Bioelectrochemistry (v.58, #1)
Editorial by R.I Zhdanov; T Hianik (1).
DNA–lipid interactions in vitro and in vivo by V.V Kuvichkin (3-12).
The data on lipid–nucleic interactions and their role in vitro and in vivo are presented. The results of study of DNA–lipid complexes in absence and in presence of divalent metal cations (triple complexes) are discussed. The triple complexes represent the generation of cellular structures such as pore complexes of eucaryotes and “Bayer's junctions” of procaryotes. The participation of triple complexes in the formation of structure of bacterial and eucaryotic nucleoid and nuclear matrix is analysed. A model of formation of triple complexes and cellular structures and their role in DNA–lipid interactions are discussed.
Keywords: DNA; DNA–membrane complexes; Cations; Triple complexes;
Functional role of phospholipids in the nuclear events by A.V Alessenko; E.B Burlakova (13-21).
This review presents the structural and functional role of phospholipids in chromatin and nuclear matrix as well as the difference in composition and turnover compared to those present in the nuclear membrane. Nuclei have a very active lipid metabolism which seems to play an important role in the transduction of the signals to the genome in response to agonists acting at the plasma membrane level. The evidence on the presence of phospholipid-calcium-dependent protein kinase C (PKC) in nuclei and enzymes of phospholipids turnover is given. Protein kinase C interacts with nuclear phosphoinositol and sphingomyelin cycles products. This fact evidences about possibility that signal transduction events could also occur at the nuclear level during induction of cell proliferation, differentiation and apoptosis.
Keywords: Nuclei; Chromatin; Nuclear matrix; Lipid metabolism; Phospholipids signal transduction; Apoptosis;
DNA-bound lipids of normal and tumor cells: retrospective and outlooks for functional genomics by V.A Struchkov; N.B Strazhevskaya; R.I Zhdanov (23-30).
By very soft phenol method, the high-molecular-mass natural DNA complexes (108–109 Da), which contain 1–3% specific lipids, were isolated from different eukaryotic and prokaryotic cells. Two pools of DNA-bound lipids were isolated: loosely bound (extracted with 35% ethanol) and tightly bound lipids (extracted after additional treatment DNAse I). The composition of these two lipid pools of different sources (rat thymus, liver, regenerating liver, loach sperm, pigeon erythrocytes, Zajdel ascites hepatoma, Ehrlich ascites carcinoma, sarcoma 37, Escherichia coli B, T2 phage) was studied. The DNA-bound lipid pools consist of neutral lipids (NL) and phospholipids (PL), moreover NL is always in a few fold more than PL. The composition of these lipid pools of eukaryotes distinguishes between themselves, mainly, by free cholesterol (minor fraction), cardiolipin (major fraction), and by phosphatidylcholine. Only the tightly bound lipid pool was present in T2 phage DNA. The dramatic redistribution effect between all fractions of NL pools (free and ester cholesterol, free fatty acids, diglycerides) was observed in DNA synthesis phase of cell cycle on the background of the unchanged composition of PL pools. Comparative analysis of DNA-bound lipid pools of normal and cancer cells was carried out. The DNA-bound lipid pools of transformed cells significantly differ from the same normal cells both by PL composition (cardiolipin) and by the presence of additional fractions (mono- and triglycerides) as well. The possible functions of DNA-bound lipid pools, especially of cardiolipin and cholesterol at the attachment of DNA loops to the nuclear matrix, DNA replicon organization, replication, and transcription are discussed.
Keywords: Two pools of DNA-bound lipids; Lipid composition and function; DNA-bound cardiolipin; DNA-bound cholesterol; Functional genomics;
Nuclear and chromatin lipids: metabolism in normal and γ-irradiated rats by I.K Kolomiytseva; T.P Kulagina; L.N Markevich; V.I Archipov; L.V Slozhenikina; L.A Fialkovskaya; N.I Potekhina (31-39).
The data on nuclear and chromatin lipid metabolism are reviewed. The amount of neutral lipids and phospholipids in nuclei of rat thymus, liver and neocortex neuron as well as the amount of lipids in rat thymus and liver chromatin are described. The metabolic responses of nuclear and chromatin lipids from thymus to different doses and dose rates of γ-irradiation of rats are discussed. In most cases, the nuclear and chromatin lipid responses are distinct. Changes in nuclear and chromatin lipid metabolism in response to γ-irradiation are suggested to connect with the signal transduction pathway and the regulation of the transcriptional and replicative chromatin activity. The influence of β-carotene and picrotoxin on rat liver nuclear lipids and neocortex neuronal nuclear lipids, respectively, was analyzed. The possible involvement of the lipid traffic in the chromatin lipid responses to γ-irradiation and other agents is suggested.
Keywords: Lipids; Nuclei; Chromatin; Liver; Thymus; γ-Radiation;
Role of lipid membrane–nucleic acid interactions, DNA–membrane contacts and metal (II) cations in origination of initial cells and in evolution of prokaryotes to eukaryotes by R.I Zhdanov; V.V Kuvichkin; A.S Shmyrina; A.R Jdanov; V.A Tverdislov (41-46).
The problems of the origin of primary cells and eukaryotic cells are discussed in terms of possible role of interactions between nucleic acids with lipid membrane according to corresponding original hypothesis. We propose that there are two main hypotheses of the origin of primary cells: (a) RNA appeared before proteins and DNA [Nature 213 (1967) 119]; (b) it is needed for the appearance of a primary cell, the volume closed by the lipid membrane. There was no information about the ways on how RNA appeared inside that volume for saving the reaction products around. Our hypothesis suggests that one of the starting points in the origination of primary cells was the interaction of nucleic acid and lipid membrane bubbles in the presence of metal (II) ions (which existed in high concentrations in prebiotic conditions), and this resulted in the enclosing of the pro-RNAs inside the lipid membrane. This hypothesis is formulated by us on the basis of experimental biochemical and biophysical studies of the DNA/RNA–phospholipid vesicles interactions in the presence of metal ions (II) fulfilled in the Institute of Biomedical Chemistry, RAMS, Moscow and Institute of Biophysics, RAS, Pushchino. Our belief is that DNA–membrane contacts (DNA–MCs) played an important role in the prokaryotes-to-eukaryotes transition. The model of the confluence of four prokaryotic cells may explain the prokaryotes-to-eukaryotes transition by the way of eukaryotic nuclear pore formation from prokaryotic Bayer' contacts. The main requirement for the following fusion of prokaryotic cells must be their mutual orientation. After possible association, the division of the formed cell is begun. The great advantage of the model of four prokaryotic cells is the profit in the metabolism and the possibility of the intensive growth of intercellular membrane structures.
Keywords: Initial cells; Prokaryotic cells; Eukaryotic cells; Lipid–nucleic acid interaction and recognition; DNA–membrane contacts; Cell fusion; Bayer's junctions; Prokaryote-to-eukaryote transition;
DNA–phospholipid recognition: modulation by metal ion and lipid nature. Complexes structure and stability calculated by molecular mechanics by P.N D'yachkov; B.B Fedorov; R Bischoff; G Bischoff; R.I Zhdanov (47-51).
The structures and formation energies of nucleic acid–phospholipid complexes both in the absence and in the presence of Mg2+ ions were calculated taking double-stranded trinucleoside diphosphates NpNpN or heptanucleotides ApAp(NpNpN)pApA, composed of 64 possible combinations of genetic code, and phosphatidylcholine (PC) and sphingomyelin (SM) as model compounds. The dependence of intramolecular interactions on the primary structure of nucleic acid molecules and on the presence of a cationic bridge was revealed. The formation energies and structure of oligonucleotides were found by molecular mechanics calculations with the AMBER force field. The structures of phospholipid and MgCl2 molecules were calculated by the semiempirical PM3 method, while the energies of phospholipid–oligonucleotide complexes were calculated by the molecular mechanics method. Calculations of complexes were carried out with consideration of solvation effects. Considerable gain in the formation energy of triple complexes is achieved due to the presence of the electroneutral metal bridge. A tendency toward increasing the stability of “triple” PC complexes (but not SM ones), containing guanosine- and cytidine-enriched triplets was revealed. Depending on the structure of NpNpN trinucleotides, the formation energy values of NpNpN–MgCl2–PC and ApAp(NpNpN)pApA–MgCl2–PC complexes differ by 1.7–2.6 kcal mol−1, which can be considered as the atomic-scale manifestation of the recognition phenomenon. Presence of metal (II) ion bridge results in a greater stabilization of the phospholipid–nucleic acid complexes for SM in comparison to PC (the total energy difference equals to 4–16 kcal mol−1). Depending on the structure of NpNpN trinucleotides, the formation energies of NpNpN–MgCl2–SM and ApAp(NpNpN)pApA–MgCl2–SM complexes differ by 1.7–2.1 kcal·mol−1, which is essential at physiological conditions and can also be considered as the recognition effect.
Keywords: Phospholipid–nucleic acids complexes; Formation energy; Computer simulation; Molecular mechanics; Quantum-chemical calculations;
Cationic lipid–DNA complexes—lipoplexes—for gene transfer and therapy by R.I Zhdanov; O.V Podobed; V.V Vlassov (53-64).
Cationic lipid-mediated gene transfer and delivery still attract great attention of many gene therapy laboratories. From the point of view of the most important characteristics of lipoplex particles, e.g. its charge and size, we reviewed recent studies available. In general, the paper deals with non-viral systems of gene transfer into eukaryotic cell based on various lipids. Having usually less efficiency in gene transfer, lipid-based gene transfer vehicles (lipoplexes/genosomes) are characterized with certain advantages even over viral ones: they are less toxic and immunogenic, could be targetable and are easy for large-scale production, a size of transferred DNA being quite high. Conditions of DNA condensation during interactions with lipids are described. Results of the studies of mechanism of DNA–lipid complex interactions with the cell membrane and their transport into the nucleus are summarized. Dependence of efficiency of gene transfer on lipoplex structure and physical–chemical properties is reviewed. Advantages and disadvantages of different macromolecule complexes from the point of view of transfection efficiency, possibility of use in vivo, cytotoxicity and targeted gene transfer in certain organs and tissues are also discussed. Results of transfection of different cells using neutral, anion and cation liposomes are reviewed. The conclusion reached was that efficiency and specificity of gene transfer may grow considerably when mixed macromolecule lipid systems including polycations and glycolipids are used.
Keywords: DNA–(phospho)lipid complexes; Genosome; Lipoplex; In vitro and in vivo gene transfer; Cationic lipids; Polycations; Hydrophobic polycations; Non-viral targeted gene delivery;
An experimental approach for direct observation of the interaction of polyanions with sphingosine-containing giant vesicles by N.I Hristova; M.I Angelova; I Tsoneva (65-73).
A new approach for direct optical microscopy observation of polyanion interactions with bilayers of giant cationic liposomes (GUVs) was suggested. Polyanions as DNA, dextran sulfate (DS), heparin (H) and polyacrylic acids (PA) were locally delivered by a micropipette to a part of a giant unilamellar vesicle membrane. The phenomena were directly observed under optical microscope. GUVs, about 100 μm in diameter, formed of phosphatidylcholines and up to 33 mol% of the natural bioactive cationic amphiphile sphingosine (Sph), were prepared by electroformation. The effects of water-soluble molecules with high negative linear charge density as dextran sulfate (DS), heparin (H) polyacrylic acids (PA) and adenosine-5′-triphosphoric acid (ATP) were compared with those of DNAs. The resulting membrane topology transformations were monitored in phase contrast, while the DNA distribution was followed in fluorescence. DNA-induced endocytosis-like membrane morphology transformation due to the DNA/lipid membrane local interactions was observed. The DS, H and PA induced membrane topology transformations similar to those of the DNAs, while ATP did not cause any detectable ones. The endocytosis mechanism involves the formation of ordered domains in the GUV membrane where some surface and charge asymmetries between the two membrane monolayers were created. The sizes of created polyanionic/cationic membrane domains depend on the form, length and elasticity of the adsorbed highly charged molecules. Endosome-including capacities of polyanionic molecules depend heavily on the high linear negative charge at a certain length.An original method for direct studying of the DNA/membrane interactions in autoadaptable giant liposome system imitating biological membrane interactions was forwarded. The model observations could also help for understanding events associated with cationic liposome/DNA complex formation in gene transfer processes.
Keywords: DNA and polyanion interactions; Cationic GUV; Microinjection; Sphingosine; Endocytosis;
Structural and thermodynamic features of complexes formed by DNA and synthetic polynucleotides with dodecylamine and dodecyltrimethylammonium bromide by A.I Petrov; D.N Khalil; R.L Kazaryan; I.V Savintsev; B.I Sukhorukov (75-85).
Complex formation of native and denatured DNA, single-stranded polyribonucleotides poly(A) and poly(U), as well as double-stranded poly(A)·poly(U) with dodecylamine (DDA) and dodecyltrimethylammonium bromide (DTAB) has been studied by UV-, CD-, IR-spectroscopy and fluorescence analysis of hydrophobic probe pyrene. DDA and DTAB were shown to bind cooperatively with DNA and polyribonucleotides, resulting in the formation of complexes containing hydrophobic micelle-like clusters. Critical aggregation concentration (CAC) of DDA and DTAB shifts sharply to lower values (30–50 times) in the presence of DNA and polynucleotides as compared to critical micelle concentration (CMC) of free DDA and DTAB in solution. The analysis of binding isotherms within the frame of the model of cooperative binding of low-molecular ligands to linear polymers allowed us to determine the thermodynamic parameters of complex formation and estimate the contribution of electrostatic interaction of positively charged heads of amphiphiles with negatively charged phosphate groups of DNA and polyribonucleotides, and hydrophobic interaction of aliphatic chains to complex stability. Electrostatic interaction was shown to make the main contribution to the stability of DNA complexes with DDA, while preferential contribution of hydrophobic interactions is characteristic of DTAB complexes with DNA. The opposite effect of DDA and DTAB on the thermal stability of DNA double helix was demonstrated from UV-melting of DNA—while DTAB stabilizes the DNA helix, DDA, to the contrary, destabilizes it. The destabilizing effect of DDA seems to originate from the displacement of intramolecular hydrogen bonds in complementary Watson–Crick A·T and G·C base pairs with intermolecular H-bonds between unsubstituted DDA amino groups and proton-accepting sites of nucleic bases.
Keywords: DNA; Polyribonucleotides; Cationic amphiphile; Fluorescence; Binding isotherm; Thermodynamics;
Condensed lamellar phase in ternary DNA–DLPC-cationic gemini surfactant system: a small-angle synchrotron X-ray diffraction study by Daniela Uhrı́ková; Gert Rapp; Pavol Balgavý (87-95).
We report on a small-angle synchrotron X-ray diffraction study of dilauroylphosphatidylcholine (DLPC) liposomes aggregated with high molecular DNA in the presence of 1,4-butanediammonium-N,N′-dilauryl-N,N,N′,N′-tetramethyl gemini surfactant cations (C12GS). The aggregates prepared at the DLPC/C12GS/DNA phosphate group=2:1:1.6 molar ratio in 0.0015 mol l−1 NaCl aqueous solution exhibit Bragg reflections due to lamellar lipid bilayer stacking and the Bragg reflection typical of one-dimensional DNA lattice with parallel strands intercalated between lipid bilayers. In this condensed fluid lamellar L α c phase, the interactions between DNA and charged bilayers damp the thermally induced bilayer undulations. The diffraction data obtained with the mixture of DLPC liposomes and DNA (at DNA phosphate group/DLPC=0.8:1 molar ratio) indicate a DNA–lipid interaction in the absence of C12GS.
Keywords: Cationic liposome; DNA; Small-angle X-ray diffraction; 1,2-Dilauroyl-sn-glycero-3-phosphorylcholine; 1,4-Butanediammonium-N,N′-dilauryl-N,N,N′,N′-tetramethyl dibromide; Transfection;
Electrostriction of supported lipid films at presence of cationic surfactants, surfactant–DNA and DNA–Mg2+ complexes by T Hianik; A Labajova (97-105).
The method of electrostriction has been applied to study the physical properties of supported lipid membranes (sBLM) during membrane formation at application of negative potential. Application of negative potential −350 mV to the sBLM during its formation resulted in more compact membrane structure as revealed by higher elastic modulus in comparison with sBLM formed without application of this potential. We also studied interaction with sBLM cationic surfactant hexadecylamine (HDA), HDA–DNA and DNA–Mg2+ complexes. Interaction of HDA with sBLM resulted in decrease of membrane capacitance and two-directional effect on elasticity modulus (increase or decrease), which can be caused by different aggregation state of surfactant at the surface of sBLM. In contrast with effect of HDA, the complexes of HDA–DNA resulted, in most cases, increase of elasticity modulus and increase of membrane capacitance, which can be caused by incorporation of these complexes into the hydrophobic interior of the membrane. Certain part of these complexes can, however, be adsorbed on the sBLM surface. DNA itself does not cause substantial changes of physical properties of sBLM; however, addition of bivalent cations Mg2+ to the electrolyte-contained DNA caused substantial increase of elasticity modulus and surface potential. These changes are, however, much slower than that observed for HDA–DNA complexes, which can be caused by slow competitive exchange between Na+ and Mg2+ ions.
Keywords: Electrostriction; Capacitance; Cationic surfactants; Surfactant–DNA complexes; DNA–Mg2+–lipid complexes;
A minisensor for the rapid screening of atenolol in pharmaceutical preparations based on surface-stabilized bilayer lipid membranes with incorporated DNA by Dimitrios P Nikolelis; Syrago-Styliani E Petropoulou; Maria V Mitrokotsa (107-112).
This work describes an electrochemical technique that is suitable for the rapid and sensitive screening of atenolol based on surface-stabilized bilayer lipid membranes (s-BLMs) composed from egg phosphatidylcholine (PC). The interactions of atenolol with s-BLMs produced electrochemical ion current increases that reproducible appeared within a few seconds after the exposure of the membranes to the drug. The current signal increase was related to the concentration of atenolol in bulk solution in the micromolar range. The present lipid film-based sensor provided fast response (i.e. on the order of a few seconds) to alterations of atenolol concentration (20 to 200 μM) in electrolyte solution. ssDNA incorporated into s-BLMs can interact with atenolol, and decreased the detection limit of this drug by one order of magnitude. The oligomers used were single stranded deoxyribonucleic acids: thymidylic acid icosanucleotide terminated with a C-16 alkyl chain to assist incorporation into s-BLMs (5′-hexadecyl-deoxythymidylic acid icosanucleotide, dT20-C16). The electrochemical transduction of the interactions of atenolol with s-BLMs was applied in the determination of these compounds in pharmaceutical preparations by using the present minisensor.
Keywords: Biosensors; Electrochemistry; Surface-stabilized bilayer lipid membranes; Atenolol; ssDNA;
Electrochemical DNA biosensor for analysis of wastewater samples by F Lucarelli; A Kicela; I Palchetti; G Marrazza; M Mascini (113-118).
The application of a disposable electrochemical DNA biosensor to wastewater samples is reported. The DNA biosensor is assembled by immobilising double-stranded calf thymus DNA on the surface of a disposable, carbon screen-printed electrode (SPE). The oxidation signal of the guanine base, obtained by a square wave voltammetric scan, is used as analytical signal. The presence of compounds with affinity for DNA is measured by their effect on the guanine oxidation. The comparison of the results with a toxicity test based on bioluminescent bacteria has confirmed the applicability of the method to real samples.
Keywords: DNA biosensor; Screen-printed electrodes; Wastewater samples; Toxalert® 100;
DNA and PNA sensing on mercury and carbon electrodes by using methylene blue as an electrochemical label by Dilsat Ozkan; Pinar Kara; Kagan Kerman; Burcu Meric; Arzum Erdem; Frantisek Jelen; Peter E Nielsen; Mehmet Ozsoz (119-126).
Described here are the electrochemical parameters for MB on binding to DNA at hanging mercury drop electrode (HMDE), glassy carbon electrode (GCE), and carbon paste electrode (CPE) in the solution and at the electrode surface. MB, which interacts with the immobilized calf thymus DNA, was detected by using single-stranded DNA-modified HMDE or CPE (ssDNA-modified HMDE or CPE), bare HMDE or CPE, and double-stranded DNA-modified HMDE or CPE (dsDNA-modified HMDE or CPE) in combination with adsorptive transfer stripping voltammetry (AdTSV), differential pulse voltammetry (DPV), and alternating current voltammetry (ACV) techniques. The structural conformation of DNA and hybridization between synthetic peptide nucleic acid (PNA) and DNA oligonucleotides were determined by the changes in the voltammetric peak of MB. The PNA and DNA probes were also challenged with excessive and equal amount of noncomplementary DNA and a mixture that contained one-base mismatched and target DNA. The partition coefficient was also obtained from the signal of MB with probe, hybrid, and ssDNA-modified GCEs. The effect of probe, target, and ssDNA concentration upon the MB signal was investigated. These results demonstrated that MB could be used as an effective electroactive hybridization indicator for DNA biosensors. Performance characteristics of the sensor are described, along with future prospects.
Keywords: DNA; Biosensor; Methylene blue; Peptide nucleic acid; Hybridization; Point mutation;