The role of hydrophobic interactions in catalysis of RNA cleavage by 1,4-diazabicyclo[2.2.2]-octane based artificial ribonucleases

Molecular interactions of RNA cleaving compounds-conjugates of 1,4-diazabicyclo[2.2.2.]octane substituted at the bridge position with tetradecamethylene fragment and imidazole were investigated using light scattering and small angle x-ray scattering methods. The compounds are known to efficiently cleave RNA and one source of the activity could result from micellar catalysis. It was found that the compounds indeed are capable of forming complex aggregates in solution. However, maximal efficacy of RNA cleavage by the conjugates is observed at concentrations well below the concentration required for micelle formation.     

Effect of nucleotides on the oligomeric state of human lactoferrin

Lactoferrin (LF) is a multifunctional acute-phase protein involved in nonspecific defense against bacteria, viruses, and cancer diseases and is present in human barrier fluids, blood, and milk. Small-angle X-ray scattering (SAXS) and light scattering (LS) demonstrated for the first time that LF occurs in the form of oligomers, with a high monomer unit number in the solution. The degree of LF oligomerization depends on the LF concentration and the storage period of non-frozen neutral LF solutions. The average inertial radius of scattering particles (R _g) reaches 100–450 Å at LF concentrations comparable with those in human milk, while R _g of LF monomers is 26.7 Å. LF forms complexes with various nucleotides and hydrolyzes them. The addition of ATP or AMP to LF solutions accelerates LF oligomerization and increases R _g to 600–700 Å, regardless of the initial degree of LF oligomerization. According to the different models (sphere, plate, and cylinder) of LF aggregates, its complexes with such R _g presumably contain several tens to thousands of LF monomers. The possible role of oligomeric complexes in multiple biological functions of LF is discussed.     

Aggregation of tryptophanyl-tRNA synthetase depending on temperature. Study by a low-angle scatter x-ray method

By means of small angle X-ray scattering, an aggregation of beef pancreas Trp-tRNA synthetase (EC 6.1.1.2) was observed at physiological temperatures. A Trp-tRNA synthetase preparation which is homogeneous after PAGE in beta-ME-SDS was found to be heterogeneous in particle sizes even at low (4-8 degrees C) temperature. At heating up to 30-45 degrees C, the oligomer sizes increased as well as its proportion depending on the incubation time and temperature; very large aggregates were observed 10 times exceeding the sizes of initial particles. Cooling to 20 degrees C caused no disaggregation due to disulphide bond formation between associated subunits of Trp-tRNA synthetase. A hypothesis is proposed that the aggregation of bovine Trp-tRNA synthetase evaluated in vitro and not observed earlier with any aminoacyl-tRNA synthetases of unicellular organisms might serve as one of the mechanisms of its compartmentation in pancreas.     

Small-angle X-ray characterization of the nucleoprotein complexes resulting from DNA-induced oligomerization of HIV-1 integrase

HIV-1 integrase (IN) catalyses integration of a DNA copy of the viral genome into the host genome. Specific interactions between retroviral IN and long terminal repeats (LTR) are required for this insertion. To characterize quantitatively the influence of the determinants of DNA substrate specificity on the oligomerization status of IN, we used the small-angle X-ray scattering (SAXS) technique. Under certain conditions in the absence of ODNs IN existed only as monomers. IN preincubation with specific ODNs led mainly to formation of dimers, the relative amount of which correlated well with the increase in the enzyme activity in the 3′-processing reaction. Under these conditions, tetramers were scarce. Non-specific ODNs stimulated formation of catalytically inactive dimers and tetramers. Complexes of monomeric, dimeric and tetrameric forms of IN with specific and non-specific ODNs had varying radii of gyration (R_g), suggesting that the specific sequence-dependent formation of IN tetramers can probably occur by dimerization of two dimers of different structure. From our data we can conclude that the DNA-induced oligomerization of HIV-1 IN is probably of importance to provide substrate specificity and to increase the enzyme activity.     

The Effect of Tetrahydrocortisol–Apolipoprotein A-I Complex on the RNA Polymerase Interaction with Eukaryotic DNA and the Rate of Protein Biosynthesis in Hepatocytes

A mechanism of activation of protein biosynthesis in hepatocytes was proposed as effected by the conditioned medium of nonparenchymal liver cells incubated in the presence of high density lipoproteins, cortisol, and lipopolysaccharides. It was found that the increase in the biosynthesis rate was associated with the formation of the tetrahydrocortisol–apolipoprotein A-I (THC–apoA-I) complex in macrophages, which display 5- and 5-reductase activity and are constituents of nonparenchymal liver cell. Using the small-angle X-ray scattering technique, it was shown that the THC–apoA-I–eukaryotic DNA interaction may break hydrogen bonds between pairs of complementary nucleic bases and cause the formation of single-stranded DNA fragments capable of binding to DNA-dependent RNA polymerase. The interaction is highly cooperative and has a saturating mode, up to six enzyme molecules being bound per DNA molecule.     

Interaction of tetrahydrocortisol-apolipoprotein A-I complex with eukaryotic DNA and with single-stranded oligonucleotides

The complex formed by tetrahydrocortisol (THC) and apolipoprotein A-I (ApoAI) specifically interacts with eukaryotic DNA from rat liver. Taken together, physical and chemical data and the results of small-angle X-ray scattering analysis show that interaction of the THC-ApoAI complex with eukaryotic DNA results in deformation of the DNA double helix. Single-stranded fragments were demonstrated to cause deformation of the double helix. In this state DNA forms complexes with DNA-dependent RNA polymerase. This interaction is cooperative and of saturating type; up to six enzyme molecules bind with one DNA molecule. The putative site of complex binding with DNA is the sequence CC(GCC)n found in many genes including the human ApoAI gene. An oligonucleotide of this type was synthesized. Its association constant (Ka) was 1.66 x 10(6) M-1. Substitution of THC with cortysol considerably decreases the Ka. We suggest that THC interacting with GC pairs of the binding site forms hydrogen bonds with cytosine, inducing rupture of the bonds within the complementary nucleic base pair.     

Features of interaction of complexes cortisol-apolipoprotein A-I and tetrahydrocortisol-apolipoprotein A-I with eukariotic DNA

On primary culture of hepatocytes it is shown, that a complex cortisol-apolipoprotein A-I did not change rate of biosynthesis DNA and protein, whereas the complex tetrahydrocortisol-apolipoprotein A-I (THC-apoA-I) essentially raised rate of incorporation 3H-thymidine in DNA and 14C-leucine into protein. By a method of small-angle X-ray scattering it is shown, that appreciable interaction with eukariotic DNA is marked only in case of use of a complex THC-apoA-I, thus there is local fusion of DNA. The most probable region of interaction of the given complex with DNA is repetition (GCC)n the type, included in structure of many genes eukariot, including the human. It is synthesized oligonucleotid (duplex) of this type. It is shown, that at his interaction with complex THC-apoA-I there is a formation of more difficult complex, which breaks up with formation of complementary chains of oligonucleotides. The last also enter interaction with complex THC-apoA-I. It is given of kinetic this multiphasic process. Interaction of a complex cortisol-anoA-I with a duplex is less specific and does not result reduce in decay of the duplex and in formation of complementary oligonucleotides.     

Strong changes in lipoproteins and autoantibodies of blood serum of the Tundra Nency population as a result of environmental radiation on the territory they inhabit

As a result of large-scale nuclear tests on the Novaya Zemlya test site (1955-62) the Tundra Nentsy population of Yamal-Nentsy autonomous region (YNAR) fell under the constant influence of incorporated radioactive isotopes (137Cs and 90Sr). Therefore, it is very important to analyze a possible spectrum of diseases of Tundra Nentsy population.     

Characteristics of lipids imbalance in patients with tick-borne encephalitis

Using a novel approach, we have analyzed 30 parameters characterizing detailed spectrum and fractional content of LPs in plasma of patients with tick-borne encephalitis (TBE). The blood plasma of all TBE patients (30 patients), as compared with that of healthy individuals (120 patients), is characterized by decreased concentrations of many LP subfractions and of the total concentration of all plasma LPs (hypolipoproteinemia). The observed difference in some parameters was statistically significant. Using computer-assisted factor analysis, we have shown that according to these 30 parameters TBE patients are similar to patients with multiple sclerosis and systemic lupus erythematosus. The results provide grounds for using data on blood plasma LPs as additional criteria for diagnosis of TBE.