[Theoretical study of hydrophobicity and hydrophilicity of uracil and its dimers].

The influence of hydrophilic and hydrophobic properties of the uracil elementary nucleic acids bases on its solubility and structure in aqueous solution was studied. Complexes of uracil with water molecules (from 1 to 14) were then calculated. The geometrical parameters of the hydrogen bridge of uracil and the changes in the frequency of valence vibrations of the bonds participating directly in hydrogen bond formation were calculated. It is shown that for the hydrogen bonds O(w)...HN(1) and O(w)...HN3 the hydrogen atom can tear, it may lead to tautomeric transformation of uracil. The results obtained having calculated the structure of uracil dimers, formed with the hydrogen bonds, in an isolated state and water solution, energy, dipole moments and the hydrogen bridge parameters made it possible to explain low solubility of uracil in water at room temperature. It is shown that water molecules with increase in their number are located mainly at one side of the plane of a pyrimidine uracil ring, that leads to the formation of stacking. Of two possible variants of stacking formation, the most profitable grouping is when a dipole moment of the formed dimer is equal to zero (anti-parallel stacking).

[Murine peritoneal neutrophil activation upon tungsten nanoparticles exposure in vivo].

Two examples of tungsten carbide nanoparticles (d = 15 nm, 50 nm) and tungsten carbide nanoparticles with 8% cobalt (d = 50 nm) have been found to induce the neutrophil activation 3 h and 36 h after intraperitoneal administration in the doses 0.005; 0.025; 0.05; 0.25; 0.5; 1; 2.5 and 5 microgram per 1 gram body weight to FVB mice. Neutrophil activation was calculated based on the CD11b and S100 antigen expression. Effect of nanoparticles is bimodal for all tested examples.

[Structural-functional characteristics of cervical vessels in hypertensive patients under changed atmospheric pressure].

The ultrasonic location technique was used to measure intima-media thickness (IMT), as well as internal systolic diameter of and linear blood velocity in the cervical arteries in people with initial hypertension. Correlation analysis elicited a temporal contingency between these parameters and daily average values of atmospheric pressure. Thus, common carotid artery IMT tended to increase on high-pressure days. Besides, diameters of the common and internal carotid arteries, and vertebral artery were narrowed and, consequently, linear blood velocity in these vessels increased. This relationship is more evident in men than women and in elderly subjects than young. These results are suggestive of a vasoconstrictive action of high atmospheric pressure on these arteries. The relationship is not universal, as it is nonlinear for diameter of the internal carotid artery and inverse for the external one. This implies different sensitivity of arteries to the factor under study and possible blood redistribution in the arterial basin depending on external pressure. The relationship was observed equally on the day of investigation and previous days, which points to its temporal stability.

[On the possibility of determination of stereological characteristics of interactions of biological micro objects with nanoparticles based on analysis of 2-dimensional images of cytological and histological specimens].

In this article the method for studying 3-dimensional (stereological) characteristics of interactions of biological micro objects with nanoparticles on a basis of morphodensitometry analysis of standard 2-dimensional images of cytological and histological specimens is proposed. The performance of the task of determining the distance from registered nanoparticle to the center of the nucleus of the cell is described in detail. It is shown that using specific nanoparticles the results obtained may find application in science and diagnostics. Furthermore, it is possible to employ these results in nanotoxicology, in particular for determination of quantity characteristics of translocation of nanoparticles of different types in biological structures.

[The use of biologically controlled ultrasound therapy for the combined treatment of primary open-angle glaucoma].

The present study included 160 patients (206 eyes) presenting with the initial and advanced stages of primary open-angle glaucoma. 120 patients (157 eyes) were treated using biologically controlled ultrasound therapy besides traditional medicamentous therapy, the remaining 40 ones (49 eyes) were given only conventional medication. Results of the treatment were evaluated based on a set of commonly used hydrodynamic and electrophysiological characteristics of the quality of visual function. Biologically controlled massage of trabecular structures of the affected eyes made it possible to stabilize and improve major hydrodynamic characteristics of the eyeball drainage system, visual function, and electrophysiological parameters. Biologically controlled ultrasound therapy proved to produce a more pronounced beneficial effect on ocular hydrodynamics than the traditional ultrasonic treatment. The positive action of biologically controlled therapy persisted during 8 months; its repeated sessions prolonged this period up to 2 years.

[Effects of silver and silicon dioxide nanopowders on the development of herpesvirus infection in vitro].

Estimation of the potential ability of nanoparticles (NP) to affect human health has generated a need for developing rapid, sensitive, and efficient laboratory tests of the toxicity of nanomaterials. The purpose of the investigation was to study the cytotoxic effect of NP of silver (Ag) and silicon dioxide (SiO2). The transplantable Vero cells treated with NP at different concentrations were used as target cells. Some experiments examined the combined effects of nanopowders and herpes simplex virus type 2 (HSV-2) on Vero cell viability and the direct effect of NP on the reproductive potential of HSV-2 in the culture. SiO2 NPs at concentrations of 1.0 to 0.1 mg/ml were found to cause a marked cytotoxic effect that was in the complete destruction of the cell monolayer. Ag HPs were more toxic than silicon nanopowders and induced a complete degradation of the cell monolayer at substantially lower concentrations. The results of the study formed the basis for the development of a rapid (24-48-hour), reliable, and efficient test for the toxicity of nanomaterials, by using the cultured cells in the laboratory setting. It was also shown that silicon NPs did not noticeably affect the reproductive potential of HSV-2 while nano silver suppressed the capacity of HSV-2 for multiplication, by significantly reducing viral progeny titer in the cell culture.

A continuous cell-free translation system capable of producing polypeptides in high yield.

A cell-free translation system has been constructed that uses a continuous flow of the feeding buffer [including amino acids, adenosine triphosphate (ATP), and guanosine triphosphate (GTP)] through the reaction mixture and a continuous removal of a polypeptide product. Both prokaryotic (Escherichia coli) and eukaryotic (wheat embryos, Triticum sp.) versions of the system have been tested. In both cases the system has proven active for long times, synthesizing polypeptides at a high constant rate for tens of hours. With the use of MS2 phage RNA or brome mosaic virus RNA 4 as templates, 100 copies of viral coat proteins per RNA were synthesized for 20 hours in the prokaryotic or eukaryotic system, respectively. With synthetic calcitonin messenger RNA, 150 to 300 copies of calcitonin polypeptide were produced per messenger RNA in both types of continuous translation systems for 40 hours.

[A theoretical investigation of tautomerism of adenine, purine, guanine, and cytosine].

The parameters of tautomeric equilibrium and stability of lactam-lactim, amino-imino, and N9H <--> N7H tautomeric forms of adenine, purine, guanine, and cytosine for isolated states have been calculated. It was found that the Ade-N9H tautomeric form is most stable; in this case the intramolecular proton transfer and tautomeric transformation Ade-N9H --> Ade-N7H can occur. The parameters of tautomeric equilibrium and stability for seven tautomeric forms of guanine were determined. For cytosine, the simultaneous existence of the amino-hydroxy, imino-oxo and valence amino-hydroxy of tautomeric forms was predicted.

[Occupational hygiene and hygienic regulations during the manufacture, use and utilization of nanomaterials].

This paper gives information on the action of nanoparticles when interacting with biological objects and penetrating into them. The high chemical activity of nanoparticles determines their enhanced biological activity against living organisms. Due to their special properties and sizes, the nanoparticles can penetrate across the cell membranes or between the cells and spread to various organs and systems by different mechanisms (endocytosis and transcytosis). In vitro and in vitro investigations of human exposure to nanopowders strongly suggest the occurrence of inflammatory processes, oxidative stresses in the lung and peripheral organs when such powders are inhaled. A few days after intracheal administration, the nanoparticles (30-60 nm in size) penetrate into the cells of the liver, kidney, and gonads and, moreover, primarily impair the mitochondria and penetrate into the nucleus, carrying the viruses on their arms into the cell nucleus. By taking into account the penetrating capacity of nanoparticles into various human organs and tissues and into cell substances, there are a number of problems and questions as both positive in the use of nanoparticles in medicine and other branches of industry as materials that can deliver a drug to the involved organs, by improving the properties of many materials, and negative, such as the development of new forms of abnormalities that are still unknown medicine. In this connection, it is necessary to develop technical regulations on their manufacture, use, storage, transportation, and utilization and those on the realization of various imported materials containing the nanoparticles and obtained by nanotechnologies.

Multiple cellular antigen detection by ICP-MS.

There is a great need in cell biology for the simultaneous detection of many intracellular and extracellular proteins within single cells. Current optical methods based on fluorescence activated flow cytometry are difficult to multiplex. We have developed a novel application of ICP-MS-linked metal-tagged immunophenotyping which has great potential for highly multiplexed proteomic analysis. Expression of intracellular oncogenic kinase BCR/Abl, myeloid cell surface antigen CD33, human stem cell factor receptor c-Kit and integrin receptor VLA-4 were investigated using model human leukemia cell lines. Antigens to which specific antibodies are available and are distinguishably tagged can be determined simultaneously, or multiplexed. Four commercially available tags (Au, Sm, Eu, and Tb) conjugated to secondary antibodies enable a 4-plex assay assuming that the primary antibodies are not cross-reactive. Results obtained by ICP-MS were compared with data from FACS. ICP-MS as an analytical detector possesses several advantages that enhance the performance of immunoassays, which are discussed in detail. Although multiplexing using metal-conjugated reagents is in a very early stage of research and feasibility studies, it is already apparent that more than four antigens could be accurately detected simultaneously using the ICP-MS instrument.

Translocation makes the ribosome less compact.

Translating ribosomes of Escherichia coli were prepared either in the pre-translocation or in the post-translocation states by a special technique based on the use of poly(U)-Sepharose columns where the template was coupled to the matrix through splittable -S-S- bridges. Elongation factors were absent from the final preparations. A neutron scattering study of the translating ribosomes in the two functional states was performed at different contrasts (various 1H2O/2H2O mixtures). Under conditions of a high contrast for the protein constituent the radius of gyration of the post-translocation-state ribosomes was found to be slightly greater than that of the pre-translocation-state ribosomes. Using the results of this study the conclusion can be drawn that translocation is accompanied by a spatial displacement of some parts of the ribosome with a magnitude of several ångström units.

Gene expression in a cell-free system on the preparative scale.

A cell-free system for preparative gene expression is described. It is composed of DNA-free Escherichia coli extract and added plasmid DNA; coupled transcription-translation proceeds with a continuous flow of the feeding solution containing nucleoside triphosphates and amino acids. The system works at a high constant rate for tens of hours. The yield of synthesised proteins after 20-50 h is hundreds of micrograms from 1 ml of the reaction mixture. Electrophoretic analysis of translation products confirms synthesis of proteins of the expected molecular mass.

Does the channel for nascent peptide exist inside the ribosome? Immune electron microscopy study.

MS2 phage RNA-directed synthesis of an N-terminal polypeptide of the phage coat protein on Escherichia coli 70 S ribosomes was initiated in a cell-free system with the N-dinitrophenyl derivative of methionyl-tRNAFMet) and performed in the absence of tyrosine, lysine, cysteine and methionine. As a result, the translating ribosomes carried peptides up to 42 amino acid residues in length with the dinitrophenyl hapten at the N-ends. Using the immune electron microscopy technique the positions of the nascent peptide N-ends on the 70 S ribosomes have been visualized. It has been found that (i) the N-ends of nascent peptides of these lengths are accessible to antibodies, (ii) the exit site of a nascent peptide is the pocket between the base of the central protuberance and the L1 ridge on the 50 S subunit, i.e. presumably its peptidyl transferase center, and (iii) the further pathway of a nascent peptide seems to proceed along the groove on the external surface of the 50 S subunit.

Structural study of translating 70 S ribosomes from Escherichia coli. I. Electron microscopy.

Translating 70 S ribosomes of Escherichia coli either in the pre-translocation or in the post-translocation state have been prepared by using the cell-free translation system in poly(U)-S-S-Sepharose columns [Methods Enzymol. (1979) 59, 382-398]. Electron microscopy study of the preparations has demonstrated that: (1) the mutual orientation of the ribosomal subunits in the translating ribosomes is the same as proposed by Lake for routine 30 S.50 S couples [J. Mol. Biol. (1976) 105, 111-130]; (2) the L7/L12 stalk of the 50 S subunit sticks out from the 70 S particle and does not join the 30 S subunit; (3) pre-translocation and post-translocation state ribosomes do not differ in mutual orientation of the subunits and in the position of the L7/L12 stalk, within the limits of electron microscopy resolution.

Is the three-site model for the ribosomal elongation cycle sound?

The release of deacylated tRNA from the ribosome as a result of translocation has been studied. Translating ribosomes prepared with poly(U)-S-S-Sepharose columns have been used. It has been shown that deacylated tRNA released from the ribosomal P site as a result of translocation rebinds with the vacated A site. Consistent with the known properties of the A site of the ribosome, this interaction is reversible, Mg2+-dependent, codon-specific and is inhibited by the antibiotic tetracycline. It has been concluded that the proposed three-site model of the ribosomal elongation cycle (Rheinberger and Nierhaus (1983) Proc. Natl. Acad. Sci. USA 80, 4213-4217) is not sound: the experimentally observed 'retention' of the deacylated tRNA on the ribosome after translocation can be explained by a codon-dependent rebinding to the A site, rather than by its transition to the 'E site', i.e., in terms of the classical two-site model.

PCR assay of DNA damage and repair at the gene level in brain and spleen of gamma-irradiated young and old rats.

The PCR amplification of fragments of transcribed (beta-actin, p53) and nontranscribed (IgE, heavy chain) genes in brain and spleen DNA from gamma-irradiated and unirradiated 2- and 28-month-old rats was studied. The amplification levels of fragments of these genes in DNA from old rats were substantially lower than those from young rats, which suggested that these gene fragments in old-rat DNA contained lesions blocking thermostable polymerase in PCR. The beta-actin and IgE gene fragments of spleen DNA from old rats exhibited a significantly higher level of lesions inhibiting Tth polymerase compared to analogous fragments of brain DNA from the same animals. DNA from the tissues of gamma-irradiated rats showed the amount of damage inhibiting amplification to be dependent on animal age and the postirradiation time before DNA isolation. As judged from the changes in the amplification level of gene fragments, there was no preferential fast repair of lesions in the actively transcribed gene beta-actin compared to the nontranscribed gene IgE (heavy chain) in the brain and spleen of gamma-irradiated young and old rats. The amplification results suggest that equal amounts of DNA lesions were repaired in the brain of both old and young rats during the first 0.5 h of the postirradiation time (fast-repair phase), whereas in the subsequent postirradiation period over 5 h (slow-repair phase), the efficiency of damage elimination in the brain DNA of old rats was markedly lower. As for the spleen tissue, the elimination of lesions blocking Tth polymerase was much lower in old gamma-irradiated animals for both of the repair phases.

[Structural studies of translating ribosomes. II. Comparative sedimentation analysis of pre- and post-translocation states]. / Strukturnye issledovaniia transliruiushchikh ribosom. II. Sravnitel'nyi sedimentatsionnyi analiz pre- i post-translokatsionnykh sostoianii.

Using the solid-phase translation system technique where template poly(U) is covalently coupled to Sepharose through cleavable disulfide bridges translating monoribosomes carrying a polypeptide (polyPhe) of 10 to 20 amino acids long have been isolated. Both pre-translocation state and post-translocation state ribosomes have been obtained. It has been shown that the sedimentation coefficient of the pre-translocation state ribosomes exceeds that of the post-translocation state ribosomes by a magnitude of about 1S. This difference is independent on the sedimentation rate (hydrostatic pressure) in the range of 20 000 to 40 000 rev/min and, most likely, is not a direct contribution of the increase of the particle mass at the expense of an additional tRNA in the pre-translocation state ribosomes. Together with other data, this result suggests that translating ribosomes in the pre-translocation state are more compact than post-translocation state ribosomes.

[Structural analysis of translating ribosomes. Proton magnetic resonance method]. / Strukturnye issledovaniia transliruiushchikh ribosom. III. Metod protonnogo magnitnogo rezonansa.

Comparison has been made of the proton magnetic resonance (PMR) spectra of translating ribosomes in the pre-translocation and post-translocation states as well as of the complexes of translating ribosomes with elongation factors Tu (EF-Tu) or G (EF-G) in the presence of the uncleavable analogue of GTP--guanylyl-imidodiphosphate (GMP-PNP). It is shown that proteins L7/L12 within the translating ribosomes possess a high intramolecular mobility both in the pre-translocation and in the post-translocation states. The interaction of EF-G with translating ribosomes results in a decrease of the mobility of the L7/L12 proteins. The interaction of EF-Tu with translating ribosomes leads to slight changes in the PMR spectra different from the changes caused by EF-G.

[Preparation of translating ribosomes by using a column with immobilized polyuridylic acid]. / Poluchenie transliruiushchikh ribosom s pomoshch'iu kolonok, soderzhashchikh immobilizovannuiu poliuridilovuiu kislotu.

An improved method for preparation of translating ribosomes using columns with immobilized polyuridylic acid is described. A peculiarity of the method is that, first, optimal ratios of the components are used in the translation system for obtaining high yields of translating ribosomes. Second, purification of translating ribosomes from admixtures of non-translating particles is achieved by passing the buffer containing 5 mM MgCl2 and 250 mM NH4Cl through the column. The purity of the translating ribosomes is no less than 95%, the yield of active ribosomes is 5-20% of the initial amount of the ribosomes. Reagent expenditure is cut 15 to 20 times.

[Use of diagnostic DNA probes for detection of the provirus of bovine leukemia virus in infected cows]. / Ispol'zovanie diagnosticheskikh DNK-zondov dlia obnaruzheniia provirusa virusa bych'ego leikoza u infitsirovannykh korov.

A method of detection of BLV proviral DNA in the peripheral blood leukocytes of cattle is reported. Leukocytes were used without preliminary cultivation. Cell DNA was dot-hybridized with P-labeled plasmid containing a fragment of BLV proviral DNA. The sera samples taken from the cattle under study were also tested using immunodiffusion assay (ID). The results of comparison showed that dot-hybridization assay is a more sensitive diagnostic test than ID, because it detects BLV infection in apparently normal cattle, which was seronegative in ID.