Publications (5)16.04 Total impact
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Article: A reliable method to concentrate circulating DNA.
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ABSTRACT: Concentration of circulating DNA probes is required to increase the amount of DNA involved in subsequent study (by polymerase chain reaction, sequencing, and microarray). This work was dedicated to the comparison of five different methods used for concentration of DNA circulating in blood. Precipitation of circulating DNA with acetone in the presence of triethylamine provides minimal DNA loss, high reproducibility, and at least three times higher DNA yield in comparison with the standard ethanol protocol.Analytical Biochemistry 01/2011; 408(2):354-6. · 3.00 Impact Factor -
Article: Inhibition of metastasis development by daily administration of ultralow doses of RNase A and DNase I.
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ABSTRACT: Recent data on the involvement of miRNA and circulating tumor-derived DNA in regulation of tumorigenesis showed a great prospect for these molecules as a novel class of therapeutic targets and gave a new start for the study of enzymes cleaving nucleic acids as potential antitumor and antimetastatic agents. In the present paper using two murine tumor models with pulmonary or liver metastases we studied the antimetastatic potential of RNase A and DNase I and performed a search for possible molecular targets of the enzymes. Herein, we show for the first time that daily administration of ultralow doses of RNase A (0.5-50 μg/kg) and DNase I (0.02-2.3 mg/kg) inhibits the development of metastasis to 60-90% and RNase A exerts 30% retardation of tumor growth. Remarkably, the increase in RNase A dose from 50 μg/kg to 10mg/kg leads to a disappearance of antitumor and antimetastatic effects. Simultaneous treatment of tumor-bearing animals with RNase A and DNase I leads to an additive effect and results in almost total absence of metastases. The use of RNase A as an adjuvant in conjunction with conventional cytostatic cyclophosphamide results in a reliable enhancement of antitumor and antimetastatic effect of the therapy compared with the use of these agents individually. The search for possible molecular mechanism of antimetastatic effect of nucleases showed that daily administration of the enzymes reduced the pathologically increased level of extracellular nucleic acids and increased nuclease activity of the blood plasma of tumor-bearing mice back to the level of healthy animals. Thus, we unequivocally show that the proposed protocol of treatment of tumor-bearing animals with RNase A and DNase I has a general systemic and immunomodulatory effect, leads to a drastic suppression of metastasis development, and in perspective may become an effective component of intensive complex therapy of cancer.Biochimie 12/2010; 93(4):689-96. · 3.02 Impact Factor -
Article: Immunochemical assay for deoxyribonuclease activity in body fluids.
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ABSTRACT: We have developed two microtiter plate assays to quantify the deoxyribonuclease activity in biological fluids. Both assays are based on hydrolysis of biotinylated and fluorescein-labeled DNA substrates, with subsequent immunochemical detection of non-digested DNA. The assay based on hydrolysis of 974 bp PCR product labeled with biotinylated forward and fluorescein-labeled reverse primers is more sensitive (0.05 U/ml) and convenient for quantifying the DNase activity in biological fluids than the assay based on hydrolysis of double-labeled 20 bp oligonucleotide. The DNase activity in urine and blood plasma of healthy donors was measured using the PCR product-based assay. Urine samples revealed greater activity, 1.49+/-1.41 U/ml; blood plasma DNase I-like activity was 0.36+/-0.20 U/ml. DNase II-like activity was not detected in the plasma samples. The data obtained confirm that DNase I-like enzymes are responsible for the majority of deoxyribonuclease activity in blood plasma.Journal of Immunological Methods 09/2007; 325(1-2):96-103. · 2.20 Impact Factor -
Article: Animal model of drug-resistant tumor progression.
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ABSTRACT: Experimental animal model of tumor progression based on mice lymphosarcoma (LS) and resistant lymphosarcoma (RLS) has been developed. LS tumor displays high sensitivity to cyclophosphamide, which is widely used in anticancer therapy. RLS tumor was derived from LS by passaging in mice receiving low concentration of cyclophosphamide (20 mg/kg) and display resistance to cyclophosphamide (up to dose 150 mg/kg). The primary cultures of LS and RLS tumors display different expression levels of the genes related to apoptosis and multiple drug-resistant phenotype: in RLS tumor high levels of mdr1b and bcl-2 genes and low level of p53 gene expression were found. A total of 10% of cells in RLS primary culture display multiple drug-resistant phenotype and survive even at high dose of cytostatics. Cultivation of RLS primary culture in the presence of increasing vinblastine concentrations gives RLS(40) cell culture, which exhibits high levels of mdr1a/1b genes expression as compared to RLS and 20-fold increase of resistance to cytostatics. Drug-resistant RLS(40) cells were transplanted into CBA mice and sensitivity of the tumors to anticancer drugs was tested. RLS(40) tumors were resistant to a number of cytostatics used in anticancer therapy (cyclophosphamide, cysplatin, vinblastine, rubomycinum). Thus, RLS(40) tumor can be used as model, which corresponds to tumor status observed in patients after one or several courses of chemotherapy and can be useful for testing conventional therapy alone or together with newly developed gene-targeted therapeutics.Annals of the New York Academy of Sciences 01/2007; 1091:490-500. · 3.15 Impact Factor -
Article: Knock down of cytosolic phospholipase A2: an antisense oligonucleotide having a nuclear localization binds a C-terminal motif of glyceraldehyde-3-phosphate dehydrogenase.
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ABSTRACT: We have previously shown that an antisense, effective in the knock down of cytosolic phospholipase A2 (cPLA2), localizes mainly in the nucleus of human endothelial cells and monocytes and that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is involved in its nuclear localization. In this study, we clarify how GAPDH participates in the nuclear localization of this antisense oligodeoxynucleotide (ODN) directed against cPLA2 mRNA. A central TAAAT motif providing specificity and high affinity binding was assumed to interact with the enzyme Rossmann fold region on the basis of competition to this site by NAD+. To asses whether the TAAAT motif interacts directly with the enzyme Rossmann fold region, we evaluated the binding to GAPDH of different oligonucleotides and the effect of competitors such as NAD+, NADH, mononucleotides, DNA, polyribonucleic acids and polyanions. We found that the dissociation constant for TAAAT containing oligonucleotides was three--to fivefold higher with respect to oligo not containing this motif. By covalently linking 32P-labeled cPLA2p(N)16 to GAPDH and after executing hydrolysis with hydroxylamine, the labeling was exclusively found in the C-terminal domain (aa 286-334). These results indicate that the antisense oligonucleotide interacts with a site not having a defined function but which can be negatively allosterically regulated when NAD+ or polynucleotides are bound to Rossmann fold.Biochimica et Biophysica Acta 04/2004; 1636(2-3):129-35. · 4.66 Impact Factor
