Biomedical subjects

Taras Shevchuk

Publications and source records attributed to Taras Shevchuk.

5 recordsLinked to original sources

PubMed2005

The use of prokaryotic DNA methyltransferases as experimental and analytical tools in modern biology.

Prokaryotic DNA methyltransferases (MTases) are used as experimental and research tools in molecular biology and molecular genetics due to their ability to recognize and transfer methyl groups to target bases in specific DNA sequences. As a practical tool, prokaryotic DNA MTases can be used in recombinant DNA technology for in vitro alteration and enhancing of cleavage specificity of restriction endonucleases. The ability of prokaryotic DNA MTases to methylate cytosine residues in specific sequences, which are also methylated in eukaryotic DNA, makes it possible to use them as analytical reagent for determination of the site-specific level of methylation in eukaryotic DNA. In vivo DNA methylation by prokaryotic DNA MTases is used in different techniques for probing chromatin structure and protein-DNA interactions. Additional prospects are opened by development of the methods of DNA methylation targeted to predetermined DNA sequences by fusion of DNA MTases to DNA binding proteins. This review will discuss the application of prokaryotic DNA MTases of Type II in the methods and approaches mentioned above.

Animals↗

PubMed2005

Construction of ordered protein arrays.

Artificially ordered protein arrays provide a facile approach to a variety of problems in biology and nanoscience. Current demonstration systems use either nucleic acid tethers or methyltransferase fusions in order to target proteins or peptides of interest to nucleic acid scaffolds. These demonstrations point to the large number of useful devices and assemblies that can be envisioned using this approach, including smart biological probes and drug delivery systems. In principle, these systems are now capable of imitating the earliest forms of prebiotic organisms and can be expected to reach the complexity of a small virus in the near future. Third-generation methyltransferase inhibitors provide an example of a smart chemotherapeutics that can be constructed with this approach. We describe the use of mechanistic enzymology, computer-aided design, and microfluidic chip-based capillary electrophoresis in assessing the final assembly and testing of designs of this type.

Deoxyribonucleases, Type II Site-Specific↗

PubMed2004

Prostate cancer molecular markers GSTP1 and hTERT in expressed prostatic secretions as predictors of biopsy results.

OBJECTIVES: To develop noninvasive diagnostic tools for the early detection of prostate cancer (PCa). Current screening for PCa lacks sensitivity and specificity. Two molecular markers, telomerase activity and aberrant methylation of the glutathione S-transferase P1 (GSTP1) promoter, are found in more than 90% of PCa specimens. Additionally, these markers can be detected in bodily fluids such as urine and postprostatic massage urethral washes. METHODS: Expressed prostatic secretions (EPS) from men being evaluated for PCa were analyzed for human telomerase reverse transcriptase (hTERT) expression (the critical factor for telomerase activity) and GSTP1 methylation status. The results were compared with the prostate needle biopsy findings. RESULTS: EPS could be obtained from 86% of all subjects, and 90% of these samples yielded sufficient RNA and/or DNA for assaying. hTERT expression from EPS (n = 49) had 36% sensitivity and 66% specificity, and GSTP1 methylation from EPS (n = 58) had 46% sensitivity and 56% specificity for the detection of PCa. The combined analysis (n = 32) of hTERT and GSTP1 had 73% sensitivity and 43% specificity, giving a positive predictive value of 40% and a negative predictive value of 75%. CONCLUSIONS: These results demonstrate that EPS can be successfully obtained and easily tested for hTERT expression and GSTP1 methylation. Tests with a high negative predictive value, such as our combination assay results, could be useful in augmenting current PCa diagnostic procedures. For example, the examination of EPS for hTERT and GSTP1 methylation in patients with an elevated prostate-specific antigen level might be used in predicting which patients will have negative biopsies. The use of this assay could potentially eliminate up to 30% of costly and invasive needle biopsies.

Adenocarcinoma↗

PubMed2003

Methods for the design and analysis of oligodeoxynucleotide-based DNA (cytosine-5) methyltransferase inhibitors.

Several second-generation inhibitors of DNA (cytosine-5) methyltransferases based on studies of modified synthetic oligodeoxynucleoides have been described. As an aid to studies of these inhibitors, we present an electronic structure-based algorithm that can be used as a method for predicting the nature of the expected inhibition by any noncytosine nucleotide target. Targeting by the major human enzyme (hDnmt1) is governed by the presence of a three-nucleotide motif. In hemimethylated DNA, this motif consists of a 5-methylcytosine targeting signal that causes the enzyme to probe the opposite strand for a normally paired guanosine or inosine residue and attempt to methylate the residue 5' to that site. As a demonstration of the method, we apply these rules to the design and characterization of a novel oligodeoxynucleotide inhibitor of hDnmt1. This inhibitor takes advantage of the three-nucleotide recognition motif characteristic of hDnmt1 and shows that the enzyme is inhibited in vitro by non-CG methylation which targets the enzyme to normally basepaired but unproductive nucleotides such as dG, dA, and dT. Kinetic analysis at constant S-adenosyl-L-methionine concentration shows that representative inhibitory oligodeoxynucleotides are best viewed as weakly productive components of systems containing two DNA substrates. This model suggests that the most effective inhibitors are those with very low apparent Vmax and very low Km values. Oligodeoxynucleotides containing mispaired and unproductive targets such as dG, dA, dT, and dU are also inhibitory as secondary substrates for the human enzyme. Biologically, fail-safe mechanisms identified by the ab initio approach appear to be active in preventing potentially mutagenic deamination of dihydrocytosine and enzymatic methylation of dU.

Base Sequence↗

PubMed2003

Mobility-shift analysis with microfluidics chips.

Electrophoretic mobility shift analysis (EMSA) is a well-characterized and widely used technique for the analysis of proten-DNA interaction and the analysis of transcription factor combinatorics. Currently implemented EMSA generally involves the time-consuming use of radiolabeled DNA and polyacrylamide gel electrophoresis. We are studying the bionanoscience of self-assembling supramolecular protein-nucleic nanostructures. We have undertaken these studies because they promise to enhance our understanding of assemblies formed during prebiotic evolution, provide tools for analysis of biological processes like DNA recombination, and may lead to the development of nanoscale biosensors designed for site-specific molecular targeting. During the course of that work, we noted that EMSA of these complex structures could be effectively implemented with microfluidics chips designed for the separation of DNA fragments. In this report we compare the two techniques and demonstrate that the microfluidics system is also capable of resolving complex mixtures produced by decorating DNA recombination intermediates with mixtures of DNA binding proteins. Moreover, the microfluidics chip system improves EMSA by permitting analysis with smaller samples, avoiding the use of radiolabeling, and reducing the time involved to a matter of minutes.

Computer Simulation↗