confocal laser scanning microscopy revealed that the polycation/DNA/SPG ternary complexes showed high uptake efficiency when the complexes were exposed to macrophage-like cells (J774.A1). IL-12 secretion was enhanced when CpG-DNA was added as the ternary complex. These features can be ascribed to the fact that J774.A1 has a SPG recognition site called Dectin-1 on the cellular surface and the ternary complex can be ingested by this pathway.     

Steric stabilization of poly-L-Lysine/DNA complexes by the covalent attachment of semitelechelic poly[N-(2-hydroxypropyl)methacrylamide

The concept of steric stabilization was utilized for self-assembling polyelectrolyte poly-L-lysine/DNA (pLL/DNA) complexes using covalent attachment of semitelechelic poly[N-(2-hydroxypropyl)methacrylamide] (pHPMA). We have examined the effect of coating of the complexes with pHPMA on their physicochemical stability, phagocytic uptake in vitro, and biodistribution in vivo. The coated complexes showed stability against aggregation in 0.15 M NaCl and reduced binding of albumin, chosen as a model for the study of the interactions of the complexes with plasma proteins. The presence of coating pHPMA had no effect on the morphology of the complexes as shown by transmission electron microscopy. However, results of the study of polyelectrolyte exchange reactions with heparin and pLL suggested decreased stability of the coated complexes in these types of reactions compared to uncoated pLL/DNA complexes. Coated complexes showed decreased phagocytic capture by mouse peritoneal macrophages in vitro. Decreased phagocytosis in vitro, however, did not correlate with results of in vivo study in mice showing no reduction in the liver uptake and no increase in the circulation times in the blood. We propose that the rapid plasma elimination of coated pLL/DNA complexes is a result of binding serum proteins and also of their low stability toward polyelectrolyte exchange reactions as a consequence of their equilibrium nature.     

Zinc-chelated poly(1-vinylimidazole) and a carbohydrate ligand polycation form DNA ternary complexes for gene delivery

Zinc-chelated poly(1-vinylimidazole) (PVIm-Zn) and a carbohydrate ligand polycation, a poly(l-lysine) conjugated with lactose molecules (PLL-Lac), have formed DNA ternary complexes for gene delivery. The particle size of the PVIm-Zn/DNA complexes with negative zeta potential was decreased by the addition of the PLL-Lac. The resulting PLL-Lac/PVIm-Zn/DNA ternary complexes, which exhibited the pH-dependent dissociation of the PLL-Lac, mediated more gene expression than the PVIm/DNA binary complexes. The PLL-Lac/PVIm-Zn/DNA complexes with the specific recognition of cell surface receptors mediated the highest gene expression without cytotoxicity at a relatively lower charge ratio (positive/negative = 2.5). These results suggest that the pH-dependent dissociation of the carbohydrate ligands after the recognition of cell surface receptors, including the physicochemical and biochemical function of PVIm-Zn, played an important role in gene expression.     

An in vitro study of interactions between insulin-mimetic zinc(II) complexes and selected plasma components

The speciations of some potent insulin-mimetic zinc(II) complexes of bidentate ligands: maltol and 1,2-dimethyl-3-hydroxypyridinone with (O,O) and picolinic acid with (N,O) coordination modes, were studied via solution equilibrium investigations of the ternary complex formation in the presence of small relevant bioligands of the blood serum such as cysteine, histidine and citric acid. Results show that formation of the ternary complexes, especially with cysteine, is favoured at physiological pH range in almost all systems studied. Besides these low molecular mass binders, serum proteins among others albumin and transferrin can bind zinc(II) or its complexes. Accordingly, the distribution of zinc(II) between the small and high molecular mass fractions of the serum was also studied by ultrafiltration. Modelling calculations relating to the distribution of zinc(II), using the stability constants of the ternary complexes studied and those of the serum proteins reported in the literature, confirmed the ultrafiltration results, namely, the primary role of albumin in zinc(II) binding among the low and high molecular mass components of the serum.     

Hydrogen Peroxide Dependent Oxidative Degradation of DNA by Copper Epinephrine

The hydrogen peroxide dependent oxidation of the epinephrinecopper complex to adrenochrome is mediated by free copper ions. The oxidation is enhanced by chloride ions and by the presence of serum albumin. The reaction is not inhibited by SOD or by hydroxyl radical scavengers.

The 2:1 epinephrine or dopamine:Cu(II) complexes are able to bind to DNA and to catalyze its oxidative destruction in the presence of hydrogen peroxide. The DNA-epinephrine-Cu(II) terenary complex has characteristic spectral properties. It has the capacity to catalyze the reduction of oxygen or H₂O₂ and it preserves the capacity over a wide range of comp1ex:DNA ratios. The rate of DNA cleavage is proportional to the rate of epinephrine oxidation and the rate determining step of the reaction Seems to be the reduction of free Cu(II) ions. The ability to form redox active stable DNA ternary complexes, suggests that under specific physiological conditions, when “free” copper ions are available. catecholamina may induce oxidative degradation of DNA and other biological macromolecules.     

Supramolecular gene delivery vectors showing enhanced transgene expression and good biocompatibility

Soluble supramolecular inclusion complexes were formed by threading alpha-cyclodextrin (alpha-CD) molecules over poly(ethylene glycol) (PEG) and poly(epsilon-caprolactone) (PCL) chains of ternary block copolymers of PEG, PCL and polyethylenimine (PEI). Characteristic shifts of PCL absorptions in FTIR, (1)H NMR and UV spectra strongly suggest that alpha-CD is threaded over PEG and PCL blocks. Due to the reduced hydrophobic interaction between PCL blocks, the resulting supramolecular complexes displayed a dramatically increased solubility, in comparison with the ternary block copolymers. Their ability to complex DNA was almost as efficient as that of branched PEI 25 kDa, as shown in the ethidium bromide fluorescence quenching experiments. Resulting DNA polyplexes displayed a size of around 200 nm and a neutral surface charge. Microscopy studies in 3T3 fibroblasts revealed an efficient cellular uptake. Transfection efficiencies of inclusion complexes were in the same order of magnitude as PEI. In contrast to PEI a 100x lower toxicity was observed by MTT-assay, allowing the administration of nitrogen-to-phosphate ratios of up to 20. These new gene delivery systems merit further characterization under in vivo conditions.     

Hydrogen Peroxide Dependent Oxidative Degradation of DNA by Copper Epinephrine

The hydrogen peroxide dependent oxidation of the epinephrinecopper complex to adrenochrome is mediated by free copper ions. The oxidation is enhanced by chloride ions and by the presence of serum albumin. The reaction is not inhibited by SOD or by hydroxyl radical scavengers.

The 2:1 epinephrine or dopamine:Cu(II) complexes are able to bind to DNA and to catalyze its oxidative destruction in the presence of hydrogen peroxide. The DNA-epinephrine-Cu(II) terenary complex has characteristic spectral properties. It has the capacity to catalyze the reduction of oxygen or H₂O₂ and it preserves the capacity over a wide range of comp1ex:DNA ratios. The rate of DNA cleavage is proportional to the rate of epinephrine oxidation and the rate determining step of the reaction Seems to be the reduction of free Cu(II) ions. The ability to form redox active stable DNA ternary complexes, suggests that under specific physiological conditions, when “free” copper ions are available. catecholamina may induce oxidative degradation of DNA and other biological macromolecules.     

Transfer of recA protein from one polynucleotide to another. Kinetic evidence for a ternary intermediate during the transfer reaction

We have analyzed the transfer kinetics of recA protein from one polynucleotide to another by monitoring the change in fluorescence of a modified single-stranded M13 DNA, referred to as etheno M13 DNA, that accompanies recA protein dissociation. The observed rate of transfer is dependent on the concentration of competitor polynucleotide, polythymidylic acid (poly(dT]; increasing the poly(dT) concentration increases the rate of transfer. These data are inconsistent with the rate-limiting step in the transfer mechanism occurring by a simple dissociation process. Under certain conditions, the apparent rate constant displays plateau behavior at high poly(dT) concentrations. This result is indicative of transfer occurring through a ternary intermediate including etheno M13 DNA and poly(dT). The transfer reaction was found to occur through two kinetically distinct species of transferring recA protein X DNA complexes. The relative amounts of these two species was affected by both the MgCl2 and protein concentration, suggesting that the two kinetic components reflect different aggregation states of the recA protein X DNA complex. Because etheno M13 DNA and poly(dT) contain no complementary sequences, we have concluded that recA protein has the intrinsic ability to form a kinetic ternary intermediate with two separate single-stranded DNA molecules in the absence of homology.     

Circular dichroism and hydrodynamic measurements of ternary protein--two ligand systems: serum albumin-bilirubin-drug or alcohol.

The effects of various ligands on bilirubin-serum albumin complexes in aqueous solution were investigated at pH 7.4 and 27 °C by circular dichroism (CD) measurements. The ligands included various penicillins, benzoic acid derivatives, and various lower aliphatic alcohols, using a molar excess of charcoal-treated human or bovine serum albumin with respect to bilirubin. In all cases investigated, significant changes in the visible-range CD spectra of the bilirubin-serum albumin complexes occurred within a certain range of added ligand concentrations. For several such ligand systems, analogous CD effects could be measured on both diluted and undiluted human blood serum or plasma. For part of the isolated albumin-ligand systems, significant dissociation of the bilirubin from the albumin was demonstrated by electrophoretic and analytical ultracentrifugation measurements, while other systems did not reveal measurable dissociation under the conditions used, indicating the formation of a ternary complex. A scheme of equilibria among all complex components is proposed, which includes both dissociation of the bilirubin and ternary complex formation in which the bilirubin conformation appears to be modified. At least two different sets of binding sites (competitive and noncompetitive) for added ligands are assumed. Values of apparent parameters describing the formation of ternary complexes from the bilirubin-albumin complex are estimated for a number of systems. Some relationships between the chemical structure of a ligand and its effect on the bilirubin-serum albumin complex are deduced. The relevance of the results obtained for the isolated protein-ligand complexes with respect to in vivo conditions is evaluated.     

Methodologies for monitoring nanoparticle formation by self-assembly of DNA with poly(l-lysine)

DNA self-assembly with polycations produces nanoparticles suitable for gene delivery, although there is no standard methodology to measure particle formation and stability. Here we have compared three commonly used assays, namely, light scattering, inhibition of ethidium bromide fluorescence, and modified electrophoretic mobility of DNA. Analysis by light scattering and loss of ethidium bromide fluorescence both showed poly(l-lysine) (pLL)/DNA nanoparticles form over the lysine/phosphate ratio range 0.6-1.0, although retardation of DNA electrophoretic mobility commenced at lower lysine/phosphate ratios. This probably indicates that the first two assays monitor DNA collapse into particles, while the electrophoresis assay measures neutralization of the charge on DNA. Gel analysis of the complexes showed disproportionation during nanoparticle formation, probably reflecting cooperative binding of the polycation. The assays were used to examine stability of complexes to dilution in water and physiological salts. Whereas all pLL/DNA nanoparticles were stable to dilution in water, the presence of physiological salts provoked selective disruption of complexes based on low-molecular-weight pLL. Polyelectrolyte complexes for targeted application in vivo should therefore be based on high-molecular-weight polycations, or should be stabilized to prevent their dissociation under physiological salt conditions.     

Determination of the stability constants and oxidation susceptibility of nickel(II) complexes with 2'-deoxyguanosine 5'-triphosphate and L-histidine

The formation of binary Ni(II) complexes with 2'-deoxyguanosine 5'-triphosphate (dGTP, L) as well as ternary complexes thereof with L-histidine (His, A) was studied with the use of potentiometry and electronic absorption spectroscopy. In the binary and ternary systems, the complexes with stoichiometries NiH2L-, NiHL2-, NiL3- and NiH2LA2-, NiHLA3-, NiLA4- respectively, were detected. The ternary complexes are very stable at pH 7.4 and thus may constitute biologically relevant Ni(II) carriers in the cell. In the presence of hydrogen peroxide, the binary and ternary systems both generate hydroxyl radical-like species and undergo dGTP degradation with the formation of the 8-oxo-dGTP intermediate. The latter, along with dGTP complexation and degradation, may lead to mutagenesis and carcinogenesis due to base-mispairing properties of 8-oxoguanine and the disturbance in the physiological balance among the four canonical triphosphodeoxynucleotide substrates for DNA synthesis.     

Degradable-brushed pHEMA-pDMAEMA synthesized via ATRP and click chemistry for gene delivery

Brushed polymers composed of a backbone of poly(hydroxyethyl methacrylate) (pHEMA) onto which poly(2-(dimethylamino)ethyl methacrylate)s (pDMAEMAs) was grafted via a hydrolyzable linker were synthesized and evaluated as nonviral gene delivery vectors. Both pDMAEMA and pHEMA polymers with controlled molecular weights and narrow distributions were synthesized by controlled atom transfer radical polymerization (ATRP). The azide initiator was used to ensure complete and monoazide functionalization of the pDMAEMA polymer chains. Click reaction between pHEMA with alkyne side groups and the azide end group in the pDMAEMA resulted in a high-molecular-weight polymer composed of low-molecular-weight constituents via an easily degradable carbonate ester linker. The length of the pDMAEMA grafts as well as the number of grafts of the brushed pHEMA-pDMAEMA can be easily varied. At physiological conditions (pH 7.4 and 37 degrees C), the brushed polymer degraded by hydrolysis of the carbonate ester with a half-life of 96 h. The molecular weights of the formed degradation products was very close to that of the starting pDMAEMA, which is likely below the renal excretion limit (<30 kDa). It was shown that the degradable brushed pHEMA-pDMAEMAs were able to condense plasmid DNA into positively charged nanosized particles. The resulting polyplexes were able to transfect cells efficiently in the presence of the endosomal membrane disrupting INF-7 peptide, and all these degradable polymers showed lower cellular toxicity compared to a high-molecular-weight pDMAEMA reference. On the other hand, the low-molecular-weight pDMAEMA used for the grafting to pHEMA was neither able to condense the structure of DNA nor able to transfect cells. This study demonstrates that grafting a low-molecular-weight cationic polymer via a hydrolyzable linker to a neutral hydrophilic polymer is an effective approach to modulate the transfection activity and toxicity profile of gene delivery polymers.     

Investigation of Ternary Complex Formations of Polyacrylic Acid with Bovine Serum Albumin in the Presence of Metal Ions by Fluorescence and Dynamic Light Scattering Measurements

In this paper, the complex formation of bovine serum albumin (BSA) and polyacrylic acid (PAA) in the presence metal ions at pH = 7 has been examined by using fluorescence and dynamic light scattering measurements. It has been observed that the most stable complexes of polyacrylic acid and bovine serum albumin have occurred in the presence of copper(II) ions. The other ions have the ability to form weak complexes between polyions and bovine serum albumin. To prior characterizing the interaction between bovine serum albumin and polyacrylic acid, the dynamic light scattering technique have been applied to determine the intensity-size distributions of the solutions of bovine serum albumin, polyacrylic acid, and ternary mixtures containing various molar ratios of bovine serum albumin to polyacrylic acid (the molar ratios of bovine serum albumin to polyacrylic acid has been taken equal to 0.5, 1.0, 1.5, 2.0 and 2.5) prepared at different molar ratios of copper(II) ions/acrylic acid unit. When the molar ratio of copper(II) ions to acrylic acid in the ternary mixtures has been lower than and equals to 0.3, two peaks have been observed in the curves of the intensity-size distributions due to contents of free bovine serum albumin and ternary complexes of polyacrylic acid-copper(II)-bovine serum albumin whereas when the molar ratio of copper(II) ions to acrylic acid equals to 0.4, the hydrodynamic diameter has pointed out only one peak. This result indicates that soluble and stable ternary complexes has occurred when the molar ratio of copper(II) ions to acrylic acid has been taken equal to 0.4.     

DNA complexes with polycations useful for delivery of DNA into cells

Generation and physicochemical properties of complexes formed by high-molecular thymus DNA and plasmid DNA with synthetic polymers of (dimethyl amino)ethyl methacrylate, (diethyl amino)ethyl methacrylate, and poly(vinyl amine) were studied in solutions of different ionic strength using low-gradient viscometry, electrophoresis, circular dichroism, spectrophotometry, and dynamic light scattering. The complexes were tested for toxicity with T98G cell cultures. Condensation of DNA was shown to occur when the ratio of charged groups in the polycations and DNA exceeded unity. This condensation manifested itself as an increase in the optical density of DNA solutions. Condensation-associated changes in the dimensions of DNA molecules were determined, and phase diagrams of DNA-polycation systems were analyzed in the presence of NaCl. MTT analysis revealed no toxicity of these complexes.     

RecO-mediated DNA homology search and annealing is facilitated by SsbA

Bacillus subtilis RecO plays a central role in recombinational repair and genetic recombination by (i) stimulating RecA filamentation onto SsbA-coated single-stranded (ss) DNA, (ii) modulating the extent of RecA-mediated DNA strand exchange and (iii) promoting annealing of complementary DNA strands. Here, we report that RecO-mediated strand annealing is facilitated by cognate SsbA, but not by a heterologous one. Analysis of non-productive intermediates reveals that RecO interacts with SsbA-coated ssDNA, resulting in transient ternary complexes. The self-interaction of ternary complexes via RecO led to the formation of large nucleoprotein complexes. In the presence of homology, SsbA, at the nucleoprotein, removes DNA secondary structures, inhibits spontaneous strand annealing and facilitates RecO loading onto SsbA–ssDNA complex. RecO relieves SsbA inhibition of strand annealing and facilitates transient and random interactions between homologous naked ssDNA molecules. Finally, both proteins lose affinity for duplex DNA. Our results provide a mechanistic framework for rationalizing protein release and dsDNA zippering as coordinated events that are crucial for RecA-independent plasmid transformation.