Phase-change related epigenetic and physiological changes in <Emphasis Type="Italic">Pinus radiata</Emphasis> D. Don

DNA methylation and polyamine levels were analysed before and after Pinus radiata D. Don. phase change in order to identify possible molecular and physiological phase markers. Juvenile individuals (without reproductive ability) were characterised by a degree of DNA methylation of 30-35% and a ratio of free polyamines to perchloric acid-soluble polyamine conjugates greater than 1, while mature trees (with reproductive ability) had 60% 5-methylcytosine and a ratio of free polyamines to perchloric acid-soluble polyamine conjugates of less than 1. Results obtained with trees that attained reproductive capacity during the experimental period confirmed that changes in the degree of DNA methylation and polyamine concentrations found among juvenile and mature states come about immediately after the phase change. We suggest that both indicators may be associated with the loss of morphogenic ability during ageing, particularly after phase change, through a number of molecular interactions, which are subsequently discussed.     

Polyamines and novel polyamine conjugates interact with DNA in ways that can be exploited in non-viral gene therapy

As a part of our continuing studies on 'Polyamines and their role in human disease' we are investigating how polyamines, and especially how novel polyamine conjugates, interact with DNA. We are studying how these conjugates interact with circular plasmids in order to produce nanometre-sized particles suitable for transfecting cells. Our considerations of structure--activity relationships (SAR) within naturally occurring and synthetic polyamines have shown the significance of the inter-atomic distances between the basic nitrogen atoms. As these atoms are typically fully protonated under physiological conditions, they exist in equilibrium as polyammonium ions. The covalent addition of a lipid moiety, typically one or two alkyl or alkenyl chains, or a steroid, allows much greater efficiency in DNA condensation and in the cellular transfection achieved. Thus efficient DNA condensation and subsequently drug delivery (i.e. with DNA as the drug) can be brought about using novel polyamine conjugates. Taking further advantage of the functionalization of specific steroids (e.g. cholesterol and certain bile acids), we have designed and prepared novel fluorescent molecular probes as tools to throw light on the problematic steps in non-viral gene delivery which still impede efficient gene therapy. Thus, the current aims of our research are to understand, design and prepare small-molecule lipopolyamines for non-viral gene therapy (NVGT). The rational design and practical preparation of non-symmetrical polyamine carbamates and amides, based on steroid templates of cholesterol and the bile acid lithocholic acid as the lipid moiety, provides fluorescent molecular probes that condense DNA. These novel lipopolyamine conjugates mimic the positive charge distribution found in the triamine spermidine and the tetra-amine spermine alkaloids. After optimizing their SAR, these fluorescent probes will be useful in monitoring gene delivery in NVGT.     

In vitro morphogenic potential of differently aged Pinus radiata trees correlates with polyamines and DNA methylation levels

DNA methylation and polyamine content have been analysed in juvenile, mature vegetative and mature reproductive Pinus radiata trees. Juvenile individuals were characterised by a DNA methylation degree of 30% and a high ratio of free versus PCA (perchloric acid) soluble polyamine conjugates, while mature trees showed 60% methylcytosine and low ratio of free versus PCA-soluble polyamine conjugates. We propose that both indicators are related with the lost of morphogenic ability during pine ageing and so with the inability of mature trees to in vitro establishment through specific interactions. These interactions are discussed.     

Transition metal derivatives of peptide nucleic acid (PNA) oligomers-synthesis, characterization, and DNA binding

This work describes the first automated solid-phase synthesis of metal derivatives of peptide nucleic acid (PNA) oligomers and their interaction with DNA and PNA. PNA constitutes a relatively young and very promising class of DNA analogues with excellent DNA and RNA binding properties. However, PNA lacks a suitable handle that would permit its sensitive detection on its own as well as when hybridized with complementary oligonucleotides. Metal complexes, on the other hand, offer high potential as markers for biomolecules. In this paper, we describe the synthesis of PNA heptamers (tggatcg-gly, where gly is a C-terminal glycine carboxylic acid amide) with two covalently attached metal complexes at the PNA N-terminus, namely a ferrocene carboxylic acid derivative and a tris(bipyridine)ruthenium(II) derivative. We show how all synthesis steps may be carried out with high yield on a DNA synthesizer, including attachment of the metal complexes. The conjugates were characterized by HPLC (>90% purity) and ESI-MS. Binding studies of the purified Ru-PNA heptamer to complementary DNA and PNA and comparison to the isosequential metal-free acetyl PNA heptamer proves that the attached metal complex has an influence on the stability (UV-T(m)) and structure (CD spectroscopy) of the conjugates, possibly by disruption of the nearby A:T base pair.     

Synthesis, DNA binding and cleavage activities of the copper (II) complexes of estrogen-macrocyclic polyamine conjugates

A series of the copper (II) complexes 5a-d of estrogen-macrocyclic polyamine conjugates were synthesized and characterized. The interactions of complexes 5a-d with DNA were studied by fluorescence spectroscopy and gel electrophoresis under physiological conditions. The results indicate that the conjugated estrogens have increased the cleavage efficiency of Cu[cyclen](2+) while the conjugates display poor binding affinities. The functional groups of D-ring of estrogens may play a key role in deciding binding and cleavage extent of the complexes to DNA.     

Gene transfection activities of amphiphilic steroid-polyamine conjugates

The design and evaluation of a novel potent class of DNA delivery agents based on steroid-polyamine conjugates bearing a flexible linker are reported. The hydrophobic regions are based on steroids, i.e. chlolestane and lithocholic acid motifs. The linker, which couples a hydrophobic steroid and a hydrophilic polyamine, in this study can be regarded as a two-atom extension of the conventional carbamate linker. We found that the gene transfection activity of the steroid-polyamine conjugates is influenced by the polyamine chain length and steroid structure. Molecular modeling of the relevant amphiphilic molecules revealed low-energy structures in which the polyamine chains are folded rather than stretched. This work suggests a significant effect of space-filling, i.e. the shape and orientation of the hydrophilic and hydrophobic regions, upon the efficiency of gene transfection.     

DNA structure control by polycationic species: polyamine, cobalt ammines, and di-metallo transition metal chelates

Many polycationic species bind to DNA and induce structural changes. The work reported here is the first phase of a program whose long-term aim is to create a class of simple and inexpensive sequence-selective compounds that will enable enhanced DNA structure control for a wide range of applications. Three classes of molecule have been included in this work: the polyamine spermine (charge: 4(+)) and spermidine (charge: 3(+)) (which are known to induce a wide range of DNA conformational changes but whose binding modes are still not well understood); cobalt (III) amine transition metal complexes as potential polyamine mimics and [Fe(H(2)O)(6)](3+); and the first member of a new class of di-metallo tris-chelated cylinders of helical structure (charge 4(+)). Temperature-dependent absorption, circular dichroism, linear dichroism, gel electrophoresis, and molecular modeling data are presented. The cobalt amines prove to be effective polyamine mimics, although their binding appears to be restricted to backbone and major groove. All the ligands stabilize the DNA, but the 4(+) di-iron tris-chelate does so comparatively weakly and seems to have a preference for single-stranded DNA. All the molecules studied bend the DNA, with the di-iron tris-chelate having a particularly dramatic effect even at very low drug load.     

DNA photocleavage by porphyrin–polyamine conjugates

A series of polyamine–porphyrin conjugates bearing two (cis or trans position) or four units of spermidine or spermine was synthesized. We studied the binding of these cationic porphyrins to calf thymus DNA by the means of UV–vis spectroscopy and we investigated their ability to cleave plasmid DNA in the presence of light. DNA binding and DNA photocleavage abilities were found to depend on structural characteristics as (a) the relative positions of the side chains on the porphyrin ring and (b) the nature of the attached side chains (spermidine or spermine). DNA cleavage was also studied in the presence of a singlet oxygen quencher (NaN₃) and in the presence of a hydroxyl radical scavenger (mannitol). Singlet oxygen was the major species responsible for the cleavage of DNA previously observed. Collectively, these data show that polyamine–porphyrin conjugates could be promising phototherapeutic agents.     

Synthesis of cholesteryl polyamine carbamates: pK(a) studies and condensation of calf thymus DNA

Novel polyamine carbamates have been designed and prepared from cholesterol. Our synthesis uses an orthogonal protection strategy based upon trifluoroacetyl and Boc-protecting groups. These unsymmetrical polyamine carbamates have been prepared from symmetrical (e.g., spermine and thermine) polyamines. Detailed interpretations of (1)H and (13)C NMR spectroscopic data led to the unambiguous assignment of these polyamine carbamates. These target conjugates contain a variety of positive charges distributed along methylene chains. Their pK(a)s have been determined potentiometrically for conjugates substituted with up to five amino functional groups. Condensation of calf thymus DNA into particles was monitored using light scattering at 320 nm. Salt-dependent binding affinity for calf thymus DNA was determined using an ethidium bromide fluorescence quenching assay. These cholesteryl polyamine carbamates are models for lipoplex formation with respect to gene delivery (lipofection), a key first step in gene therapy.     

Synthesis and bioevaluation of aryl-guanidino polyamine conjugates targeting the polyamine transporter

Aryl-guanidino polyamine conjugates were prepared to evaluate their recognition for polyamine transporter (PAT) via a-difluoromethylornithine (DFMO) and spermidine (SPD)-treated B16 cell lines. The potent synergistic effects of DFMO on guanidino polyamine conjugates indicated that the presence of DFMO strongly facilitates the transport of conjugates into cells via PAT on cell membrane. The apoptotic mechanisms of triamine conjugates 10 and 1 (with and without guanidine groups) revealed that they induced apoptosis of Hela cells through the mitochondrial pathway associated with lysosomes, while DFMO strongly synergizes the function of 10 without changing the apoptotic route. Taken together, guanidino polyamine conjugates can target PAT for transport as normal polyamine ones, and the presence of guanidine in the polyamine vectors does not seem to alter the cellular targets of the conjugates, which may depend mainly on the pharmacophore.     

Measurements of polyamines and their acetylated derivatives in cell extracts and physiological fluids by use of an amino acid analyzer

A fast and sensitive method for the determination of free polyamines and their acetylated derivatives is presented. The separation is carried out on a Durrum DC-6A cation-exchange resin with an automated amino acid analyzer. The determination is based on a stepwise elution with a sodium chloride—sodium citrate buffer system. Detection is done by fluorescence of the o-phthaldialdehyde—polyamine conjugates. The sensitivity is in the picomole range. No prior purification step is needed. The method has been applied to cell extracts and urine samples.     

Chiral multinuclear macrocyclic polyamine complexes: synthesis, characterization and their interaction with plasmid DNA

A series of multinuclear macrocyclic polyamine metal (Zn(2+), Cu(2+), Co(2+)) complexes containing chiral dipeptide linkage were synthesized and used as artificial nuclease enzyme model. The interaction between the complexes and plasmid DNA (pUC19) was studied, and the results revealed that these complexes could act as powerful catalysts for the cleavage of plasmid DNA under physiological conditions.     

Application of the Poisson Boltzmann polyelectrolyte model for analysis of thermal denaturation of DNA in the presence of Na+ and polyamine cations

The Poisson Boltzmann (PB) cell model of polyelectrolyte solution has been used for calculation of the electrostatic free energy difference, Delta G(el), between double- and single-stranded DNA. The calculations have been performed for conditions relevant to describe the DNA helix-coil transition in NaCl solution in the presence of the natural polyamines putrescine(2+), spermidine(3+), spermine(4+) and their synthetic homologs with different spacing between the charged amino groups, for which experimental values of the DNA 'melting' transition temperature (T(m)) are available. Using the PB theory and the polyamine ion radius as an adjusting parameter provides quantitative agreement between experimental and theoretical T(m)--salt concentration dependencies only by using physically unreasonable radii for the polyamine. Thus, modeling the linear and flexible polyamines as charged spheres within the PB cell model is an implausible oversimplification. We propose another explanation for the experimental observations, still within the frame of the 'primitive' PB polyelectrolyte theory. This explanation is based on an analysis of the Delta G(el) dependence on the stoichiometry of polyamine-polyanion binding to double- and single-stranded DNA.     

Effect of common sage plant treatment with polyamines on the contents of proline and free and conjugated polyamines under oxidative stress

Common sage (Salvia officinalis L.) plants grown in water culture to the stage of 4–5 true leaves were treated with paraquat (PQ) ( 1 ml of the solution containing 0.1 μM PQ in 0.05% Tween 80), putrescine (Put), spermidine (Spd), or Spermine (Spm) (all polyamines (PA) at the concentration of 0.5 mM in 0.05% Tween 80) or PA plus PQ for 6, 12, 24, 36, or 48 h. Under normal conditions, treatment with individual PA did not induce any changes in the content of free proline. Under oxidative stress induced by PQ, oxidation of proline and free PA by ROS resulted in their spending and demanded restoration of free PA due to hydrolysis of their insoluble conjugates. As distinct from treatment with PQ only, its combination with PA in the light was accompanied by proline accumulation. Treatment with Put plus PQ induced accumulation of intracellular Put and its soluble and insoluble conjugates. Treatments with Spd and Spm in combination with PQ resulted similarly in the increase in the levels of their intracellular soluble and insoluble conjugates. In treatments with these high-molecular PA, polyamine oxidase was activated and diaminopropan formation was observed. It seems likely that, for restoration of high-molecular PA homeostasis, their degradation by polyamine oxidase or production of insoluble conjugates is induced. Thus, the amount of free and conjugated particular PA is under strict control and is maintained at a definite level. A decrease in the content of a particular free PA induces primarily a decrease in the content of its soluble and then insoluble conjugates. The data obtained demonstrate the effects of exogenous PA on the content of intracellular proline under oxidative stress but do not allow a conclusion about direct regulation of its content by PA.     

Targeting DNA mismatches with rhodium intercalators functionalized with a cell-penetrating peptide

Cell-penetrating peptides are widely used to deliver cargo molecules into cells. Here we describe the synthesis, characterization, DNA binding, and cellular uptake studies of a series of metal-peptide conjugates containing oligoarginine as a cell-penetrating peptide. d-Octaarginine units are appended onto a rhodium intercalator containing the sterically expansive chrysenequinone diimine (chrysi) ligand to form Rh(chrysi)(phen)(bpy)(3+)-tethered oligoarginine conjugates, where the peptide is attached to the ancillary bpy ligand; some conjugates also include a fluorescein or thiazole orange tag. These complexes bind and with photoactivation selectively cleave DNA neighboring single-base mismatches. The presence of the oligoarginines is found to increase the nonspecific binding affinity of the complexes for both matched and mismatched DNA, but for these conjugates, photocleavage remains selective for the mismatched site, as assayed using both gel electrophoresis and mass spectrometry experiments. Significantly, the rhodium complex does not interfere with the delivery properties of the cell-penetrating peptide. Confocal microscopy experiments show rapid nuclear localization of the metal-peptide conjugates containing the tethered fluorescein. Mass spectrometry experiments confirm the association of the rhodium with the HeLa cells. These results provide a strategy for targeting mismatch-selective metal complexes inside cell nuclei.     

Proline controls the level of polyamines in common sage plants under normal conditions and at UV-B irradiation

Common sage (Salvia officinalis L.) plants grown in water culture to the stage of 4–5 true leaves were treated for 12, 24, 36, or 48 h with proline added to nutrient medium to a final concentration of 5 mM, or irradiated with UV-B light (12.3 kJ/m² for 10 min), or subjected to combined action of these factors. In these plants, activity of proline dehydrogenase (PDH), the content of proline, and the contents of free and conjugated polyamines were determined in the leaves and roots. It was shown that, in control plants, the content of endogenous proline was close to zero. In the presence of proline in medium, its total content in the roots was 9 μmol/g fr wt in 12 h of exposure, whereas in the leaves the content of proline increased only in 24 h and achieved only 1 μmol/g fr wt. The content of free putrescine increased in the leaves and especially in the roots after 10-min irradiation with UV-B light. The biosynthesis of putrescine was induced in the presence of proline in medium and was observed earlier than after UV-B irradiation. UV-B irradiation affected not only the synthesis of putrescine but also that of spermidine and spermine; it also induced accumulation of their soluble conjugates. Exogenous proline enhanced putrescine synthesis but inhibited the formation of polyamine soluble conjugates. At combined treatment of the two factors, the content of free putrescine in the leaves displayed a tendency to the rise and in the roots, to the decrease. At the same time, the content of polyamine free conjugates increased in both leaves and roots. All these facts could be considered as an indirect indication of relationship between proline and polyamine biosyntheses. We can also state that an artificially created high proline concentration in common sage tissues characterized of its low constitutive level resulted in disturbances in the homeostasis of low-molecular cell metabolites and induced a requirement in its restoration by diverse ways. This agrees with activation of PDH, a key enzyme of proline degradation. Induction of polyamine biosynthesis and changes in the content of their soluble conjugates might be one of the ways for such restoration. Under stress conditions, the high proline concentration is not toxic for plants because polyamines and proline are the components of the plant defense system, thus weakening damaging effects of abiotic stressors.     

Monitoring of the formation and dissociation of polyethylenimine/DNA complexes by two photon fluorescence correlation spectroscopy

Polyethylenimines (PEI) constitute efficient nonviral vectors for gene transfer. However, because free PEI shows some cytotoxicity and because intracellular dissociation of PEI/DNA complexes seems to be required for efficient transfection, it is important to monitor the concentrations of free and bound partners in the mixtures of DNA and PEI used for transfection. To reach this objective, we used fluorescence correlation spectroscopy with two-photon excitation to characterize the complexes formed with either rhodamine-labeled 25 kDa PEI or DNA plasmid molecules. At the molar ratios of PEI nitrogen atoms to DNA phosphate usually used for transfection, we found that approximately 86% of the PEI molecules were in a free form. The PEI/DNA complexes are composed on the average by 3.5 (+/-1) DNA plasmids and approximately 30 PEI molecules. From this composition and the pK(a) of PEI, it could be inferred that in contrast to DNA condensation by small multivalent cations, only a limited neutralization of the DNA phosphate groups is required for DNA condensation by PEI. Moreover, DNA appears only poorly compacted in the PEI/DNA complexes. As an application, fluorescence correlation spectroscopy was used to monitor the purification of PEI/DNA complexes by ultrafiltration as well as the heparin-induced dissociation of the complexes.     

Oligonucleotide probes containing polylysine residues for fabrication of DNA chips on various solid surfaces

Various materials, such as glass, plastic, metals, etc., are utilized for preparing DNA chips. In each particular case special approaches are used for immobilization of different oligonucleotide derivatives on the solid supports. We describe a general technique for DNA chips preparation on various unmodified surfaces using one type of oligonucleotide derivative, polylysine-oligonucleotide conjugates (PL-oligo). A long polyamine spacer in the PL-oligo conjugates provides a durable irreversible non-covalent immobilization onto a variety of solid supports and enough distance between oligonucleotides and the surface. The resulting DNA chips were shown to be useful for the detection of PCR DNA fragments and to be sensitive to single nucleotide discrepancies. They represent a promising instrument for revealing genetic diseases, genotyping viruses and bacteria, and for displaying their drug-resistant strains.     

Conjugated polyamines and reproductive development: Biochemical, molecular and physiological approaches

Whole tobacco plants containing the root-inducing, left-hand transferred DNA (Ri TL-DNA) display a transformed phenotype, that includes alterations in a number of developmental processes, such as floral induction, flowering and reproduction. We show that the entire Ri TL-DNA is responsible for repression of ornithine and tyrosine decarboxylases while it exerts no effect on transferase and the methyl transferase activities. Evidence is provided that two genes from the Ri TL-DNA, rolA and rolC, alter polyamine metabolism as well as floral induction and flowering. Thus, plants transformed by the rolC gene (under the control of the 35S promoter from cauliflower mosaic virus) were male-sterile (non-viable pollen) and female fertility was reduced by approximatively 80%. A constitutive overexpression of the rolC gene may directly or indirectly cause inhibition of the accumulation of water-insoluble amine conjugates located in the anthers and all the methyl transferases, leading to increases of ornithine decarboxylase, phenylalanine ammonia lyase and putrescine caffeoyl-CoA transferase. The results suggest that male sterility is associated with catabolic processes exerted at the level of water-insoluble amine conjugates and support the view that diamine oxidase may be involved in the regulation of the amine concentration during sexual differentiation, a factor that should be considered when attempting to decipher the mechanisms of control of sexual differentiation. The rolC gene could be useful in determining the role of diamine oxidase in the physiology of flowering. These results suggest that elevated free polyamine and water-soluble polyamine levels (located in the ovaries) contribute to abnormal floral development. The transformed phenotype due to P_35S-rolA(the rolA gene fused to the 35S promoter) consisted of inhibited or delaved flowering, and altered floral morphology in the form of flower abortion. The effects of P_35S-rolA on flowering and fertility are closely correlated with limitations in the accumulation of the water-soluble and -insoluble amine conjugates and increase in accumulation of free amines, indicating that amine conjugates (via transferases) have important functions in floral induction, floral evocation and reproduction. Spermidine availability as well as tyramine availability (in conjugated forms) could be limiting factor(s) in sexual development in tobacco.