A family of DNA aptamers with varied duplex region length that forms complexes with thrombin and prothrombin.

Structural properties determine binding affinities of DNA aptamers specific to thrombin. Our paper is the first to focus on a family of eight G-quadruplex-based aptamers with varied duplex region length (from two to eight base pairs). We have shown that the duplex, which is not the main binding domain, greatly influences the interaction with thrombin and prothrombin. Furthermore, the affinity of an aptamer to thrombin and prothrombin increases (respectively from 2.7×10⁻8 M to 5.6×10⁻¹° M and from 1.8×10⁻5 M to 7.1×10⁻9 M) with an increase in the number of nucleotide pairs in the duplex region.

[Toxicological assessment of nanostructured silica. I. Integral indices, adducts of DNA, tissue thiols and apoptosis in liver].

Nanostructured amorphous silica (SiO2) is widely used in food additives, pharmaceuticals and cosmetics. Available data on the oral toxicity of this nanomaterial (NM) in vivo, obtained in acute and subacute experiments are contradictory. The purpose of this study is evaluation of some parameters of toxicity of nanostructured SiO2 when orally administered to rats for 3 months. We used commercial SiO2 preparation, obtained by gas-phase hydrolysis of tetrachlorosilane with a size of the primary nanoparticles close to 5-30 nm, which was characterized as NM by several independent methods. SiO2 in the form of sonicated aqueous dispersion was administered to male rats with initial weight of 80 +/- 4 g for the first 30 days by intragastric gavage and then for 62 days with consumed diets in daily dose of 0,1; 1,0; 10 and 100 mg/kg body weight. The control animals received vehicle--deionized water. Weight gain, relative mass of internal organs, intestinal permeability to protein macromolecules (determination of ovalbumin level in blood serum by solid-phase bivalent immunoassay), urinary excretion of oxidative degradation product of DNA 8-oxo-2-deoxyguanosine (8-oxo-G) (by reversed phase HPLC), the level of thiol compounds in liver (spectrophotometrically), liver cell apoptosis (flow cytometer), fixing efficiency of passive avoidance (CRPA) have been measured. It has been shown that three-month administration of nanostructured SiO2 in all doses resulted in animal body weight decrease by 10-15%; a significant increase in adrenal weight was noticed under doses of 1 and 10 mg/kg and urinary 8-oxo-G excretion was significantly reduced at the dose 10 mg/kg. At the maximum dose of NM, 100 mg/kg, after 2 months of administration the number of animals decreased that entered the dark compartment of the experimental setup at initial testing of CRPA. The rest of the studied indices did not experience any significant changes depending on the dose of NM. It is concluded that no toxic effect were expressed in indices studied under the influence of nanostructured SiO2 in rats at daily doses up to 100 mg per kg body weight for 3 months.

[Toxicological assessment of nanostructured silica. The acute oral toxicity].

Nanostructured amorphous silica (SiO2) is one of the priorities of nanomaterials, exposing human to the ever-increasing scale as a component of food additives, drugs and cosmetic products. According to numerous publications SiO2 nanoparticles (NPs) possess deleterious effects on animal and human cells in vitro and also exhibit inhalation toxicity. However, the biological effects in vivo of silica NPs taken orally are studied insufficiently. This article represents the first section of this study which aim is identification of silica preparation as nanomaterial and estimating of acute toxicity after oral administration in the form of aqueous suspension. Studies of size and shape of the particles in aqueous suspension of silica used in the study by electron and atomic force microscopy, spectroaqustic analysis and dynamic laser light scattering showed that the test substance is a nanomaterial. Estimation of acute toxicity of an aqueous suspension of nanostructured silica with a single intragastric gavage to male BALB/C mice allowed to conclude that the test material has LD50 by the oral route greater than 10 000 mg/kg and consequently belongs to class IV (low danger agents). Pathological changes in jejunum and colon of mice in the acute experiment (at a dose of 10 000 mg/kg) haven't been identified. Thus SiO2 NPs possess low toxicity when administered in the gastrointestinal tract. The available data, however, do not exclude possible presence of adverse effects under their long-term administration by oral way.

[Bioaccumulation of silver and gold nanoparticles in organs and tissues of rats by neutron activation analysis].

Bioaccumulation of silver (Ag) and gold (Au) nanoparticles (NPs) with mean sizes of 6 nm and 35 nm, respectively, has been studied after their intragastric administration to rats at a dose of 100 µg/kg of body weight for 28 or 14 days. The organs and tissues (liver, kidney, spleen, heart, gonads brain, and blood) were subjected to thermal neutron activation, and, then, the activity of the 110mAg and 198Au isotopes generated was measured. The NPs of both metals were detected in all biological samples studied, the highest specific weight and content of Ag NP being found in the liver, and those of Au being found in kidneys of animals. The content of Ag NPs detected in the brain was 66.4 ± 5.6 ng (36 ng/g tissue), no more than 7% ofthese NPs being localized in the lumen of brain blood vessels. The content of Ag and Au NPs found in organs and tissues of rats could be regarded as nonhazardous (nontoxic) in accordance with the known literature data.

Structural changes in R-phycoerythrin upon CdS quantum dot synthesis in tunnel cavities of protein molecules.

Structural changes in R-phycoerythrin used as a matrix for the synthesis of CdS quantum dots have been analyzed by circular dichroism spectrometry. In deionized water, quantum dot synthesis in the tunnel cavity of the R-phycoerythrin molecule proved to be accompanied by uncoiling of α-helices and changes in the conformation of its chromophore groups, with consequent decay of protein fluorescence. Since R-phycoerythrin fluorescence is important for practical applications, conditions for quantum dot synthesis have been optimized by replacing deionized water with 0.01 M MES buffer, pH 5.7. Under such conditions, the size of the CdS quantum dots (determined from atomic force microscopy images) remains the same as in deionized water, but quantum dots cause only minor structural changes in protein molecules, as follows from circular dichroism and absorption spectra. The thermostability of R-phycoerythrin is enhanced, as indicated by an increase in the experimental activation energy for denaturation (from 140.8 to 149.9 kJ/mol) and the intensity of R-phycoerythrin fluorescence is also enhanced approximately twofold.

Effect of cadmium sulfide quantum dots on physical properties of R-phycoerythrin as a protein matrix.

The synthesis and analysis of nanostructures in the cavities of protein molecules is a promising research field in the industry of nanosystems. In this study, atomic force microscopy (AFM) has been used to evaluate the properties of CdS quantum dots synthesized in the tunnel cavities of R-phycoerythrin, a 290 kDa water-soluble pigment protein responsible for light harvesting in red algae. It has been shown that R-phycoerythrin dissolved in deionized water to a concentration of 50 µg/ml is prone to self-organization into regular spatial structures upon adsorption on the surface of mica, but no such structuring takes place in films prepared from R-phycoerythrin solutions diluted tenfold. In the latter case, protein molecules are deformed, as judged from the analysis of the surface profile. R-phycoerythrin with CdS quantum dots in protein cavities (the concentration of the preparation was (48 µg/ml) loses the self-organization ability and is not deformed upon adsorption on the mica surface. Analysis of AFM images by flicker-noise spectroscopy has shown that incorporation of CdS quantum dots into R-phycoerythrin molecules provides for "smoothing" of the protein surface, with various irregularities being leveled off. Conversely, the irregularity of the protein surface increases when R-phycoerythrin molecules are arranged into three-dimensional branching structures. It is concluded that CdS quantum dots interfere with protein-protein interactions and restrain the conformational mobility of the protein. The anomalously rigid structure of R-phycoerythrin in the presence of CdS is due to its conformational rearrangements during the synthesis of quantum dot.

Iron-dependent superoxide dismutase from novel thermoacidophilic crenarchaeon Acidilobus saccharovorans: from gene to active enzyme.

A gene encoding superoxide dismutase was revealed in the genome of the thermoacidophilic crenarchaeon Acidilobus saccharovorans. A recombinant expression vector was constructed and transformed into E. coli cells. The novel recombinant superoxide dismutase was purified and characterized. The enzyme was shown to be an iron-dependent superoxide dismutase able to bind various bivalent metals in the active site. According to differential scanning calorimetric data, the denaturation temperature of the enzyme is 107.3°C. The maximal activity of the Fe(II) reconstituted enzyme defined by xanthine oxidase assay is 1700 U/mg protein. Study of the thermal stability of the superoxide dismutase samples with various metal contents by tryptophan fluorescence indicated that the thermal stability and activity of the enzyme directly depend on the nature of the reconstituted metal and the degree of saturation of binding sites.

Application of atomic force microscopy for characteristics of single intermolecular interactions.

Atomic force microscopy (AFM) can be used to make measurements in vacuum, air, and water. The method is able to gather information about intermolecular interaction forces at the level of single molecules. This review encompasses experimental and theoretical data on the characterization of ligand-receptor interactions by AFM. The advantage of AFM in comparison with other methods developed for the characterization of single molecular interactions is its ability to estimate not only rupture forces, but also thermodynamic and kinetic parameters of the rupture of a complex. The specific features of force spectroscopy applied to ligand-receptor interactions are examined in this review from the stage of the modification of the substrate and the cantilever up to the processing and interpretation of the data. We show the specificities of the statistical analysis of the array of data based on the results of AFM measurements, and we discuss transformation of data into thermodynamic and kinetic parameters (kinetic dissociation constant, Gibbs free energy, enthalpy, and entropy). Particular attention is paid to the study of polyvalent interactions, where the definition of the constants is hampered due to the complex stoichiometry of the reactions.

[Toxicological sanitary characterization of silver nanoparticles introduced in gastrointestinal tract of rats].

Water suspensions of silver nanoparticles were introduced intragastrically to growing male Wistar rats daily for 28 days. There were studied animals mass gain, relative masses of viscera, intestinal barrier resistance against protein macromolecules, DNA oxidative damage, tissue non-protein thiol levels, first and second phase of xenobiotic detoxication system activity, lysosomal membranes stability in liver together with routine blood biochemical and hematological indices and caecal's microbiocenose state. The data testifying to possible toxic risks, connected with reaction of silver's nanoparticles is obtained.

[Methods of detection and identification of manufactured nanoparticles].

The actual methods of detection and identification of manufactured nanoparticles in both simple and complex multi-component matrix for assessing biological effects and safety of nanotechnology products have been reviewed. The detection of priority species of biologically active nanoparticles, which include fullerenes, single- and multi-walled carbon nanotubes, nanoparticles of silver, gold, titanium oxide, aluminum, cerium, zinc and silicon, has been given a special attention. The requirements for sample preparation have been discussed. The results of the successful application for the detection of manufactured nanoparticles in bioassays with methods of scanning and transmission electron microscopy, confocal laser scanning microscopy, atomic force microscopy, scanning tunneling microscopy, size exclusion chromatography, field-flow fractionation, electrophoretic, light scattering, spectrophotometry, fluorescent spectroscopy, X-ray and other spectrometry, mass spectrometry, "particle counters", immunochemistry have been reviewed. The possibilities and limitations of different techniques, and their complementarity have been analyzed.

Interaction of plum pox virus with specific colloidal gold-labeled antibodies and development of immunochromatographic assay of the virus.

Two monoclonal antibodies (mABs) raised against plum pox virus (PPV) were shown to recognize its D, M, and C strains. Conjugates of the antibodies with colloidal gold (CG) nanoparticles averaging 26 nm in diameter were synthesized. The binding constants of PPV with both the native and conjugated mABs were determined using a Biacore X device. The complexes between the CG-mAB conjugates and plum pox virions were examined by means of transmission electron and atomic force microscopy. Using the conjugates with optimal component ratio, an express immunochromatographic assay of PPV was developed with a detection limit of 3 ng/ml and duration of 10 min. The assay was tested for PPV detection in samples of stone fruit tree leaves and demonstrated a good compatibility with the data obtained by "sandwich"-ELISA. The developed assay can be used in the field and applied for monitoring viral infection and for quarantine purposes.

[Development of immunochromatographic test systems for express detection of plant viruses].

Express immunochromatographic test-strip assays were developed for detection of five plant viruses varying in shape and size of virions: spherical carnation mottle virus, bean mild mosaic virus, rod-shaped tobacco mosaic virus, and filamentous potato viruses X and Y. Multimembrane composites (test strips) with immobilized polyclonal antibodies against viruses and colloidal gold-conjugated antibodies were used for the analysis. The immunochromatographic test strips were shown to enable the detection of viruses both in purified preparations and in leaf extracts of infected plants with a sensitivity from 0.08 to 0.5 microg/ml for 10 min. The test strips may be used for express diagnostics of plant virus diseases in field conditions.