Role of changes in serum chitotriosidase activity in mice under conditions of hyperlipidemia and lipid-lowering effect of carboxymethylated (1-3)-ß-D-glycan.

Enhanced expression and activity of chitotriosidase in humans is regarded as a marker of atherosclerosis. However, it remains unclear, whether this increase is related to lipemia or enhanced secretion of the enzyme by activated macrophages in the atherosclerotic plaques. It was shown that acute lipemia in mice caused by single administration of poloxamer 407 (P-407) in a dose of 300 mg/kg is accompanied by an increase in serum chitotriosidase activity (24 h) that correlated with elevated content of total cholesterol and triglycerides. Preliminary administration of (1-3)-ß-D-glycan prevented the P-407-induced increase in chitotriosidase activity, probably due to the hypolipidemic action of (1-3)-ß-D-glycan. The relationship between changes in chitotriosidase activity with atherogenic fractions and subfractions of serum lipoproteins during lipemia is discussed.

Comparative characteristics of lipemia models induced by injections of Triton WR-1339 and poloxamer 407 in mice.

Lipemia modeled by injections of Triton WR-1339 (500 mg/kg) and poloxamer 407 (500 mg/kg) to mice were compared. LP fraction and subfraction compositions were compared by small-angle X-ray scattering on a diffractometer. Both compounds in the same dose caused a sharp increase in serum concentrations of total cholesterol (CH) and triglycerides (TG), the increases in response to poloxamer 407 being more pronounced. The differences in the models consisted in the levels of atherogenic fractions: CH-VLDL (subfractions CH-VLDL1-2) and CH-LDL, which were higher under the effect of poloxamer 407. Similar increases were observed for atherogenic fractions: TG-VLDL (subfractions TG-VLDL1-2) and TG-LDL (subfractions TG-LDL1-3). A specific feature of the model induced by poloxamer 407 was elevation of the concentrations of antiatherogenic CH-HDL and TG-HDL (subfractions CH-HDL2 and TG-HDL2). Both models exhibited high similarity, but changes in atherogenic fractions were more pronounced under the effect of poloxamer 407.

Synthesis of nanoscale TiO2 and study of the effect of their crystal structure on single cell response.

To study the effect of nanoscale titanium dioxide (TiO(2)) on cell responses, we synthesized four modifications of the TiO(2) (amorphous, anatase, brookite, and rutile) capable of keeping their physicochemical characteristics in a cell culture medium. The modifications of nanoscale TiO(2) were obtained by hydrolysis of TiCl(4) and Ti(i-OC(3)H(7))(4) (TIP) upon variation of the synthesis conditions; their textural, morphological, structural, and dispersion characteristics were examined by a set of physicochemical methods: XRD, BET, SAXS, DLS, AFM, SEM, and HR-TEM. The effect of synthesis conditions (nature of precursor, pH, temperature, and addition of a complexing agent) on the structural-dispersion properties of TiO(2) nanoparticles was studied. The hydrolysis methods providing the preparation of amorphous, anatase, brookite, and rutile modifications of TiO(2) nanoparticles 3-5 nm in size were selected. Examination of different forms of TiO(2) nanoparticles interaction with MDCK cells by transmission electron microscopy of ultrathin sections revealed different cell responses after treatment with different crystalline modifications and amorphous form of TiO(2). The obtained results allowed us to conclude that direct contact of the nanoparticles with cell plasma membrane is the primary and critical step of their interaction and defines a subsequent response of the cell.

Fractional composition of blood serum lipoproteins in mice and rats with Triton WR 1339-induced lipemia.

We compared fractional composition of blood serum lipoproteins (LP) in female ICR mice and Wistar rats induced by single administration of a nonionic detergent Triton WR 1339 in doses of 300 and 500 mg/kg. Lipemia in animals of both species was characterized by a sharp increase in the concentration of cholesterol and, particularly, of triglycerides in blood serum lipoproteins by the 24th hour after administration of the detergent. We revealed a significant increase in the concentrations of atherogenic VLDL cholesterol (due to VLDL2), intermediate density lipoproteins, and LDL. These changes were more pronounced in rats. The model of lipemia can be used to study the role of fractional composition of lipoproteins and, particularly, of triglycerides in the pathogenesis of atherosclerosis. Moreover, this model holds much promise for evaluation of the efficiency of hypolipidemic drugs (statins and fibrates) in normalizing the increased level of atherogenic cholesterol of VLDL and LDL.

[DNA and oligosaccharides stimulate oligomerization of human milk lactoferrin].

Lactoferrin (LF), the glycoprotein transferring Fe+ ions, is contained in barrier liquids, human blood and milk. LF is an acute phase protein and one of the most important factors of nonspecific defense. The protein has unique set of biological functions. Using the methods of small-angle X-ray scattering and light-scattering it was shown for the first time that addition of DNA and oligosaccharides to LF with different level of initial oligomerization leads to an enhance in the oligomerization rate. 1M NaCl stimulates almost a complete dissociation of LF oligomeric complexes obtained in the pesence of DNA, oligosaccharides, or early founded oligomerization effectors (nucleotides). It was shown that LF oligomeric complexes obtained in the presence of different oligomerization effectors have different stability. Incubation with 50 mM MgCl2 leads to complete destruction of the protein complexes formed by ATP and oligosaccharide but partially dissociate the complexes with following formation of new in the case of AMP- and d(pT)10-dependent associates, which possess higher stability i n presence of the salt. A possible role of LF oligomerization for different biological functions of the protein is discussed.

Changes in the secondary structure of highly polymeric DNA and CC(GCC)n-type oligonucleotides under the action of steroid hormones and their complexes with apolipoprotein A I.

A small-angle X-ray scattering study showed that the action of tetrahydrocortisol (THC) in complex with apolipoprotein A I (ApoA-I) on DNA leads to local melting of DNA. The most probable site of interaction between this complex and DNA is the (GCC)n-type sequence. Oligonucleotides (duplexes) of this type have been synthesized. It was demonstrated that the interaction of this oligonucleotide with the THC-ApoA-I complex leads to dissociation into complementary oligonucleotides. The latter ones also interact with the THC-ApoA-I complex. The kinetics of this multistep process is presented. The mechanism of interaction between hormones or their ApoA-I complexes and duplex CC(GCC)5.GG(CGG)5Li2 was studied using IR spectroscopy. It was shown that the interaction with THC or the THC-ApoA-I complex leads to the formation of hydrogen bonds between the OH group of the hormone A-ring and the C=O group of cytosine or guanine. Interaction with cortisol or the cortisol-ApoA-I complex leads to the formation of a hydrogen bond with the NH group of cytosine; in addition, THC and cortisol form hydrogen bonds with the PO2 group of the duplex and with the OH group of the monosaccharide. The interaction of ApoA-I with the duplex is accompanied by the formation of hydrogen bonds between the protein NH2 group and the C=O group of cytosine and the P=O group. The order-to-order structural transition takes place in the duplex under the action of THC or cortisol, with THC causing a higher ordering as compared to cortisol. The order-to-disorder structural transition occurs in the duplex under the action of the THC-ApoA-I, cortisol-ApoA-I, or ApoA-I complexes. Shifting the pH of the medium from 7.2 to 6.0 also leads to an order-to-disorder-type structural transition.

[Cold-induced changes in blood lipoproteins in normotensive and hypertensive rats].

It is shown that in thermoneutral conditions ISIAH (Inherited Stress-Induced Arterial Hypertension) hypertensive rats had a lower level of high-density lipoproteins (HDLP) in plasma and a higher atherogenic coefficient compared to normotensive Wistar rats. After cooling there were different changes in fractional composition of plasma lipoproteins both in normo- and hypertensive rats. These changes depended on the cooling rate and were more pronounced after slow cooling. Slow cooling resulted in a more significant increase of plasma HDLP and in a greater decrease in LDLP and atherogenic coefficient in hypertensive rats compared to normotensive ones.

[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.

[The effect of nucleotides on oligomeric state of human lactoferrin].

A polyfunctional protein lactoferrin (LF) which is present in human barrier fluids, blood and milk and this protein of acute phase is responsible for nonspecific cells defense against microbial and viral infection and cancer diseases. Using the methods of small-angle X-ray scattering and light-scattering it was shown that LF in solution exists in oligomeric state. The level of LF oligomerization depends upon its concentration and time of keeping of no frozen neutral protein solutions. At the concentrations comparable with those in human milk (1-6 mg/ml) the average inertial radius values (Rg) of LF can reach 100-450 angstroms, while Rg for monomer LF form is 26.7 angstroms. LF was shown to complex with different nucleotides and hydrolyze them. The addition of ATP and AMP to LF demonstrating any level of oligomerization leads to increase of oligomerization processes and enhancement of the Rg values up to 600-700 angstroms According to different models of LF monomer association to its oligomeric forms (sphere, plate, cylinder) the oligomeric complexes demonstrate high Rg values which can contain from several tens up to several thousands of LF monomers. A possible role of LF oligomerization for different biological functions of the protein is discussed.

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.

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.

Tetrahydrocortisol-apolipoprotein A-I complex specifically interacts with eukaryotic DNA and GCC elements of genes.

Tetrahydrocortisol stimulates DNA and protein biosynthesis in hepatocytes only when it enters the complex with apolipoprotein A-I. Tetrahydrocortisol-apolipoprotein A-I (THC-apoA-I) complex specifically interacts with eukaryotic DNA isolated from rat liver. In the process of interaction, rupture of hydrogen bonds between the pairs of nitrous bases occurs with the formation of single-stranded DNA structures. In such state DNA forms complexes with DNA-dependent RNA-polymerase. The most probable site of binding the tetrahydrocortisol-apolipoprotein A-I complex with DNA is the sequence of CC(GCC)(n) type entering the structure of many genes, among them the structure of human apolipoprotein A-I gene. Oligonucleotide of this type has been synthesized. Association constant (K(ass)) of it with tetrahydrocortisol-apolipoprotein A-I complex was shown to be 1.66 x 10(6)M(-1). Substitution of tetrahydrocortisol for cortisol in the complex results in a considerable decrease of K(ass). It was assumed that in the GC-pairs of the given sequence tetrahydrocortisol itself participates in the formation of hydrogen bonds with cytosine, favoring their rupture with complementary base-guanine.

Slow cooling improved blood lipoprotein composition in hypertensive rats.

Under normal thermal conditions, hypertensive NISAG rats are characterized by lower plasma levels of high-density lipoproteins and increased coefficient of atherogenicity compared to normotensive Wistar rats. Slow cooling significantly modified fractional composition of plasma lipoproteins in hypertensive rats: decreased the content of low-density lipoproteins, markedly increased the content of high-density lipoproteins, and normalized coefficient of atherogenicity. Our results demonstrated the possibility of correcting disturbances in lipoprotein spectrum in essential hypertension by using thermal exposures.

Effect of tetrahydrocortisol-apolipoprotein A-I complex on the secondary structure of eukaryotic DNA and its interaction with RNA-polymerase.

The in vitro effect of tetrahydrocortisol-apolipoprotein A-I complex on native adult rat liver DNA results in the formation of S1 nuclease sensitive fragments that are irregularly distributed throughout a genome. Low-angle X-ray scattering showed that after the interaction with the tetrahydrocortisol-apolipoprotein A-I complex, DNA can bind to RNA-polymerase with a high and dose-dependent cooperativity. This indicates that the effect of tetrahydrocortisol-apolipoprotein A-I complex on secondary eukaryotic DNA structure causes a local denaturation of the double helix, promoting high cooperativity of binding to RNA-polymerase. The reduced form of the hormone, tetrahydrocortisol, previously considered as an inactive metabolite, when complexed with apolipoprotein A-I, promotes a biological function similar to that of a transcription factor.

[Determination of fractional and subfractional blood lipoprotein compositions with small angle X-ray scattering (a comparison with a biochemical method)]. / Opredelenie fraktsionnogo i subfraktsionnogo sostavov lipoproteinov krovi metodom malouglovogo rentgenovskogo rasseianiia (sravnenie s biokhimicheskim metodom).

Highly accurate and fast method for determination of quantitative and qualitative compositions of serum lipoprotein (LP) fractions and subfractions, using small-angle X-ray scattering on the "Siemens" difractometr, has been developed. The method allows to determine the concentrations of serum cholesterol (CH), triglycerides (TG), high-density lipoprotein (HDL)-CH, low-density lipoprotein (LDL)-CH, phospholipides (FL) and total lipids. It allows to determine the levels of CH, TG, FL in LP subfractions and concentrations of 5 very-low-density lipoprotein, intermedius-density lipoprotein, 3 LDL (large, intermedius and small dense particles) and 5 HDL subfractions. The method is based on the elaborated united model of all LP fractions and subfractions as a result of generalization and analysis of literature data about LP composition and size. New method was proved to be highly reproductive. Strong positive correlation exists between serum levels of CH, TG, HDL-CH determined by enzymatic biochemical and our methods. The method is simple to perform and of high diagnostic value, and can, therefore, be applied to clinical purposes.

[Interaction of tetrahydrocortisol-apolipoprotein A-I complex with eukaryotic DNA and with single-stranded oligonucleotides]. / Vzaimodeistvie kompleksa tetragidrokortizol-apolipoprotein A-I c éukarioticheskoi DNK i odnotsepochechnymi oligonukleotidami.

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.

[Assessment of the status of biological health of the population of tundra Nentsy inhabiting northwestern Siberia from data on analysis of autoantibodies and lipoproteins from blood donors]. / Otsenka sostoianiia biologicheskogo zdorov'ia populiatsii tundrovykh nentsev Iamalo-Nenetskogo avtonomnogo okruga po dannym analiza autoantitel i lipoproteidov krovi donorov.

As a result of large-scale nuclear explosion on Novaya Zemlya test site (1955-62) the Tundra Nentsy population of Yamal-Nentsy autonomous region (YNAR) fell under 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. We have developed recently a new method for determination of concentrations of all main fraction and subfraction of lipoproteins (LP, 30 parameters) in human sera using small-angle X-ray scattering, and a general mathematical model to describe LP composition in human blood. The analysis of the 30 parameters characterizing fine spectrum of LPs in 374 YNAR natives showed that only approximately 10% of the donors are normal, while the indices for approximately 90% of the test subjects fall into the range of different pathologies (3-8% incidence in normal population, according to epidemiological studies). Moreover, we found that approximately 41% of European and approximately 56% of Tundra Nentsy have high level of autoantibodies to DNA and cardiolopine like the same for autoimmune diseases patients.

[Effect of tetrahydrocortisol-apolipoprotein A-I complex on RNA polymerase interaction with eukaryotic DNA and the rate of protein biosynthesis in hepatocytes]. / Vliianie kompleksa tetragidrokortizol-apolipoprotein A-I na vzaimodeistvie RNK polimerazy s éukarioticheskoi DNK i na skorost' biosinteza belka v gepatotsitakh.

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 lypoproteins, cortisol, and lypopolysaccharides. 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 alpha- and 5 beta-reductase activity and are constituents of nonparenchymal liver hepatocytes. 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 eukaryotic DNA with apolipoprotein A-I and its complexes with glucocorticoids]. / Vzaimodeistvie éukarioticheskoi DNK s apolipoproteinom A-I i ego kompleksami s gliukokortikoidami.

A biochemically active complex of apolipoprotein A-I with tetrahydrocortisol was revealed, which increases gene expression in hepatocytes. It was shown by the method of fluorescent probe that titration of rat liver DNA by the apolipoprotein A-I-tetrahydrocortisol complex leads to the appearance of single-stranded fragments. The effect of the complex on the secondary structure of native DNA was confirmed by the method of small-angle X-ray scattering. It was shown that approximately 54 apolipoprotein A-I molecules carrying tetrahydrocortisol as a ligand bind to one molecule of isolated native DNA, inducing a break of hydrogen bonds between the pair of nitrous bases. It is concluded that the cooperative effect of high-density lipoproteins and cortisol in the regulation of gene expression in hepatocytes with the participation of resident liver macrophages is accomplished by a new biochemical mechanism. This mechanism makes itself evident as a result of the interaction of DNA with the apolipoprotein A-I-tetrahydrocortisol complex, the appearance of single-stranded DNA regions in binding sites, and subsequent initiation of gene transcription.