[The latex agglutination with video digital registration: the enhancement of diagnostic significance of conventional technique].

The rapid semiquantitative latex-tests, because of their analytic characteristics and convenient application, became widespread in the practice of laboratory diagnostics. Though, in spite of high sensitivity and specificity, their diagnostic effectiveness is lower that it could be mainly because of the impossibility to document the results of latex agglutinative re4actions and to manage the objective quality control. The application of systems of video digital registration permits to enhance the clinical significance of these analyses. By means of scanner systems (control and program complex "Expert Lab") the image of analytic objects is received with the results of latex agglutination reaction. The application of program techniques (the programs "Expert Lab - Agglutination" and "Expert Lab - Agglutination - Micros") in data processing permits to get the precise qualitative characteristics of active reactions, to ensure the automatic interpretation of results and gives an opportunity to proceed with the internal laboratory quality control. The saving of analytic object image in computer memory after termination of reaction favors the formation of data base, the implementation of retrospective evaluation of obtained results, additional consultations in dubious cases, including on-line. The application of complex "Expert Lab" permitted to develop the miniaturizes matrix systems permitting to decrease the withdrawal of latex reagents, to increase the productivity of analytical stage of operation preserving all analytical characteristics of method.

[Analytical characteristics micromatrix semiquantitative method used in serial tests with video digital registration].

A multiplex analytical system has been developed to carry out microformat latex agglutination tests with video digital registration. The system makes it possible to apply less than 1 microl drops of latex and samples in the matrix format to a carrier and to make the test in 30 points at once, as well as to record and to interpret the results of the test, by keeping analytical information for each sample. Comparison of the results of determination of the serum levels of C-reactive protein, rheumatoid factor, and antistreptolysine in the system developed by the authors with the results obtained in the macroagglutination test by the immunoturbidimetric technique leads to the conclusion that the methods are comparable. The proposed type of a latex agglutination test based on a matrix approach and miniaturization assures efficient production and the quality of laboratory studies.

[A scanner-based system for documentation, objective assessment, and recording of the results of latex-agglutination tests].

The present paper describes the use of the scanner "Expert-Lab Agglutination" system to record the results of latex-agglutination tests in making a reaction on the lids of 96-well immunoassay plates. The described system provides a possibility of having a primary image of all tests carried out on a carrier. Software offers the prospect of contrasting the images of individual and control samples, increasing the size of images, and comparing the latter. The use of special image treating techniques may also yield objective figures characterizing the rate of a reaction. The possibility of archiving primary information and retrospectively appraising the correctness of the result of an analysis at any required moment is of fundamental importance to the latex-agglutination test.

[Effect of supporting substrates on the structure of DNA and DNA-trivaline complexes studied by atomic force microscopy]. / Vliianie podlozhek pri atomno-silovoi mikroskopii DNK i kompleksov DNK s trivalinom na ikh strukturu.

Linear DNA, circular DNA, and circular DNA complexes with trivaline (TV), a synthetic oligopeptide, were imaged by atomic force microscopy (AFM) using mica as a conventional supporting substrate and modified highly ordered pyrolytic graphite (HOPG) as an alternative substrate. A method of modifying the HOPG surface was developed that enabled the adsorption of DNA and DNA-TV complexes onto this surface. On mica, both purified DNA and DNA-TV complexes were shown to undergo significant structural distortions: DNA molecules decrease in height and DNA-TP displays substantial changes in the shape of its circular compact structures. Use of the HOPG support helps preserve the structural integrity of the complexes and increase the measured height of DNA molecules up to 2 nm. AFM with the HOPG support was shown to efficiently reveal the particular points of the complexes where, according to known models of their organization, a great number of bent DNA fibers meet. These results provide additional information on DNA organization in its complexes with TV and are also of methodological interest, since the use of the modified HOPG may widen the possibilities of AFM in studying DNA and its complexes with various ligands.

[The hierarchy of complexes and compact structures of trivaline with nucleic acids. III. Complexes of trivaline with trinucleotides forms a rod-like structure with length of about 1000 A in solution]. / Ob ierarkhii kompleksov i kompaktnykh struktur trivalina s nukleinovymi kislotami. III. Kompleksy trivalina s trinukleotidami obrazuiut v rastvore palochkoobraznye struktury dlinoi 1000 A]

We demonstrated the ability of trivaline in the course of interaction with certain trinucleotides in solution to form extended fibre-like structures with lengths of up to several thousand angstroms. Such structures were observed for complexes of trivaline with both deoxyribo- and ribonucleotides with homopurine, homopyrimidine, or random sequences, with or without terminal 5'-phosphate. A model of organization of such structures is proposed. It is based on tetramer complex of trivaline with short nucleotides, two structural units of which, consisting of trivaline tetramer and two trinucleotides, form the octamer complex. It has three perpendicular axes of symmetry of the second order. The spatial location of bases in this structure is additionally fixed by nucleopeptide interactions. The latter create favourable conditions for arranging hydrogen bonds between trinucleotides belonging to different tetramer complexes and stacking interactions between the bases of each nucleotide. Octamer complexes are able to form regular aggregates in the form of a "stack", consisting of dozens of elementary units. These aggregates can be electron microscopically visualized as extended fibre-like structures.

[Electron microscopic study of DNA compactization under the effect of putrescine]. / Elektronno-mikroskopicheskoe izuchenie kompaktizatsii DNK pod deistviem putrestsina.

DNA-putrescine complexes were studied by electron-microscopy with the use of protein-free method. The latter gives the opportunity to investigate the interaction of DNA molecules spread on the surface layer of hypophase and the polyamine molecules in the thick layer of hypophase. Polyamine concentration varied from 5 x 10(-4) mM to 5 x 10(-1) mM. Under the low concentration of putrescine the complexes are represented by agglomerations of kinked knobbed fibres 10 to 20 nm thick, consisting of several fibres of duplex DNA. Upon increasing of putrescine concentration from 5 x 10(-4) to 1.5 x 10(-1) mM, the fibres become more thick (up to 25 nm), highly twisted and have the appearance of cylinders. Very often in the composition of complexes, it is possible to encounter the circular structures, which were formed at the expense of intermolecular interaction of different parts of the complex. The circular structures can serve as "embryos" of toroids of different sizes, that is of different degree of saturation with DNA and putrescine. At the concentration of putrescine 5 x 10(-1) mM the complexes have the appearance of toroids and structures on the basis of toroids, cylinders. The scheme of possible transitions of fibres of various thickness is proposed. The regularities of the compactization process, stimulated by polyamines, don't depend on the degree of compactization (the thickness of compacting fibre), that is they are similar for duplex DNA and for the fibres 25 nm thick, consisting of dozens of DNA molecules.

[Electron microscopic study of compactization of individual DNA molecules in films during the interaction with histone H1]. / Elektronno-mikroskopicheskoe izuchenie kompaktizatsii individual'nykh molekul DNK v plenkakh pri vzaimodeistvii s gistonom H1]

The protein-free method was applied for the investigation of histone H1 DNA complexes formation. The main advantage of this method is the possibility to get intramolecular compact structures at interaction of individual spread molecules of DNA with histone H1. It was shown that in the presence of 0.2-5 micrograms/ml of histone H1 in hypophase there are three types of structures on electronmicroscopic preparations: fibres of non-compacted DNA, compact fibres with twisted strands of duplex DNA and compacted rod-like and circular structures where separate fibres of duplex DNA could not be distinguished. The study of compact structures morphology allows to conclude that they are formed by side-by-side association of DNA fibres, as it takes place in the case of triple rings formation at the compactization of circular DNA due to trivaline binding. At increasing ionic strength there is a tendency for transition from second type structures to the third type structures. The latter can be explained by transition from non-cooperative to cooperative binding of histone H1 to DNA.

[Interaction of trivaline with single-stranded polyribonucleotides]. / Vzaimodeistvie trivalina s odnonitevymi poliribonukleotidami.

Binding of tripeptide H-Val3-(NH)2-Dns (TVP) to polyribonucleotides was studied by fluorescence methods, circular and flow linear dichroism, equilibrium dialysis and electron microscopy. It was found that TVP binds to poly(U) in monomer, dimer and tetramer forms with binding constants of about 10(3), 40, 18.10(4) M, respectively. The cooperativity parameter for peptide dimer binding is 2000. The peptide forms tetramer complexes with poly(A), poly(C), poly(G) also. The formation of a complex between the peptide tetramer and nucleic acid is accompanied by a significant increase in the fluorescence intensity. The cooperative binding of TVP dimers to poly(U), poly(A), poly(C) is accompanied by a dramatic decrease in the flexibility of polynucleotide chains. However, it has a small effect (if any) on the flexibility of the poly(G) chain. The observed similarity of thermodynamic, optical and hydrodynamic++ properties of TVP complexes with single-stranded and double-stranded nucleic acids may reflect a similarity in the geometries of peptide complexes with nucleic acids. Electron microscopy studies show that peptide binding to poly(U) and dsDNA leads to compactization of the nucleic acids caused by interaction between the peptide tetramers bound to a nucleic acid. At the first stage of the compactization process the well-organized rod-like particles are formed, each consisting of one or more single-stranded polynucleotide fibers. Increasing the peptide concentration stimulates a side-by-side association and folding of the rods with the formation of macromolecular "leech-like" structures with the thickness of 20-50 nm.

[Serum trophoblastic beta 1-glycoprotein and alpha fetoprotein levels in physiological pregnancy]. / Soderzhanie trofoblasticheskogo beta1-glicoproteina i alpha- fetoproteina v syvorotke krovi pri fiziologicheski protekaiushchei beremennosti.

Measurements of maternal blood serum trophoblastic beta 1-glycoprotein and alpha-fetoprotein, carried out over the course of normal pregnancy, have demonstrated a progressive increase of trophoblastic beta 1-glycoprotein up to the 36th week, though its level somewhat reduced during the 28th and 32nd weeks. After week 39 the level of this protein in maternal blood serum progressively lowered. alpha-Fetoprotein level was increasing over the course of pregnancy as long as up to the 32nd-34th weeks, then lowering the rest of the term up to delivery. These data permit a conclusion that the time course of the afore-said specific protein concentrations in pregnancy sufficiently well reflects the processes of fetoplacental system establishment and functioning, this permitting the use of these protein measurements in monitoring the course of gestation.

[Demonstration of the influenza virus A RNA by nucleic acid molecular hybridization using biotin-treated probes]. / Vyiavlenie RNK virusa grippa A metodom molekuliarnoi gibridizatsii nukleinovykh kislot s ispol'zovaniem biotinirovannykh zondov.

A probe containing full-size DNA copy of influenza A/USSR/90/70 virus protein gene M labeled with biotin on 32P was used for influenza A virus RNA detection by dot hybridization method. For labeling with biotin, a new method of its administration by chemical modification of nucleic acid was employed. In homologous DNA:DNA hybridization the sensitivity of determinations was less than 1 pg in the biotin-treatment of the probe and 1.25 pg in its radioactive labeling. Hybridization of DNA probe with cytoplasmic RNA isolated from influenza A virus-infected (strains A/USSR/90/77 and A/Texas/77) MDCK cells revealed RNA in the dot corresponding to 4.5-5.5 1g ID50 of virus present in 2 x 10(4) cells. The probe did not bind with negative controls in any dot in all the tests. The results of the study indicate that DNA probes labeled with biotin and 32P and used in dot hybridization for influenza A virus RNA detection in infected cells show the similar sensitivity and specificity.

[Effects of trophoblastic beta 1-glycoprotein (TBG) on the functional activity of different cell lines]. / Vliianie trofoblasticheskogo beta 1-glikoproteina (TBG) na funktsional'nuiu aktivnost' razlichnykh kletochnykh linii.

The effect of TBG on the functional activity of different cell lines, spontaneous and Con A induced proliferation of PBL was studied. If concentration of TBG is higher than 50 mu kg/ml it suppresses the proliferation in many used cell lines, except choriocarcinoma and cancer of uterus. The reliable increasing of spontaneous proliferation of PBL, Jurkat and K-562 cells may be observed if concentration is more lower (0.5-15 mu kg/ml). However proliferation of other cell lines corresponds to control level, and Con A induced proliferation of PBL is inhibited. The effect was more marked at 48, as compared to 24 hours of cell incubation with TBG.

[An immunochemical study of M-HeLa cells as producers of human placental alkaline phosphatase]. / Immunokhimicheskoe issledovanie kletok linii M-HeLa kak produtsentov platsentarnoi shchelochnoi fosfatazy cheloveka.

While analyzing M-HeLa cells by IFA technique secretory and membrane-bound forms of human placental alkaline phosphatase (HPAP) were detected. Activity of secretory HPAP increased if cell density and incubation time were increased too. After short influence of heat shock (15 min at 42 degrees C) activity of secretory HPAP increased for 45% and intracellular HPAP 3 for 37%. It is proposed that HPAP take part in organization of first response to heat shock and support cellular thermotolerance.

[Structural organization of kinetoplast DNA and its compactization in a model system in vitro]. / Strukturnaia organizatsiia kinetoplastnoi DNK i ee kompaktizatsiia v model'noi sisteme in vitro.

Studies on compactization and decompactization of the genome are of great importance for elucidation of structural mechanisms taking part in the regulation of gene activity. Kinetoplast DNA (kpDNA) is a convenient model for studies of compactization processes. KpDNA represents unique structure ("network"), consisting of catenated circular molecules of two types: minicircles (900 b.p.) and maxicircles (40 000 b.p.). The compactization process of kpDNA in vitro caused by interaction with synthetic peptide-dansylhydraside trivaline was studied. It was shown that at the initial stages the hairpins are observed on minicircles as if triple rings are being organized. The formation of hairpin is probably favoured by the presence in the minicircles of bent DNA, a specific nucleotide sequence causing rigid bending of the DNA helix. The hairpin does not make contact with the neighbouring DNA segment to form a triple ring, because the sizes of minicircles are too small. The minicircles compactization is finished with a complete collapse of the minicircles with the formation of rod-like structures. The catenation causes branching of rod-like structures. As a result of their intermolecular interaction, the branched rod-like structures become thicker. The process is completed with formation of the compact network, its diameter being 3-6 times smaller compared to the initial one.

[Biosynthesis of the membrane protein of the acquired immunodeficiency syndrome virus (HIV) with a deleted hydrophobic region in E. coli cells]. / Biosintez obolochechnogo elka virusa sindroma priobretennogo immunodefitsita (HIV) s udalennoi gidrofobnoi oblast'iu v kletkakh E. coli.

Effect of the structure of cloned env gene of HIV virus on the level of its expression has been studied. The deletion of the hydrophobic region from the env gene has been shown to increase considerably the biosynthesis of antigen-specific proteins in Escherichia coli cells. At the same time, the low level of expression of the gene fragment the transcription of which is controlled by a powerful lac-promoter suggests the presence of toxic regions of nonhydrophobic nature in the synthesized antigen.