Isolation and physico-chemical properties of a complex kinetoplast DNA associate from the cells of Trypanosoma lewisi]

An associate of kinetoplast DNA (k-DNA) was isolated from the cells of Trypanosoma lewisi and characterized in terms of its sedimentation properties, melting parameters and reassociation kinetics. Electron microscopy studies showed that k-DNA isolated is a complex associate of circular molecules. The contour length of minicircular molecules is 0.77 mkm. k-DNA contains sites enriched by AT-pairs; melting of native associate and k-DNA fragments in the presence of 6.5 M sodium perchlorate results in the appearance of six zones within the temperature range of 47-64 degrees C. Data from k-DNA reassociation studies suggest that k-DNA associate constitutent molecules differ in sizes and nucleotide sequences. k-DNA is found to consist of two components with molecular weights of 1.7 . 10(6) and 17.5 . 10(6), respectively.     

Development of the myoglobin sensor based on the surface plasmon resonance

Bioaffinic sensor based on the surface plasmon resonance (SPR) with golden layer as a transducer was developed. This sensor was applied for control of antigen-antibodies (Ag-Ab) interactions. For modification of transducer surface the spontaneously organized molecular assemblies of two types of mercaptan were formed. It was shown that the created bioaffinic sensor allowed to detect minimal concentration of antibodies, about 1 ng/ml, to the human heart myoglobin, which preliminary was immobilized on the sensitive surface. The particularities of the dynamics of protein interaction were observed. Duration of incubation with the biospecific component was no more than 5 minutes. After rupturing of specific binding it was possible to obtain initial signal again during 7-9 cycles.     

Method of orienting immobilized proteins on gold surface sensory modified with rhodanide

The behavior of the biomolecules near the charged surface depending on the pH level of the solution was studied by the method of surface plasmon resonance. It was shown, that the preliminary modification of the surface by thiocyanat generating the effective negative charge at the surface allowed to immobilize protein molecule orientation depending on their charge state. Proteins keep their biological properties and form the oriented monolayer in the wide range of pH.     

Analysis of the response of planar polarization interferometer to molecular layer formation: fibrinogen adsorption on silicon nitride surface.

The most sensitive optical method of interferometry was exploited for determination of changes in the refractive index following the adsorption of biological molecules onto the solid surface. Instead of having two waveguiding arms (the main and the reference) in traditional Mach-Zhender interferometer, two ortogonal TM and TE modes propagating through the SiO(2)-Si(3)N(4)-SiO(2) waveguide structure were employed in planar polarization interferometer (PPI). Multiperiodic PPI response was, therefore, formed due to the phase shift between TM and TE modes. A matrix simulation procedure was developed in order to investigate the influence of both the refractive index and molecular layer thickness on the PPI response. Nonspecifical binding of fibrinogen to silicon nitride surface was studied as a model object for PPI testing. The results obtained are in good agreement with the known information about fibrinogen adsorption on the different surfaces. An attempt to introduce the concept of 'surface molecular concentration and molecular polariziability' instead of 'molecular layer thickness and refractivity' was undertaken.     

Orientation of proteins on the sensor surface and optimization of their immobilization by prior protection of the active center

When studying biospecific interactions with application of surface plasmon resonance, one of the main problems is reagent proper orientation to the sensor surface. Due to rather high chemical activity of molecular receptor sites, the interaction between these areas and surface may become predominant. Here we propose a technique for prevention of such orientation of bioreceptors using soybean trypsin inhibitor STI as an example. To obtain oriented STI immobilization on a modified gold surface its active site has been previously blocked through interaction with its specific partner trypsin. After conjugate immobilization on the sensor surface the components were separated using a glycine buffer (pH 2.2).     

The regulation of L-type Ca2+ currents in cardiac myocytes of hibernating animals

The action of isoproterenol and BAY K 8644 on voltage-dependent Ca2+ currents in isolated ground squirrel cardiac myocytes was studied in two (active and hibernating) states of the animal. In cardiac myocytes of active animals the effect of both drugs was shown to depend on the holding potential. At Vh of about -50 mV both isoproterenol and BAY K 8644 increased the Ca2+ current and their action was additive. At Vh of about -20 mV, both drugs inhibited the Ca2+ current. In cardiac myocytes from hibernating animals, isoproterenol increased the Ca2+ current at any holding potentials, while the effect of BAY K 8644 did not differ significantly from its effect on active animals. The combined action of the two drugs caused the inhibition of the Ca2+ current at high holding potentials. In terms of the two-site Ca2+ channel model, this means that one of the two pathways of channel phosphorylation is blocked in hibernating animal cardiac cells, and BAY K 8644 restores this pathway.     

Surface enhanced IR absorption of nucleic acids from tumor cells: FTIR reflectance study

The data on surface enhanced IR absorption (SEIRA) of nucleic acids deposited on a metal substrate were obtained using FTIR in reflectance mode. A 200-400 A thick gold film on a glass plate was the metal substrate. The approximate enhancement factors of the SEIRA for different vibrations of nucleic acids in our experimental conditions were 3-5. The roughness of the Au surface was about 50 A. Application of this method to nucleic acids isolated from tumor cells revealed some possible peculiarities of their structural organization, namely, the appearance of unusual sugar and base conformations, modification of the phosphate backbone, redistribution of the H-bond net, and so forth. This method enhanced a set of the bands, which is impossible to observe in conventional IR geometry. The SEIRA spectra of the RNA from tumor cells showed more sensitivity to the grade of tumor malignancy than the spectra of the DNA. After application of the anticancer drug doxorubicin to sensitive and resistant strains, the DNA isolated from these strains had different spectral features, especially in the region of the phosphate I and II bands. As induced by anticancer drugs, the conformational changes in the DNA from resistant and sensitive cancer strains could be characterized with different levels of structure disordering.     

Two modes of longe-range orientation of DNA bases realized upon compaction.

Formation of compact particles from linear DNA-anthracycline complexes is accompanied by appearance of intense bands in the CD spectra in the region of absorption of DNA bases (UV-region) and in the region of absorption of anthracycline chromophores (visible region). The intense (positive or negative) bands in the region of anthracycline absorption demonstrate an ordered helical location of anthracycline molecules on the DNA template. This fact, in its turn, is related to formation of the DNA superstructure in PEG-containing water-salt solutions with a long-range orientation of nitrogen bases. Possible types of DNA superstructures and the relation between the local- and the long-range order of bases in the DNA superstructure are discussed.