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Biomedical subjects

A S Krylov

Publications and source records attributed to A S Krylov.

At least 19 recordsLinked to original sources
PubMed2003

Analysis of binding specificity of disulfide bonded dimeric lambda-Cro V55C protein with generic hexamer oligonucleotide microchip.

Binding specificity of mutant V55C disulfide bonded dimeric lambda-Cro protein (CroVC) to double-stranded DNA (dsDNA) was studied using generic hexamer oligonucleotide microchip. The curves of dissociation of hybridized DNA in the presence and absence of CroVC were converted into the effective discriminant constants to assess the relevant thermodynamic equilibrium binding constants for dsDNA-protein complexes. Then, tiling of longer oligonucleotides with shorter oligomers was used to search for sequence motifs with the highest binding specificity similarly to sequencing by hybridization. The comparison of the deduced sequences with the known natural operator half-sites demonstrated the principal ability to discern and reconstruct the major parts of 7-mer motifs corresponding to the strongest binding of CroVC subunits. Our results show the applicability of generic microchips to the analysis of binding specificity in the case of multi-subunit DNA-binding proteins.

Bacteriophage lambda↗
PubMed2002

Specificity of mammalian Y-box binding protein p50 in interaction with ss and ds DNA analyzed with generic oligonucleotide microchip.

p50 protein is a member of the Y-box binding transcription factor family and is a counterpart of YB-1 protein. The generic microchip was used to analyze the sequence specificity of p50 binding to single (ss) and double-stranded (ds) oligodeoxyribonucleotides. The generic microchip contained 4,096 single-stranded octadeoxyribonucleotides in which all possible core 6-mers (4(6)=4,096) were flanked at their 3' and 5'-ends with degenerated nucleotides. The oligonucleotides were chemically immobilized within polyacrylamide gel pads fixed on a glass slide. The binding of p50 to the generic microchip was shown to be the most specific to ss GGGG motif and then to ss CACC and CATC motifs. GC-rich ds oligonucleotides of the generic microchip, and particularly those containing GGTG/CACC, GATG/CATC, and GTGG/CCAC heterogeneous motifs, were most efficiently destabilized due to interaction with p50. Gel-shift electrophoresis has shown that the protein exhibits much higher binding specificity to 24-mer oligoA-TGGGGG-oligoA containing G-rich 6-mer, in comparison with 24-mer oligoA-AAATAT-oligoA carrying A,T-rich 6-mer in full correspondence with the data obtained with the microchip. Studies of DNA-binding proteins using gel-immobilized ss and ds DNA fragments provide a unique possibility to detect low-affinity complexes of these proteins with short sequence motifs and assess the role of these motifs in sequence-specific interactions with long recognition sites.

Animals↗
PubMed2001

Massive parallel analysis of the binding specificity of histone-like protein HU to single- and double-stranded DNA with generic oligodeoxyribonucleotide microchips.

A generic hexadeoxyribonucleotide microchip has been applied to test the DNA-binding properties of HU histone-like bacterial protein, which is known to have a low sequence specificity. All 4096 hexamers flanked within 8mers by degenerate bases at both the 3'- and 5'-ends were immobilized within the 100 x 100 x 20 mm polyacrylamide gel pads of the microchip. Single-stranded immobilized oligonucleotides were converted in some experiments to the double-stranded form by hybridization with a specified mixture of 8mers. The DNA interaction with HU was characterized by three type of measurements: (i) binding of FITC-labeled HU to microchip oligonucleotides; (ii) melting curves of complexes of labeled HU with single-stranded microchip oligonucleotides; (iii) the effect of HU binding on melting curves of microchip double-stranded DNA labeled with another fluorescent dye, Texas Red. Large numbers of measurements of these parameters were carried out in parallel for all or many generic microchip elements in real time with a multi-wavelength fluorescence microscope. Statistical analysis of these data suggests some preference for HU binding to G/C-rich single-stranded oligonucleotides. HU complexes with double-stranded microchip 8mers can be divided into two groups in which HU binding either increased the melting temperature (T(m)) of duplexes or decreased it. The stabilized duplexes showed some preference for presence of the sequence motifs AAG, AGA and AAGA. In the second type of complex, enriched with A/T base pairs, the destabilization effect was higher for longer stretches of A/T duplexes. Binding of HU to labeled duplexes in the second type of complex caused some decrease in fluorescence. This decrease also correlates with the higher A/T content and lower T(m). The results demonstrate that generic microchips could be an efficient approach in analysis of sequence specificity of proteins.

Bacterial Proteins↗
PubMed2000

Structure-apoptotic potency evaluations of novel sterols using human leukemic cells.

Three oxidized analogs of cholesterol have been characterized for their ability to cause apoptotic cell death in CEM-C7-14 human leukemic cells. In addition to testing 15-ketocholestenol (K15), 15-ketocholestenol hydroxyethyl ether (CK15), and 7-ketocholesterol hydroxyethyl ether (CK7), an oxysterol of known apoptotic response, 25-hydroxycholesterol (25OHC), served as a standard for comparison. Growth studies based on dye exclusion by viable cells while using a sublethal concentration of oxysterols ranked their potency for cell kill as 25OHC > K15 > CK15 > CK7. Both the TUNEL assay (terminal deoxynucleotidyl transferase-mediated dUTP-X nick end labeling), which quantifies the amount of DNA nicks caused by a toxic agent, and the MTT assay, which measures cell metabolism and thus reflects cell viability, substantiated the same rank order. An ELISA assay for evaluating release of DNA fragments into the cytosol after treatment gave a similar potency order. The oncogene c-myc mRNA was suppressed by all three oxysterols, with 25OHC and K15 being the most potent suppressors. Hoechst and Annexin V staining documented that these oxysterols kill cells by an apoptotic pathway as evidenced by condensation of nuclear chromatin and plasma membrane inversion, respectively. From these in vitro studies, we believe that 25OHC, K15, and possibly CK15 have the potential to be chemotherapeutic agents.

Apoptosis↗
PubMed1997

[Affinity of 3-beta-(2-hydroxyethoxy)-5-alpha-cholest-8(14)-ene-15-one to oxysterol binding protein and its metabolism in HepG2 hepatoma cells].

beta-(2-Hydroxyethoxy)-5 alpha-cholest-8(14)-en-15-one, a synthetic inhibitor of cholesterol biosynthesis, was shown to exhibit a high affinity to oxysterol binding protein. This was proved by ultracentrifugation of the protein fraction from rabbit liver in the presence of the 3H-labeled inhibitor, 3 beta-(2-hydroxy-2-[3H]ethoxy)-5 alpha-cholest-8(14)-en-15-one, or by the substitution of the [3H]-25-hydroxycholesterol in its complex with the oxysterol binding protein. In human hepatoma Hep G2 cells, the inhibitor decreased activity of 3-hydroxy-3-methylglutaryl CoA reductase [ID50 (2.7 +/- 0.7) x 10(-5) M] and was transformed into 3 beta-[2-(9-Z-octadecenoyloxy)ethoxy]-5 alpha-cholest-8(14)-en-15-one.

Animals↗
PubMed1984

[Isolation and properties of beta-lactamase of the cephalosporinase type from cells of Enterobacter aerogenes 6803].

beta-Lastamase with the molecular weight of 32500 was isolated from the cells of clinical strain 6803 of Enterobacter aerogenes and purified. By the substrate profile determined microiodometrically beta-lactamase was classified as belonging to the cephalosporinase type. The activity of the electrophoretically homogenous enzyme was equal to 430 microM a minute per mg protein with respect to benzylpenicillin. The Km for benzylpenicillin, dicloxacillin, cephaloridin and cephalothin was 6.5410(-5), 3 X 10(-4), 2.1 X 10(-5) and 5.7 X 10(-5) M, respectively. The isoelectric point of the enzyme equal to 5.45 was estimated with the method of preparative isoelectrofocusing. The presence of the serine residue or residues was shown with the use of selective reagents applied to the functionally important groups. With the method of circular dichroism the ratio of alpha- and beta-structures in the enzyme molecule was determined, the slow hydrolysis of cephazolin was demonstrated and the values of Km and Kcat for this process were estimated.

Cephalosporinase↗
PubMed1984

[Determination of beta-lactamase types of clinical strains of bacteria using a modified microiodometric method].

The substrate profiles and sensitivity to dicloxacillin inhibition were studied in the enzymes of the clinical strains of Pseudomonas aeruginosa and the transconjugants of E. coli carrying the plasmids discovered earlier in P. aeruginosa. The study was performed with a modified microiodometric method for determination of the activity of beta-lactamases. According to the M. Richmond classification of beta-lactamases the enzymes detected in P. aeruginosa strains 4529, 5290 and 9902 may correspond to the 5th class, the enzymes of P. aeruginosa strain 8208 to the 2nd class and the beta-lactamases of the E. coli transconjugants to the 3rd class. Two different beta-lactamases were detected in P. aeruginosa strain 10294.

Bacteria↗
PubMed1979

Quantitative estimation of the contribution of pyrrolcarboxamide groups of the antibiotic distamycin A into specificity of its binding to DNA AT pairs.

Interaction of DNA with the analogs of the antibiotic distamycin A having different numbers of pyrrolcarboxamide groups and labeled with dansyl was studied. The binding isoterms of the analogs to synthetic polydeoxyribonucleotides were obtained. Analysis of the experimental data leads to the following conclusions: (1) the free energy of binding of the analogs to poly(dA).poly(dT) depends linearly on the number of amide groups in the molecule of the analog whereas attachment of each pyrrolcarboxamide group produces changes of 2 kcal/mole in the free energy; (2) attachment of a pyrrolcarboxamide unit to the GC pair results in the free energy change of 0.95 kcal/mole; (3) the binding of analogs to poly(dA).poly(dT) is a cooperative process, presumbly, dependent on conformational changes induced by the binding of analogs to DNA.

Adenine↗
PubMedDate not supplied

[Dye with low specificity to nucleotide sequences of DNA: use for assessing the quantity of oligonucleotides, immobilized in cells of biological microchips].

To assess the DNA amount in samples (e.g., in biological microchip gel pads) by means of fluorescent dyes, one should use the dyes whose fluorescence weakly depends on DNA composition and structure. With the ImD-310 dye created for this purpose, we have analyzed the staining of single- and double-stranded oligo- and polynucleotides of different nucleotide composition, length, and concentration both in solution and being immobilized in biological microchip gel pads. It turned out that ImD-310 has no pronounced specificity to the single- and double-stranded nucleotide sequences, while the intensity of fluorescence for the dye complexes with d(A)8, d(T)8, d(C)8, and d(G)8 at high temperatures (50 degrees C) differs by less than 25%. A linear correlation has been established between the intensity of fluorescence and the amount of oligonucleotides immobilized on a biological microchip. The plots of the intensity of fluorescence against the concentration of NaCl and the temperature were obtained. By using a generic microchip containing all 4096 hexamer oligonucleotides, it has been determined that the dye has no distinct specificity to any certain motifs of the nucleotide sequence. Thus, ImD-310 may serve as an efficient fluorescent probe to quickly estimate the amount of oligonucleotides immobilized in a microchip, in an electrophoretic gel, etc.

Base Sequence↗
PubMedDate not supplied

[The formation of liquid-crystalline dispersions of DNA-chitosan complexes under the conditions of molecular crowding].

The formation of liquid-crystalline dispersions from DNA-chitosan complexes in polyethyleneglycol-containing solutions was studied. It was shown that the molecular crowding affects neither the efficiency of binding of chitosan molecules to DNA nor the mode of spatial packing of DNA-chitosan complexes in particles of liquid-crystalline dispersions.

Chitosan↗