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The development of new film-forming compositions based on polyurethane ureas and dacarbazine will make it possible to obtain new polymer systems for local prolonged release of an anticancer drug as adjuncts to anticancer therapy. To solve the problem composite materials with 1 wt. % dacarbazine (DK) on the basis of polyurethane ureas (PUU) containing in the structure as macrochain extenders of 2-(2-aminoethoxy)ethan-1-amine, (DA1) 3,6-dioxaoctane-1,8-diamine (DA2) 3-{2-[2-(3-aminopropoxy)ethoxy]ethoxy}propan-1-amine (DA3) with a molar ratio of 4,4'-diaminodiphenylmethane (DADPh) to DA1, DA2 and DA3 as 0,7:0,3; 0,3:0,7; 0,7:0,3 were obtained The tensile strength of the compositions is within (1.21-1.27) MPa, the relative elongation is (303.9-384,2)%. Studies of thermophysical properties of synthesized PUU by DSC, TGA methods were conducted. Dacarbazine compositions are single-phase systems with glass transition temperature (Tg) from -33.55 °C to -37.06 °C. It was established that the introduction of Dacarbazine into the composition of PUU leads to a decrease in Tg and ΔCp during the second warm-up in comparison with the original PUU. Dacarbazine compositions are resistant to temperatures of 270 °C, which allows for thermal sterilization before use. The release of Dacarbazine from polymer samples into the solution was studied by the spectrophotometric method. According to the results of the study of the dynamics release of Dacarbazine, it was es tablished that for (PUU DA1)2+DK in 14 days it is 61.74 %, (PUU DA2)4+DK - 70.09 %, (PUU DA3)2+DK - 56.75 % of the total amount of immobilized DK. The resulting composites are perspective materials for medicine as means of local prolonged therapeutic action. [ABSTRACT FROM AUTHOR]

High resolution melting analysis (HRMA) using special "saturating" fluorescent dyes is a new and very effective approach to genotyping and mutation scanning. HRMA, which is carried out usually just after PCR without any intermediate manipulations (the "closed tube" format), is simple and high-throughput method excluding sample cross-contaminations. The "closed tube" format makes, however, HRMA dependent on PCR mixes and, as such, limits its capability. The "open tube" format (post-PCR amplicon shortening and optimization of the ionic medium) proposed by us earlier, although somewhat more laborious, significantly increases sensitivity of the method and makes it possible to scan mutations in the short amplicons using conventional SYBR Green I dye and a standard (not adapted specifically for HRMA) real-time PCR instrument. Detection of mutant K-RAS in DNA of clinical specimens (tumor tissues, formalin-fixed paraffin-embedded samples) reveals equal, at least, sensitivity of this method as compared with the HRMA and much higher as compared with Sanger sequencing. The problem of false-negative results in mutation scanning of K-RAS, which is highly important in some forms of cancer, is discussed.

Real-time RT-PCR using fluorescence dyes (e.g. SYBR Green I) is currently the most sensitive and precise method for investigation of RNA level and has long been widely used for absolute and relative quantification of mRNA in the cell. This highly sensitive method allows measurement of different type RNA level in the cell based on the kinetics of the corresponding double-stranded cDNA amplification. Upon its binding to the minor groove of double-stranded DNA, SYBR Green I dye increases its fluorescence about 100-fold, and this increase can be recorded even at early cycles of amplification. During the real-time RT-PCR procedure the level of amplified DNA is measured after every cycle of amplification, which permits to perform quantification at the cycles when amplification curve has not yet reached the "plateau" range and corresponds to the range of exponential increase in DNA amount. This approach makes it possible to avoid misinterpretation of data typical of conventional PCR methods "in the end point" and caused by a deficiency of one or more reaction components at the late PCR cycles. We applied for the first time real-time RT-PCR using SYBR Green I for the measurement of the class III genes RNA-product level, that is, small stable non-translated RNAs--ribosomal 5S rRNA, initiator transfer RNAiMet1, and Alu-RNA, synthesized by DNA-dependent RNA polymerase III. We investigated the level of 5S rRNA-, tRNA- and Alu-gene expression in the cell being in different states: with prolonged generation period, activated to proliferation, and apoptotic. The expression level was judged from the content of corresponding RNA-products in the total cellular RNA. The used approach enabled us to find out the specific RNA share in the total cell RNA. Human epidermoid carcinoma cells A431 were used as a model for investigating class III gene expression level in vivo. These cells expose on their surface an abnormally large amount of receptors to epidermoid growth factor (EGF), and the result of EGF action on A431 cells depends on the growth factor concentration. Low concentrations of EGF (0.1 ng/ml) cause active proliferation of A431 cells, but its high concentrations (10-100 ng/ml) cause apoptosis in these cells. Besides, upon growing in serum-free media, A431 cells continue to proliferate, but by this extending the generation period to 48 h, against 30 h on growing in serum-containing media. Hence, A431 cells can serve as a useful model for investigation of specific gene expression level in cells being in different physiological states, in both slowly and actively proliferating cells, and in apoptotic cells. For successful use of real-time RT-PCR in 5S rRNA, tRNAi(Met)1 and Alu-RNA level quantification, we optimized the amplification reaction conditions. We took into account that the share of each particular RNA in the cell may vary--the share of ribosomal RNA is high, tRNAi(Met)1--low, and Alu-RNA--very low. Moreover, the level of some small RNAs (e.g. Alu-RNA) can vary significantly in cells of different lines. This explains why the amount of cDNA, gained by reverse transcription of total cellular RNA, and the concentration of specific primers used for PCR were different in each case. We showed that the expression of different class III genes--5S rRNA-, tRNA- and Alu-genes, was not similarly regulated in response to external stimuli, causing prolongation of generation period, activation of proliferation and apoptosis. 5S rRNA level was practically the same in A431 cells both having prolonged generation period and being activated by EGF in low concentration, but in apoptotic cells this level dramatically fell about 8-fold. Alu-RNA level was equal in cells with prolonged generation period and in apoptotic cells, and increased about 2-fold in cells activated by EGF in low concentration. The initiator tRNAi(Met)1 level in cells activated by EGF in low concentration and in apoptotic cells was by almost two times higher than in cells with prolonged generation period. The data obtained testify that the real-time RT-PCR method using SYBR Green I yields highly reliable and reproducible quantification for the level of class III gene RNA-products--small stable RNAs (5S rRNA, tRNA and Alu-RNA). Examination of each specific RNA level requires individual selection for the amplification reaction conditions: the amount of cDNA and primer concentration in the sample. This is primarily caused by different expression levels in some particular class III genes within the frames of the cells, and by different levels of some small stable RNAs (e. g. Alu-RNA) in different cell lines. Special attention must be paid to the internal control for discriminating between specific RNA levels in proliferating and apoptotic cells, as in the late apoptosis RNAs of most types are degraded (for example, mRNA of "house-keeping" gene for RPLP0 protein, used as a possible internal control in our experiments). As far as the applied approach allows estimation of a specific RNA share in the total cellular RNA, we propose to chose as internal control mRNA, whose share doesn't change during the total RNA degradation in apoptosis and thus, mRNA degradation is not selective (in relation to other type RNAs). In that way, the real-time RT-PCR method, which is currently the most sensitive and precise method for quantification of RNA in the cell, holds much promise for the investigation of not only different mRNAs, but also small stable RNAs, synthesized by RNA polymerase III.

The article discusses the efficacy of using polymeric hemostatic wool and plate in operations on the lungs and pleura in experiments. From the results of experimental studies the authors concluded that a biodegradable polyamide based on hexamethylenediamine and derivatives of amino acids in a model of parenchymatous-capillary bleeding from the lungs and pleura is an effective hemostatic. The authors recommend these polymers for use in the clinic.

The method of pea ferredoxin (Fd) immobilization has been worked out. Under binding Fd with sepharose 4B activated by cyanogen bromide, a destruction took place in the Fd active center and apoprotein was bound with the matrix. The native immobilized Fd has been obtained when using P-300 biogel as a matrix. The gel was activated by glutaraldehyde and protein was linked with gel through a hexamethylenediamine spacer.

A two-step chemical method has been developed for the introduction of biotin and other low-molecular derivatives into DNA. The method is based on the interaction of aliphatic diamines with cytosine residues of the polynucleotide chain in the presence of sodium bisulfite with subsequent addition of biotin. DNA modified this way may be used as an effective probe for non-isotopic hybridization which can be used simultaneously with 32P-labelled probes.

DNA duplexes 14 bp long containing an EcoRII and MvaI restriction site in which a nucleoside is substituted by 1,3-diaminopropane or 1,3-propanediol residue have been chemically synthesized. Diaminopropane bridge was introduced by the chemical ligation, whereas the oligonucleotide containing propanediol was prepared by automatic solid phase phosphoroamidite method on "Victoria-4M" synthesizer. As CD and UV spectra show, the modification destabilises the duplex by 18-20 degrees C without essential distortion of the double helix, except for increase of the conformational mobility in the modified site.

The increase of uridine phosphorylation during the first hour after epidermal growth factor (EGF) stimulation (1.25 nM) of Swiss 3T3 cells is completely blocked by 100 microM dansylcadaverine (DC). Lack of the effect of DC on uridine transport, uridine kinase activity in cell homogenate, intracellular ATP concentration and plasma membrane permeability for phosphorylated uridine derivatives makes it possible to propose the inhibition by DC (100 microM) of the activated state of uridine kinase. The rapidity of the inhibition of EGF effect and the lack of influence of DC (in tested concentration) upon the clustering of EGF-receptor complexes, rate of their internalization (Sorkin, 1985; Nikol'skiĭ et al., 1987) and pH value of intracellular compartments (Sorkin et al., 1985; Teslenko et al., 1986) may suggest an association of DC inhibitory action with blocking of some steps of the receptor mediated endocytosis. Accumulation of DC in cell membranes, rather than in intracellular compartments with acidic pH, is a necessary factor for its blocking effect. Possibilities of DC action through the influence on calmodulin-dependent proteins or EGF-induced cell protein phosphorylation are discussed.

The chemical ligation method was used for directed introduction of aliphatic diamines or glycols into sugar-phosphate backbone of DNA. Via condensation of heptanucleotide derivatives at the terminal phosphate (aminoalkylamides or hydroxyalkyl esters) with the adjacent heptanucleotide on the corresponding template, 14 bp DNA duplexes containing residues of ethylene-, propylene- or hexamethylenediamine, as well as residues of ethylene- or propyleneglycol in one of its strands, were synthesised. In a similar way duplexes were obtained in which residues of the above diamines or glycols are substituted for a mononucleotide in one of the complementary strands. Examplified by synthesis of DNA duplexes containing ethylenediamine or ethyleneglycol residues, three methods of the phosphate group activation using carbodiimide, imidazolide and N-hydroxybenzotriazole ester were tested; the last method gave the highest yields and purity of the products. Yields of duplexes without nucleotide omissions were 50, 36 and 7% for aminoethyl, aminopropyl and aminohexylamides, and 20 and 17% for hydroxyethyl and hydroxypropyl esters, respectively, whereas duplexes with nucleotide omissions were synthesised with lower yields. Each of the modified DNA duplexes thus obtained contains a recognition site of EcoRII, SsoII or MvaI restriction nucleases, thus being a potential substrate of these enzymes.

For addressed modification of T7 phage DNA a polyalkylating derivative of T7 phage early transcript was used. The derivative was obtained by attaching polyfunctional alkylating agent N,N,N,'-tri(beta-chloroethyl)-N'-(p-formylphenyl) propylene diamine-1,3 to the transcript, their molecules being covalently bound by alkylating 4--5% of the RNA adenine and guanine residues. The polyalkylating RNA was used to form R-loops in the complementary site of T7 DNA to alkylate selectively the T7 phage early DNA. Alkylation of DNA by the modified transcript in the region of the R-loop led to the covalent binding of the transcript to the complementary site of DNA. The correct localization of this R-loop was confirmed by electron microscopy. The application of the results for the addressed mutagenesis and inactivation of selected genes is discussed.