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Insufficient drug uptake by solid tumors remains the major problem for systemic chemotherapy. Many studies have demonstrated anticancer drug effects to be dose-dependent, although dose-escalation studies have resulted in limited survival benefit with increased systemic toxicities. One solution to this has been the idea of loco-regional drug treatments, which offer dramatically higher drug concentrations in tumor tissues while minimizing systemic toxicity. Although loco-regional delivery has been most prominent in cancers of the liver, soft tissues and serosal peritoneal malignancies, survival benefits are very far from desirable. This review discusses the evolution of loco-regional treatments, the present approaches and offers rapidly reversible hydrophobization of drugs as the new future direction., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

We have investigated a rapidly reversible hydrophobization of therapeutic agents for improving first-pass uptake in locoregional drug therapy. This approach involves the attachment of a hydrophobic moiety to the drug by highly labile chemical linkages that rapidly hydrolyze upon injection. Hydrophobization drastically enhances cell-membrane association of the prodrug and, consequently, drug uptake, while the rapid lability protects nontargeted tissues from exposure to the highly active agent. Using the membrane-impermeable DNA intercalator propidium iodide, and melphalan, we report results from in vitro cellular internalization and toxicity studies. Additionally, we report in vivo results after a single liver arterial bolus injection, demonstrating both tumor targeting and increased survival in a mouse tumor model.

Background: The efficient delivery of plasmid DNA (pDNA) to hepatocytes by a hydrodynamic tail vein (HTV) procedure has greatly popularized the use of naked nucleic acids. The hydrodynamic process renders onto the tissue increased physical forces in terms of increased pressures and shear forces that could lead to transient or permanent membrane damage. It can also trigger a series of cellular events to seal or reorganize the stretched membrane. Our goal was to study the uptake mechanism by following the morphological changes in the liver and correlate these with the fate of the injected plasmid DNA., Methods: We utilized both light microscopic (LM) and electron microscopic (EM) techniques to determine the effect of the HTV procedure on hepatocytes and non-parenchymal cells at various times after injection. The LM studies used paraffin-embedded livers with hematoxylin and eosin (H&E) staining. The immune-EM studies used antibodies labeled with sub-nanometer gold particles followed by silver enhancement to identify the location of injected pDNA at the subcellular level. The level of overall damage to liver cells was estimated based on alanine aminotransferase (ALT) release and clearance., Results: Both the LM and EM results showed the appearance of large vesicles in hepatocytes as early as 5 min post-injection. The number of vesicles decreased by 20-60 min. Plasmid DNA molecules often appeared to be associated with or inside such vesicles. DNA could also be detected in the space of Disse, in the cytoplasm and in nuclei. Non-parenchymal cells also contained DNA, but HTV-induced vesicles could not be observed in them., Conclusions: Our studies suggest an alternative or additional pathway for naked DNA into hepatocytes besides direct entry via membrane pores. It may be difficult to prove which of these pathways lead to gene expression, but the membrane pore hypothesis alone appears insufficient to explain why expression happens preferentially in hepatocytes. Further study is needed to delineate the importance of each of these putative pathways and their interrelationship in enabling oligonucleotide (siRNA) activity and pDNA expression., (Copyright (c) 2006 John Wiley & Sons, Ltd.)

Background: The hydrodynamic tail vein (HTV) injection of naked plasmid DNA is a simple yet effective in vivo gene delivery method into hepatocytes. It is increasingly being used as a research tool to elucidate mechanisms of gene expression and the role of genes and their cognate proteins in the pathogenesis of disease in animal models. A greater understanding of its mechanism will aid these efforts and has relevance to macromolecular and nucleic acid delivery in general., Methods: In an attempt to explore how naked DNA enters hepatocytes the fate of a variety of molecules and particles was followed over a 24-h time frame using fluorescence microscopy. The uptake of some of these compounds was correlated with marker gene expression from a co-injected plasmid DNA. In addition, the uptake of the injected compounds was correlated with the histologic appearance of hepatocytes., Results: Out of the large number of nucleic acids, peptides, proteins, inert polymers and small molecules that we tested, most were efficiently delivered into hepatocytes independently of their size and charge. Even T7 phage and highly charged DNA/protein complexes of 60-100 nm in size were able to enter the cytoplasm. In animals co-injected with an enhanced yellow fluorescent protein (EYFP) expression vector and fluorescently labeled immunoglobulin (IgG), hepatocytes flooded with large amounts of IgG appeared permanently damaged and did not express EYFP-Nuc. Hepatocytes expressing EYFP had only slight IgG uptake. In contrast, when an EYFP expression vector was co-injected with a fluorescently labeled 200-bp linear DNA fragment, both were mostly (in 91% of the observed cells) co-localized to the same hepatocytes 24 h later., Conclusions: The appearance of permanently damaged cells with increased uptake of some molecules such as endogenous IgG raised the possibility that a molecule could be present in a hepatocyte but its transport would not be indicative of the transport process that can lead to foreign gene expression. The HTV procedure enables the uptake of a variety of molecules (as previous studies also found), but the uptake process for some of these molecules may be associated with a more disruptive process to the hepatocytes that is not compatible with successful gene delivery., (Copyright (c) 2006 John Wiley & Sons, Ltd.)

The tracking of plasmid DNA (pDNA) movement within cells requires the attachment of labels to the DNA in a manner such that: (a) the pDNA remains intact during the labeling process and (b) the labels remain stably attached to the DNA. Keeping these two criteria in mind, we have recently developed a series of alkylating reagents that facilitate the one-step, covalent attachment of compounds directly onto nucleic acids in a nondestructive manner. Using these DNA-alkylating reagents, we have attached a wide range of both fluorescent and nonfluorescent reporter molecules onto pDNAs. We now show that even with the covalent attachment of various marker compounds, the pDNA remains expression competent. The ability to create labeled, expression-competent DNA allows for the simultaneous tracking of both pDNA location and reporter gene expression within living or fixed cells.

DNA complexes of spermine and spermidine become resolubilized at very high concentrations of the oligoamine. It has been postulated that high oligoamine concentrations shift the DNA from the globule back to the coil phase. The present study indicates that DNA resolubilization at high concentrations of spermine and spermidine is explained by formation of small particles of condensed DNA that cannot be precipitated by centrifugation. The fact that DNA stays condensed during resolubilization was confirmed using a relatively new condensation assay and three independent microscopic techniques. A considerable portion of DNA was found to be in particles with diameter <100 nm. Formation of such small particles is likely to be caused by colloidal forces. The ability to form small, condensed DNA particles in solutions that contain high concentrations of oligocation should aid in the design of synthetic DNA vectors for gene transfer and gene therapy and in the handling of DNA for diagnostic studies.

The intravenous delivery of plasmid DNA complexed with either cationic lipids (CL) or polyethyleneimine (PEI) enables high levels of foreign gene expression in lung. However, these cationic DNA complexes cause substantial toxicity. The present study found that the inclusion of polyacrylic acid (pAA) with DNA/polycation and DNA/CL complexes prevented the serum inhibition of the transfection complexes in cultured cells. The mechanism mediating this increase seems to involve both particle size enlargement due to flocculation and electrostatic shielding from opsonizing serum proteins. The use of pAA also increased the levels of lung expression in mice in vivo substantially above the levels achieved with just binary complexes of DNA and linear PEI (lPEI) or CL and reduced their toxicity. Also, the use of a "chaser" injection of pAA 30 min after injection of the ternary DNA/lPEI/pAA complexes further aided this effort to reduce toxicity while not affecting foreign gene expression. By optimizing the amount of pAA, lPEI, and DNA within the ternary complexes and using the "chaser" injection, substantial levels of lung expression were obtained while avoiding adverse effects in lung or liver. These developments will aid the use of cationic DNA complexes in animals and for eventual human gene therapy.

DNA condensation and compaction is induced by a variety of condensing agents such as polycations. The present study analyzed the structure of plasmid DNA (DNA) in the small inner space of reverse micelles formed from nonionic surfactants (isotropic phase). Spectroscopic studies indicated that DNA was dissolved in an organic solvent in the presence of a neutral detergent. Fluorescent quenching of ethidium bromide and of rhodamine covalently attached to DNA suggested that the DNA within neutral, reverse micelles was condensed. Circular dichroism indicated that the DNA structure was C form (member of B family) and not the dehydrated A form. Concordantly, NMR experiments indicated that the reverse micelles contained a pool of free water, even at a ratio of water to surfactant (Wo) of 3.75. Electron microscopic analysis also indicated that the DNA was in a ring-like structure, probably toroids. Atomic force microscopic images also revealed small, compact particles after the condensed DNA structures were preserved using an innovative cross-linking strategy. In the lamellar phase, the DNA was configured in long strands that were 20 nm in diameter. Interestingly, such DNA structures, reminiscent of "nanowires," have apparently not been previously observed.

A fluorescent method is described for assessing nuclease activity. The technique is based on the preparation of quenched fluorophore-nucleic acid covalent conjugates and their subsequent dequenching due to degradation by nucleases. The resulting fluorescence increase can be measured by a spectrofluorometer and exhibits subpicogram per milliliter sensitivity level for RNase A and low picogram per milliliter level for DNase I. The method is adaptable for quantitative nuclease inhibitor testing., ((c)2001 Elsevier Science.)

Growing endothelial cells at sites of angiogenesis may be more sensitive than quiescent endothelial cells to toxin-VEGF fusion proteins, because they express higher numbers of VEGF receptors. We have constructed, expressed and purified a protein containing the catalytic A-subunit of Shiga-like toxin I fused to VEGF(121) (SLT-VEGF/L). SLT-VEGF/L inhibits protein synthesis in a cell-free translation system and induces VEGFR-2 tyrosine autophosphorylation in cells overexpressing VEGFR-2 indicating that both SLT and VEGF moieties are properly folded in the fusion protein. SLT-VEGF/L selectively inhibits growth of porcine endothelial cells expressing 2-3x10(5) VEGFR-2/cell with an IC(50) of 0.1 nM, and rapidly induces apoptosis at concentrations >1 nM. Similar results are observed with human transformed embryonic kidney cells, 293, engineered to express 2.5x10(6) VEGFR-2/cell. In contrast, SLT-VEGF/L does not affect three different types of endothelial cells (PAE/KDR(low), HUVE, MS1) expressing between 5x10(3) and 5x10(4) VEGFR-2/cell, and quiescent endothelial cells overexpressing VEGFR-2. Growth inhibition and induction of apoptosis by SLT-VEGF/L require intrinsic N-glycosidase activity of the SLT moiety, but occur without significant inhibition of protein synthesis. The selective cytotoxicity of SLT-VEGF proteins against growing endothelial cells overexpressing VEGFR-2 suggests that they may be useful in targeting similar cells at sites of angiogenesis.

DNA can be condensed with an excess of poly-cations in aqueous solutions forming stable particles of submicron size with positive surface charge. This charge surplus can be used to deposit alternating layers of polyanions and polycations on the surface surrounding the core of condensed DNA. Using poly-L-lysine (PLL) and succinylated PLL (SPLL) as polycation and polyanion, respectively, we demonstrated layer-by-layer architecture of the particles. Polyanions with a shorter carboxyl/backbone distance tend to disassemble binary DNA/PLL complexes by displacing DNA while polyanions with a longer carboxyl/backbone distance effectively formed a tertiary complex. The zeta potential of such complexes became negative, indicating effective surface recharging. The charge stoichiometry of the DNA/PLL/SPLL complex was found to be close to 1:1:1, resembling poly-electrolyte complexes layered on macrosurfaces. Recharged particles containing condensed plasmid DNA may find applications as non-viral gene delivery vectors.

The assembly of DNA into compact particles that do not aggregate in physiologic salt solution occurs naturally in chromatin and viral particles but has been challenging to duplicate using artificial constructs. Cross-linking amino-containing polycations in the presence of DNA with bisimidoester cross-linker leads to the formation of caged DNA particles that are stable in salt solutions. This first demonstration of caged DNA provides insight into how natural condensation processes avoid aggregation and a promising avenue for developing nonviral gene therapy vectors.

A fluorescent method is proposed for assessing DNA condensation in aqueous solutions with variety of condensing agents. The technique is based on the effect of concentration-dependent self-quenching of covalently bound fluorophores upon DNA collapse. The method allows a more precise determination of charge equivalency in titration experiments with various polycations. The technique's ability to determine the number of DNA molecules that are condensed together in close proximity is under further investigation., (Copyright 1999 Academic Press.)

The self-assembly of supramolecular complexes of nucleic acids and polymers is of relevance to several biological processes including viral and chromatin formation as well as gene therapy vector design. We now show that template polymerization facilitates condensation of DNA into particles that are <150 nm in diameter. Inclusion of a poly(ethylene glycol)-containing monomer prevents aggregation of these particles. The DNA within the particles remains biologically active and can express foreign genes in cells. The formation or breakage of covalent bonds has until now not been employed to compact DNA into artificial particles.

In the present work it is shown that large unilamellar lecithin/cholesterol liposomes are able to sequester small negatively charged liposomes in the presence of divalent cations. Evidence is presented suggesting that the sequestration occurs via the formation of membrane invaginations transformed further into intraliposomal vesicles.

The results of studies on interaction of oligonucleotides and polynucleotides with cell membranes are reviewed. Oligonucleotides and polynucleotides bind to lipid membranes in the presence of divalent cations that may result in spontaneous encapsulation of nucleic acids and transfer of the formed vesicles to the other side of the membrane. Oligonucleotides can enter eukaryotic cells and interact with cellular RNA and DNA. On the surface of eukaryotic cells, there are proteins capable of binding to nucleic acids that may be involved in oligonucleotide uptake. Oligonucleotides bind to cellular CD4 receptors. Efficient delivery into cells can be achieved by conjugation of oligonucleotides to lipophilic groups or by encapsulation into membrane carriers.

During experimental CCl4 cirrhosis, an increase of membrane-associated factor stimulating 3T3 cell proliferation in vitro was observed. This stimulator is a 150-kD protein similar to one previously described. In situ perfusion released growth stimulatory activity, suggesting a peripheral plasma membrane protein localizing on basolateral surfaces. The activity increased with increasing number of CCl4 treatments, reaching a maximum at the 14th intoxication. It was faster than the proliferation of connective tissues determined histologically. Cessation of treatment caused a decrease in activity to that of the level of untreated liver, although the number of fibroblastlike cells remained large. This data, taken with the results of experiments with enriched hepatocyte fraction, may serve as an evidence in favor of hepatocyte origin of the factor. A factor inhibiting fibroblast proliferation was measured in detergent extracts from membranes, suggesting an integral membrane protein. The activity of the inhibitory factor increased in acute liver lesions, but at the stage of maximal fibrogenesis this factor is reduced to levels comparable to those of the intact liver. Therefore it is unlikely that this factor is involved in CCl4-induced fibrogenesis at the final stages. These factors may be common controls for various hepatic lesions causing fibrosis, both in clinical and experimental modeling.

The outer surface of isolated metaphase chromosomes has been investigated by a method of thermally activated tritium labelling. We show that both chromosomal proteins and DNA are tritium-labelled. Fractionation of the chromosomal proteins reveals that scaffold proteins are the most labelled in condensed and EDTA-decondensed chromosomes. Exposition of some scaffold proteins on the outer surface of metaphase chromosomes is suggested.

Isolated mitotic chromosomes are able to form complexes with phosphatidylcholine liposomes in the presence and absence of Ca2+ ions, in the latter case in the presence of polyamines. Interactions with chromosomes stimulates liposome fusion. The fusion is promoted by condensed and EDTA-decondensed chromosomes.

In the course of experimental CCl4-induced cirrhosis, an increase of the membrane-associated factor stimulating 3T3 cells' proliferation in vitro was observed. Gel filtration showed an approximate molecular mass of 150 kDa. Extraction of growth stimulatory activity by liver perfusion in situ demonstrated a peripheral plasma membrane protein localization. The activity increased with an increasing number of CCl4 treatments, reaching a maximum at the tenth intoxication, faster than the proliferation of connective tissues. Cessation of treatment caused a decrease in activity to the level of untreated liver, although the amount of fibroblast-like cells remained large, which is evidence in favour of an hepatocyte origin of the factor.

High molecular mass DNA was efficiently taken up by large unilamellar vesicles exposed to a short pulse of electric field (0.1-1 ms) with an intensity as high as 12.5 kV/cm. The efficiency of uptake increased significantly in presence of Mg2+ ions and was approximately 0.6 and 1.5 micrograms of DNA per mumol of lipid for T7 DNA and plasmid pBR 322, respectively. The results presented indicate that DNA was taken up as a result of the electrostimulated formation of endosome-like vesicles rather than via field-induced membrane pores.

Polynucleotides adsorb on natural and model phospholipid membranes in the presence of Mg2+-cations. Adsorption of nucleic acids on membranes results in a considerable change of their secondary structure. The presence of model phosphatidylcholine membranes greatly stimulates the rate of the synthesis of RNA by E. coli RNA-polymerase on DNA template.

IR-spectra of poly-A phosphatidylcholine complexes in D2O were studied. It has been shown that in the absence of Mg2+ the spectrum was a superposition of spectra of poly-A and phosphatidylcholine. Introduction of MgCl2 to the concentration of 0.03 M led to the dramatic decrease of absorbance at 1629 cm-1 characteristic of backbone vibration of adenine. Further increase of the concentration of MgCl2 up to 0.08 M resulted in the additional absorbance maximum at 1627 cm-1. Under a variety of temperatures the position of the maximum did not change. It was assumed that on adding magnesium ions to the "poly-A-lipid" mixture in D2O the components formed a stable complex. At high Mg2+ concentration a partial disarrangement of stacking interactions in poly-A occurred that might be a consequence of the incorporation of nucleic bases into hydrophobic membrane phase.

A new method of the coating of isolated cell nuclei with additional phosphatidylcholine membranes is described. The additional membrane was visualized under the electron microscope using ferritin as a label. The coating diminished leakage of the content of the nuclei and restricted permeability of the nuclei for exterior macromolecules.

The antimetastatic effects of two drugs, cis-diamminedichloroplatinum(II) (DDP) and hydrocortisone (liposome-incorporated or free), were studied. The experimental models were regional and distant metastases of hepatoma A and pulmonary adenocarcinoma, which were transplanted into the footpads of A/He mice. Liposomes were prepared from phosphatidylcholine by sonic dispersion. DDP and hydrocortisone were injected sc into the region of the plantar aponeurosis of the foot with the primary tumor. This administration route was considered to be equivalent to the intralymphatic route. Evidence indicated that only liposome-incorporated DDP and hydrocortisone decreased significantly the frequency and growth rate of tumor metastases in the regional lymph nodes. The effect observed was not due to the direct action of the drugs on the primary tumors. When nonencapsulated, these drugs were ineffective. Both liposome-encapsulated and free DDP did not affect distant metastases of pulmonary adenocarcinoma. The intralymphatic administration of liposome-encapsulated antitumor agents is suggested as a method for the prophylactic treatment of tumor metastases in the lymph nodes.

A method for gene transfer by means of interphase nuclei encapsulated within lipid membranes was developed. The method was based on passage of interphase nuclei through a layer of organic solvents containing phospholipids. Evidence was obtained indicating that the nuclei become surrounded by a protective phospholipid membrane: measurements of bound labelled or non-labelled phospholipids; decrease in the permeability of lipid-encapsulated nuclei for high molecular compounds; visualization by direct electron microscopy. Lipid-encapsulated nuclei of mink fibroblasts were used for transformation of mutant mouse LMTK- cells (deficient for thymidine kinase). The frequency of occurrence of HAT-resistant colonies/recipient cell was 1.9 X 10(-5). Biochemical analysis of 14 independent clones demonstrated that they all contained TK1 of mink origin. Analysis of 15 other biochemical markers located on 12 of the mink chromosomes revealed the activities of mink galactokinase (a syntenic marker) in 5 transformed clones, and that of mink aconitase-1 (the marker of mink chromosome 12) in 1 clone. No cytogenetically visible donor chromosomes were identified in the transformed clones. Nine transformed clones were tested for the stability of the TK+ phenotype; of these, the phenotype was expressed stably in 3 and unstably in 6. The method suggested is similar to the gene transfer procedure using total DNA. Its advantage is in ensuring efficient gene transfer and donor DNA integrity.

The interaction of the isolated chromosomes with the model phospholipid membranes in the presence of divalent cations leads to chromosomal decondensation as we showed previously by using different methods. In this manuscript we report data concerning the mechanism of such membrane-promoted decondensation. Under the action of liposomes at least three proteins are released from the chromosomes. The appearance of these chromosomal proteins was detected by SDS-electrophoresis of the fraction of the liposomes after dissociation of the complexes. One of these proteins with MW 33 kDa is tightly bound with the liposomes while the other two with MW 25 and 68 kDa release without binding. We suggested that the chromosomal decondensation at mitosis is caused by the action of the newly formed nuclear envelope, in particular by its membrane part which is able to provide the release of chromosomal proteins responsible for keeping the chromosomes in the compact state.

1. Two new methods are proposed for enhancement of the binding of hydrophilic proteins by liposomes. 2. An alkylating derivative of phosphatidic acid has been obtained by its reaction with N,N,N'-tris(2-chloroethyl)-N'-(p-formylphenyl)propylene-1,3-diamine. The alkylating activity of this derivative is very low due to the electron-acceptor effect of the formyl residue. Phosphatidylcholine liposomes which contain this alkylating derivative in the lipid bilayer may be obtained. The compound residing in the outer monolayer may be reduced by NaBH4. Upon reduction, the formyl residue is transformed into a hydroxymethyl residue. Therefore, the alkylating group of the compound is activated, and proteins may be attached covalently to the outer monolayer by alkylation with such chemically reactive liposomes. 3. Reaction of alkylating liposomes with myoglobin results in covalent binding of this hydrophilic protein. Complement-mediated leakage of such myoglobin-carrying liposomes may be induced by antibodies against myoglobin. 4. Modification of hydrophilic proteins with dansyl chloride results, even at small extents of modification, in a dramatic increase of the affinity of such proteins to phosphatidylcholine liposomes.

4-(N-2-chloroethyl-N-methylamino)benzylamide of 5'-heptaadenylic acid was used for affinity labelling of the ribosome in the vicinity of its mRNA-binding centre. This derivative, similar to the free oligonucleotide, stimulates the binding of [14C]-lysyl-tRNA to ribosomes of E. coli and alkylates ribosomes both the 30S and the 50S subunits. The alkylation of ribosomes is inhibited by pre-incubation of ribosomes with polyadenylic acid, which suggests that the chemical modification is a specific one and occurs in the vicinity of mRNA-binding site. The fact, that a short oligonucleotide having an active group on its 5'end attacks the 50S subunit of ribosome may indicate that the mRNA-binding centre is located in the contact region between ribosomal subunits.

A/HeJ mice with experimental metastases of HA-1 tumor in the liver and other organs were given therapeutic doses of cisplatin, free or encapsulated in phosphatidylcholine cholesterol freeze-thawed liposomes, intravenously. Free cisplatin treatment was found to decrease the growth rate of liver metastases by half and to extend survival by 1.5 times as compared to controls. The inhibitory effect of liposome-encapsulated cisplatin on hepatic metastases growth was more pronounced than that of the free drug although it weakly affected metastatic growth in other organs.

Cells of lines of mice transplantable tumours metastasizing into the lung have been used: lung adenocarcinoma (AL), Lewis lung carcinoma (LL), melanoma B-16 (B-16), mammary tumour MMT-1 (MMT-1), malignant subline of L-cells (LS). Hybrid vesicles were obtained for each tumour. They contain fragments of cell plasmatic membranes (PM). It has been shown that AL-, LL-, LS- and B-16-liposomes were accumulated in lung, the trapping of AL- and LL-liposomes being higher than that of the other hybrid vesicles. Despite the similar dynamics and frequency of metastatic spreading into the lung for all tumours studied, no trapping of MMT-1-vesicles in the target-organ was observed. The role of specific interaction of tumour cells PM with the endothelium of the lung capillaries in the process of organotropic metastatic spreading is discussed.

Interaction of DNA with eukaryotic cells under conditions similar to those providing DNA adsorption onto liposomes was studied. It was revealed that mouse fibroblasts (line A9) and myeloma cells bind phage and plasmid DNA in 0.3 M sucrose solution containing Mg2+-ions. Additional pretreatment of the cells by trypsin did not affect DNA adsorption efficiency. The major part of the adsorbed DNA recovered by salt treatment of the cells, but 10-15% of DNA was found to be irreversible. Up to 50% of the irreversibly bound DNA molecules retain their linear size after treatment of cells with DNAse I. Efficiencies of DNA adsorption and irreversibly binding depend on the concentration of Mg2+ in the medium. The process of DNA irreversible binding is not inhibited by drugs affecting cell metabolism. It is assumed that DNA adsorbs onto the phospholipid domains of the cell membrane, and part of the adsorbed DNA is taken up into the interior of the cells.

4-(N-2-chloroethyl-N-methylamino)-benzaldehyde acetyl derivatives of penta-., hexa, hepta-, octauridylates were used for localization of the structures organizing the mRNA-binding centre of ribosomes. These derivatives, alike free oligonucleotides, stimulate the binding of phenylalanyl-tRNA to ribosomes. Within the specific complex all the oligonucleotide derivatives alkylated the 30S ribosomal subunit. Octauridylate and hexauridylate derivatives specifically alkylated also the 50S subunit of ribosomes. Polyuridylic acid protected ribosomal subunits from alkylation. In the 30S subunit the derivatives modify 16S RNA and proteins: S4, S5, S7, S9, S13, S15, S18, S21. It was found that oligouridylate derivatives of different length alkylate different proteins.

The interaction between labelled polynucleotides and the mitochondrial membrane was studied. It was shown that DNA and mRNA form a complex with the mitochondria and mitoplasts (i. e. mitochondria devoid of the outer membrane); this process does not depend on the energy state of mitochondria. Within the complex polynucleotides are accessible to the effects of nucleases. The complex formation strongly depends on the concentration of Mg2+ and is inhibited by Na+. It was found that polynucleotides are effectively bound to protein-free artificial membranes isolated from the total mitochondrial lipids. The data obtained suggest that in the presence of Mg2+ polynucleotides form a complex with the lipid components of the mitochondrial membrane.

In the present paper the interaction of metaphase chromosomes and chromatin with model and natural lipid membranes was studied. It was shown that chromatin and chromosomes are able to form complexes with membranes in the presence of divalent cations. In such complexes, the typical structure of chromosomes is altered. The character of this alteration in chromosomal structure was investigated with the use of electron microscopy and chemical modification with dimethylsulphate (DMS). The latter is possible because, according to the presented data, the condensation of chromatin into chromosomes is associated with a decrease in accessibility of N-3 in adenine (the protection of the minor groove of DNA) to modifications, and with an increased methylation of N-1 in adenine (the disarrangement of the secondary structure of DNA). It was shown that the interaction of chromosomes with liposomes provides various levels of unfolding up to the appearance of chromatin-like structures. The secondary DNA structure of decondensed chromosomes coincides with the secondary structure of chromosomal but not chromatin DNA, whereas the extent of shielding of the minor groove of DNA in such decondensed structures typical for chromatin DNA. It is possible to suggest that the chromosomal decondensation in telophase of mitosis is initiated by the action of a membrane component of the developing nuclear envelope.

The Cu(Lys)2 complex is shown to be an effective inhibitor of the oxidation of 3,4-benzpyrene and aniline in microsomes. Apparently, this is due to the dismutation of superoxide radicals involved in the metabolism of these compounds. Possible interactions between Cu(Lys)2 and model membranes have been studied using spin probes. A model has been suggested according to which Cu(Lys)2 "builds" in the membrane hydrocarbon regions.

The fluorescence of the Tb3+ complex with metaphase chromosomes of mink fibroblasts was studied. It was shown that the fluorescence intensity of this complex is 6.5 times as high as that of the Tb3+-native DNA complex. The fluorescence of the chromosome-Tb3+ complex is predominantly determined by chromosomal DNA. The high intensity of fluorescence may be due to partial disturbances in the secondary structure of DNA during folding of the metaphasic chromosome.

The effect of some olygo- and polynucleotides on the dissociation rate of the 14C-aminoacyl-tRNA - ribosome complex was investigated. Polyuridylic and polycytidylic acids were shown to accelerate significantly dissociation of the complex of lysyl- and phenylalanyl-tRNA with native ribosomes, but not to affect the complexes of these aminoacyl-tRNA's with 50S subunits. It is proposed that the template polynucleotides decrease the affinity of ribosomes to tRNA by association with the mRNA-binding site on 30S subunits.