[Effects of the H6R and D7H Mutations on the Heparin-Dependent Modulation of Zinc-Induced Aggregation of Amyloid ß].

Zinc ions and glycosaminoglycans (GAGs) are found in amyloid deposits and are known to modulate the ß-amyloid peptide (Aß) aggregation, which is thought to be a key event in the pathogenesis of Alzheimer's disease (AD). Correlation spectroscopy was used to study how the H6R and D7H mutations of the metal-binding domain (MBD) of Aß42 affect the modulation of its zinc-induced aggregation by the model GAG heparin. The H6R mutation was shown to decrease and the D7H mutation to increase the Aß42 propensity to aggregate in the presence of zinc ions. In addition, H6R diminished and D7H enhanced the modulating effect of heparin. The difference in the heparin-dependent modulation was associated with coordination of zinc ions within the MBDs of the mutant peptides. The findings indicate that anion-binding sites formed by complexes of zinc ions with the Aß MBD play an essential role in the interaction of zinc-induced Aß aggregates with heparin.

[Enzymatic Preparation of Modified DNA: Study of the Kinetics by Real-Time PCR].

The effects of modified deoxyuridine triphosphates (mod-dUTPs) with different substituents at the C5 position of the pyrimidine cycle on the kinetics of PCR with Taq and Vent (exo-) DNA polymerases are studied. Substituents in mod-dUTP include carboxamide group and groups that are part of the side chains of alanine, valine, leucine, phenylalanine, tryptophan, or tyrosine. For each mod-dUTP, the yields of the target product are measured with the full substitution of dTTP. A fragment of bacterial DNA with a certain nucleotide sequence and a synthetic combinatorial DNA library of random nucleotide sequences are used as templates for amplification. For each mod-dUTP-template-polymerase combination, the correlation between the amplification efficiencies and yields of the target product are investigated. PCR product accumulation curves are influenced by both the template used and the presence of a modified substrate. The catalytic activity of Taq polymerase is higher when mod-dUTPs with short aliphatic substituents are used and decreases when the derivatives with long aliphatic, phenyl, and indole substituents are utilized. Vent (exo-) polymerase is less sensitive to the chemical structure of mod-dUTP. The dynamic measuring of DNA accumulation may be useful for optimizing the temperature-time PCR profiles individually for each of the mod-dUTP. The derivatives may be used in combination with Vent (exo-) polymerase to obtain modified DNA sequences for the method of selection of modified aptamers (mod-SELEX).

[Preparation of Modified Combinatorial DNA Libraries via Emulsion PCR with Subsequent Strand Separation].

A modification of the enzymatic method for the preparation of combinatorial random DNA libraries, which combines amplification in isolated microvolumes with the simultaneous incorporation of modified nucleotides and subsequent separation of DNA strands, was developed. Deoxyuridine triphosphate with hydrophobic substituents such as structural analogues of amino acid side chains in the C5 position of the pyrimidine ring was used to introduce modifications into DNA. To prevent competitive amplification, which reduces the representativeness of combinatorial libraries, PCR in inverse emulsion was used. The separation of the strands of PCR products was carried out. There were six single-stranded DNA libraries with complete substitution of deoxythymidine via modified analogues with various functional groups. These DNA libraries are suitable for generating aptamers to protein targets through additional hydrophobic interactions from the introductions of appropriate modifications, and are completely compatible with the SELEX aptamer selection methodology.

[Enzymatic synthesis of high-modified DNA].

Here, we describe the synthesis and purification of six deoxyuridine triphosphate derivatives that contain protein-like functional groups and alkene linkers of various lengths. Using KOD XL and Deep Vent polymerases, these derivatives have been incorporated into single-stranded DNA, achieving a high degree of DNA modification. These polymerases are able to utilize highly modified DNA strands as templates for synthesizing unmodified DNA. The synthesized deoxyuridine triphosphate derivatives are promising as substrates for producing modified aptamers to various target proteins using, e.g., the systematic evolution of ligands by exponential enrichment (SELEX) methodology.

[Zinc-induced interactions of the metal-binding domain of beta-amyloid with nucleic acids and glycosaminoglycans].

Zinc ions form complexes with ß-amyloid peptides and play an important role in Alzheimer's disease pathogenesis. It has been demonstrated by turbidimetry and correlation spectroscopy that synthetic peptide Aß16 representing the metal-binding domain of ß-amyloid is able to interact with nucleic acids, chondroitin polysulfate, and dextran sulfates in the presence of zinc ions. The amino acid D7H substitution enhanced the peptide binding to polyanions, whereas the H6R and H6A-H13A substitutions abolished this interaction. It is suggested that the metal-binding domain may serve as a zinc-dependent site of ß-amyloid interaction with biological polyanions including DNA, RNA, and glycosaminoglycans.

[Effect of mutations and modifications of amino acid residues on zinc-induced interaction of the metal-binding domain of ß-amyloid with DNA].

Interaction of intranuclear ß-amyloid with DNA is considered to be a plausible mechanism of Alzheimer's disease pathogenesis. The interaction of single- and double-stranded DNA with synthetic peptides was analyzed using surface plasmon resonance. The peptides represent the metal-binding domain of ß-amyloid (amino acids 1-16) and its variants with chemical modifications and point substitutions of amino acid residues which are associated with enhanced neurotoxicity of ß-amyloid in cell tests. It has been shown that the presence of zinc ions is necessary for the interaction of the peptides with DNA in solution. H6R substitution has remarkably reduced the ability of domain 1-16 to bind DNA. This is in accordance with the supposition that the coordination of a zinc ion by amino acid residues His6, Glu11, His13, and His14 of the ß-amyloid metal-binding domain results in the occurrence of an anion-binding site responsible for the interaction of the domain with DNA. Zinc-induced dimerization and oligomerization of domain 1-16 associated with phosphorylation of Ser8 and the presence of unblocked amino- and carboxy-terminal groups have resulted in a decrease of peptide concentrations required for detection of the peptide-DNA interaction. The presence of multiple anion-binding sites on the dimers and oligomers is responsible for the enhancement of the peptide-DNA interaction. A substitution of the negatively charged residue Asp7 for the neutral residue Asn in close proximity to the anion-binding site of the domain 1-16 of Aß facilitates the electrostatic interaction between this site and phosphates of a polynucleotide chain, which enhances zinc-induced binding to DNA.

[Splice variants of mRNA of cytochrome P450 genes: analysis by the nanopore sequencing method in human liver tissue and HepG2 cell line]. / Splais-varianty mRNK genov tsitokhromov R450: analiz metodom nanoporovogo sekvenirovaniia v tkani pecheni cheloveka i kletkakh linii HepG2.

The analysis of cytochrome P450 transcripts was carried out by the nanopore sequencing in liver tissue samples of three donors and HepG2 line cells. It has been demonstrated that direct mRNA sequencing with a MinION nanopore sequencer (Oxford Nanopore Technologies) allows one to obtained quantitative profiles for transcripts (and their splice variants) of cytochrome P450 superfamily genes encoding isoforms involved in metabolism of the large (~80%) part of drugs. The splice variant profiles substantially differ for donors. The cytochrome P450 gene expression at the transcript level is significantly weaker in cells of the HepG2 line compared with that in the normal liver tissue. This limits the capability of the direct mRNA nanopore sequencing for studying alternative splicing of cytochrome P450 transcripts in HepG2 cells. Both quantitative and qualitative profiles of the cytochrome P450 gene expression at the transcript level are notably differ in human liver tissue and HepG2 cells.

Comparative analysis of expression of human telomerase catalytic subunit at the transcription level in cell cultures of different origin.

The expression of human telomerase catalytic subunit in HL-60 and HT-1080 malignant transformed cells and telomerized fibroblasts was studied by quantitative PCR. It was found that the number of transcripts of human telomerase catalytic subunit per cell in telomerized fibroblasts could be hundreds of times higher than in HL-60 and HT-1080 cells. Telomerized fibroblast cultures are suggested as experimental systems for selection of basal compounds for creation of anticancer drug prototypes, the molecular target of which is human telomerase catalytic subunit. The effects of human telomerase catalytic subunit expression on the fibroblast proteome are analyzed.

[PCR analysis of the expression of chromosome 18 genes in human liver tissue: interindividual variability]. / PTsR-analiz ékspressii genov khromosomy 18 v tkani pecheni cheloveka: mezhindividual'naia variabel'nost'.

Using human chromosome 18 (Ch18) genes as an example, a PCR analysis of the interindividual variability of gene expression in liver tissue was performed. Although the quantitative profiles of the Ch18 transcriptome, expressed in the number of cDNA copies per single cell, showed a high degree of correlation between donors (Pearson correlation coefficients ranged from 0.963 to 0.966), the expression of the significant number of genes (from 13% to 19%, depending on the method of experimental data normalization) varied by more than 4-fold when comparing donors pairwise. At the same time, the proportion of differentially expressed genes increased with a decrease in the level of their expression. It is shown that the higher quantitative variability of low-abundance transcripts is mainly not technical, but biological. Bioinformatic analysis of the interindividual variability of the differential expression of chromosome 18 genes in human liver tissue did not reveal any statistically significant groups of genes related to certain biological processes that indicated a rather transient nature of the interindividual variability of their expression, probably reflecting the response of cells of an individual to specific external stimuli.

[Evaluation of the diversity of random DNA-libraries by the shape of amplification curves for estimation of the efficiency of aptamer selection]. / Otsenka raznoobraziia kombinatornykh DNK-bibliotek na osnove analiza formy amplifikatsionnykh krivykh dlia monitoringa éffektivnosti selektsii aptamerov.

Using random (combinatorial) DNA-libraries with various degrees of diversity, it was shown that their amplification by polymerase chain reaction in real time resulted in appearance of a maximum on amplification curves. The relative decrease of fluorescence after passing the maximum was directly proportional to the logarithm of the number of oligonucleotide sequence variants in the random DNA-library provided that this number was within in the interval from 1 to 104 and remained practically unaltered when the number of variants was in the interval from 105 to 108. The obtained dependence was used in the course of SELEX to evaluate changes in the diversity of random DNA-libraries from round to round in selection of DNA-aptamers to the recombinant SMAD4 protein. As a result, oligonucleotides containing sequences able to form a site of SMAD4-DNA interactions known as SBE (SMAD-binding element) have been selected thus indicating that the SMAD4-SBE interaction dominates the aptamer selection.

[Use of DNA-aptamers for enrichment of low abundant proteins in cellular extracts for quntitative detection by selected reaction monitoring]. / Primenenie DNK-aptamerov dlia obogashcheniia nizkopredstavlennykh belkov v kletochnykh ékstraktakh dlia kolichestvennoi detektsii metodom monitoringa vybrannykh reaktsii.

The relationship between the amount of a target protein in a complex biological sample and its amount measured by selected reaction monitoring (SRM) mass spectrometry upon the affinity enrichment of target protein with aptamers immobilized on a solid phase was studied. Human thrombin added in known concentrations to cellular extracts derived from bacterial cells was used as model target protein. It has been demonstrated that the affinity enrichment of thrombin in cellular extracts by means of the thrombin-binding aptamer immobilized on the surface of magnetic microbeads results in an approximately 10-fold increase of the concentration of target protein and a 100-fold decrease of the low limit of a target protein concentration range where its quantitative detection by SRM is possible without an interference from other peptides present in a tryptic digest.

[PCR analysis of the absolute number of copies of human chromosome 18 transcripts in liver and HepG2 cells]. / PTsR-analiz absoliutnogo chisla kopii transkriptov khromosomy 18 cheloveka v kletkakh pecheni i linii HepG2.

Using reverse transcription in conjunction with the quantitative real-time PCR or digital droplet PCR, the transcriptome profiling of human chromosome 18 has been carried out in liver hepatocytes and hepatoblastoma cells (HepG2 cell line) in terms of the absolute number of each transcript per cell. The transcript abundance varies within the range of 0.006 to 9635 and 0.011 to 4819 copies per cell for HepG2 cell line and hepatocytes, respectively. The expression profiles for genes of chromosome 18 in hepatocytes and HepG2 cells were found to significantly correlate: the Spearman's correlation coefficient was equal to 0.81. The distribution of frequency of transcripts over their abundance was bimodal for HepG2 cells and unimodal for liver hepatocytes. Bioinformatic analysis of the differential gene expression has revealed that genes of chromosome 18, overexpressed in HepG2 cells compared to hepatocytes, are associated with cell division and cell adhesion processes. It is assumed that the enhanced expression of those genes in HepG2 cells is related to the proliferation activity of cultured cells. The differences in transcriptome profiles have to be taken into account when modelling liver hepatocytes with cultured HepG2 cells.

[Digital droplet PCR - a prospective technological approach to quantitative profiling of microRNA]. / Tsifrovaia kapel'naia PTsR - perspektivnyi tekhnologicheskii podkhod k kolichestvennomu profilirovaniiu mikroRNK.

MicroRNA is a special type of regulatory molecules governing gene expression. Circulating microRNAs found in blood and other biological fluids are considered today as potential biomarkers of human pathology. Presently, quantitative alterations of particular microRNAs are revealed for a large number of oncological diseases and other disorders. The recently emerged method of digital droplet PCR (ddPCR) possesses a number of advantages making this method the most suitable for verification and validation of perspective microRNA markers of human pathologies. Among these advantages are the high accuracy and reproducibility of microRNA quantification as well as the capability to directly measure the absolute number of microRNA copies with the large dynamic range and a high throughput. The paper reviews microRNA biogenesis, the origin of circulating microRNAs, and methods used for their quantification. The special technical features of ddPCR, which make it an attractive method both for studying microRNAs as biomarkers of human pathologies and for basic research devoted to aspects of gene regulation by microRNA molecules, are also discussed.

[Physico-chemical methods for studing ß-amyloid aggregation].

Alzheimer's disease is the most prevalent neurodegenerative pathology. According to the amyloid cascade hypothesis, a key event of the Alzheimer's disease pathogenesis is a transition of the ß-amyloid peptide (Аß) from the monomeric form to the aggregated state. The mechanism of Ðß aggregation is intensively studied in vitro, by means of synthetic peptides and various physico-chemical methods allowing evaluation of size, molecular structure, and morphology of the formed aggregates. The paper reviews both the well-known and recently introduced physico-chemical methods for analysis of Ðß aggregation, including microscopу, optical and fluorescent methods, method of electron paramagnetic resonance, electrochemical and electrophoretic methods, gel-filtration, and mass spectrometric methods. Merits and drawbacks of the methods are discussed. The unique possibility to simultaneously observe Ðß monomers as well oligomers and large aggregates by means of atomic force microscopy or fluorescence correlation spectroscopy is emphasized. The high detection sensitivity of the latter method, monitoring the aggregation process in Ðß solutions at low peptide concentrations is underlined. Among mass spectrometric methods, the ion mobility mass spectrometry is marked out as a method enabling to obtain information about both the spectrum of Ðß oligomers and their structure. It is pointed out that the use of several methods giving the complementary data about Ðß aggregates is the best experimental approach to studying the process of b-amyloid peptide aggregation in vitro.

[Atomic force microscopy fishing of gp120 on immobilized aptamer and its mass spectrometry identification].

A method of atomic force microscopy-based fishing (AFM fishing) has been developed for protein detection in the analyte solution using a chip with an immobilized aptamer. This method is based on the biospecific fishing of a target protein from a bulk solution onto the small AFM chip area with the immobilized aptamer to this protein used as the molecular probe. Such aptamer-based approach allows to increase an AFM image contrast compared to the antibody-based approach. Mass spectrometry analysis used after the biospecific fishing to identify the target protein on the AFM chip has proved complex formation. Use of the AFM chip with the immobilized aptamer avoids interference of the antibody and target protein peaks in a mass spectrum.

Capillary zone electrophoresis of rigid submicron-sized particles in polyacrylamide. Solution selectivity, peak spreading and resolution.

Submicron-sized rigid particles can be separated in a size-dependent fashion by electrophoresis in free solution. Yet it has remained unknown whether the presence of polymers in the solution confers an advantage in size-dependent separation of submicron particles and their resolution. The present study addresses that question, using capillary zone electrophoresis of carboxylate modified polystyrene latex microspheres of 55, 140 and 215 nm radius in solutions of linear polyacrylamide in the M(r) range of 0.4.10(6) to 1.14.10(6). Selectivity of particle separation increases in direct relation to the polymer concentration in the concentration range of 0 to 1% (w/v). Selectivity was found to increase with M(r) of the polymer for the particle sets of 55/140 (nm/nm) and 140/215 (nm/nm) but to decrease with polymer M(r) for the 55/215 (nm/nm) set. Peak spreading is a complex and, in the case of the largest particle, non-monotonic function of polymer concentration, with a minimum at concentrations around the entanglement threshold, c*. Consequently, resolution of the 55/215 and 140/215 (nm/nm) sets also exhibits a maximum around the entanglement threshold while resolution for the 55/140 (nm/nm) set increases with a rise of polymer concentrations above c*. Within the range of optimally resolving polymer concentrations there also occurs a maximum of resolution for all particle sets at a field strength in the range of 150 to 250 V cm-1.

Protein band spreading in capillary zone electrophoresis effects of sample zone length and presence of polymer.

It is commonly accepted that intra-column zone dispersion in CZE rests on multiple mechanisms, viz. diffusion, interaction of analyte with the capillary walls, Joule heat and conductivity differences between sample zone and the surrounding buffer. The most important extra-column contributor to bandwidth is thought to be the starting zone width. The present study shows that the length of the starting zone above 10 mm is linearly related to the bandwidth of R-phycoerythrin (M(r) 290.10(3)). Below that length, bandwidth demonstrates a plateau preceded by a slight rise. Within the 'plateau range', the ratio of bandwidth to effective capillary length is close to constant while it is independent of electric field strength in the range of 37 to 370 V cm-1 and of protein concentration in the range of 0.1 to 1000 micrograms ml-1. The experimental observations support the notion that the analyte-wall interaction is the determining source of intra-column zone dispersion. A slight rise observed at initial zone lengths of less than 2 mm was accounted for by a diffusion model taking into account a non-local initial concentration of analyte. The presence of polyethyleneglycol in the buffer within a concentration range up to 6% does not affect bandwidth. Above that concentration, the level of constant bandwidth is raised.

Asymmetry of protein peaks in capillary zone electrophoresis: effect of starting zone length and presence of polymer.

The asymmetry of R-phycoerythrin (M(r) = 240,000) peaks in capillary zone electrophoresis measured as In[(tm-t1)/(t2-tm)], where tm, t1 and t2 are migration times of the peak mode and at the intersection of the peak width at half-height with the ascending and descending limbs, respectively, was found to undergo a transition from negative to positive values with increasing starting zone length. The transition is compatible with a mathematical model of peak dispersion which assumes that an interaction of protein with the capillary walls governs the evolution of the peak during capillary zone electrophoresis. Models assuming a final peak shape defined solely by longitudinal diffusion, or by a heterogeneity with regard to mobility or by a conductivity difference between analyte zone and background electrolyte, have failed to give rise to a change in the sign of peak asymmetry when the starting zone length is varied. The presence of polyethylene glycol in the buffer within a concentration range up to 4% does not appreciably affect the peak asymmetry regardless of whether the concentration regime is dilute or semi-dilute. Above 4% of polyethylene glycol, the asymmetry becomes nearly independent of starting zone length, and progressively negative with increasing polymer concentration. The concentration range at which the transition from negative to positive asymmetry disappears coincides with that at which the average mesh size of the polymer network falls below the size of the protein.

Mechanisms of retardation of rigid spherical particles with 3 to 1,085 nm radius in capillary electrophoresis, using buffered polyacrylamide (molecular weight 5 x 10(6)) solutions.

Subjecting particles in the size range of 3 to 1085 nm radius (R) to capillary electrophoresis in buffered solution of entangled uncrosslinked polyacrylamide (M(r) 5 x 10(6)), it was found that particle size-dependent retardation ("molecular sieving") becomes electric field- and particle size range-dependent once the particle size exceeds 15-20 nm in radius. The field strength dependence of the retardation coefficient [KR = d(log mobility)/ d(polymer concentration] and the positive or negative sign of dKR/dR suggest the existence of two different mechanisms of molecular sieving depending on the particle size range: particles with diameters less than the screening length (or blob size) of the polymer network are thought to penetrate into the available spaces within a discontinuous polymer network; particles with diameters larger than the screening length (or blob size) of the polymer network are thought to undergo size-dependent retardation by exerting shear stress against polymer chains, and displacing them, so as to cause local deformations in a continuous polymer network. A limit in the separating capacity of molecular sieving, due to a sharp increase in the rate of band widening with polymer concentration, was found when the value of the retardation coefficient exceeded 60 (mL/g).

Electrophoresis of proteins in semidilute polyethylene glycol solutions: mechanism of retardation.

The retardation of proteins in the M(r) range of 15-500 kDa in capillary electrophoresis conducted in semidilute solutions of the polymer polyethylene glycol (M(r) range 0.2-8.0 X 10(6)), was measured. The purpose was to test the predictions of the scaling theory with regard to the relation of retardation to (a) the M(r) of the polymer, (b) the concentration of the polymer, and (c) the radius of the protein particles. These predictions derive from a mechanism that relates retardation to the screening length of the polymer solution, viewed as the average distance between the entanglement points of polymer chains. For the molecular weight range from 60 to 500 kDa of (near) spherical proteins, the retardation was found to be related to polymer concentration c as mu/mu(0) = exp(-Ac0.69) where mu/mu(0) is the retardation expressed as the ratio between the mobility in polymer solution and that in free solution. The value of the exponent of 0.69 is in close agreement with the value of 0.75 predicted by the scaling theory. Parameter A was found (a) to scale as the 0.04th power of M(r) (polymer), approximating the predicted value of 0; and (b) to be proportional to particle radius as predicted. All measured values of retardation were independent of electric field strength in the range of 37-370 V/cm. Thus, experimental findings are consistent with the mechanism relating electrophoretic retardation to the screening length of the polymer network in the specified molecular weight range of proteins. Under the same conditions, log(mu/mu(0)) of proteins with M(r)'s less than 60 kDa (a) scales as the -0.06th power of M(r) (polymer), and (b) is proportional to polymer concentration, suggesting a retardation mechanism that is not related to the screening length.