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Biomedical subjects

P P Laktionov

Publications and source records attributed to P P Laktionov.

At least 19 recordsLinked to original sources
PubMed2006

Cell-free and cell-bound circulating DNA in breast tumours: DNA quantification and analysis of tumour-related gene methylation.

Tumour development is characterised by the increased circulating DNA (cirDNA) concentration and by tumour-related changes in blood plasma DNA. Concentration of cirDNA and methylation of RARbeta2, RASSF1A and HIC-1 gene promoters were investigated in cell-free and cell-surface-bound fractions from healthy donors, patients with breast cancer, and patients with breast fibroadenoma. Tumour development was shown to lead to significant changes in the distribution of cirDNA between cell-free and cell-surface-bound fractions. Analysis of RARbeta2 and RASSF1A methylation in the total cirDNA provides 95% diagnostic coverage in breast cancer patients, 60% in patients with benign lesions, and is without false-positive results in healthy women. Results of the study indicate that methylation-specific PCR of RARbeta2 and RASSF1A genes based on the total cirDNA combined with the quantitative analysis of cirDNA distribution between cell-bound and cell-free fractions in blood provide the sensitive and accurate detection and discrimination of malignant and benign breast tumours.

Breast Neoplasms↗
PubMed2006

Proteins involved in binding and cellular uptake of nucleic acids.

The study of mechanisms of nucleic acid transport across the cell membrane is valuable both for understanding the biological function of extracellular nucleic acids and the practical use of nucleic acids in gene therapy. It has been clearly demonstrated that cell surface proteins are necessary for transport of nucleic acids into cells. A large amount of data has now been accumulated about the proteins that participate in nucleic acid transport. The methods for revealing and identification of these proteins, possible mechanisms of protein-mediated transport of nucleic acids, and cellular functions of these proteins are described.

Animals↗
PubMed2005

Plasma content of extracellular nucleic acids in donors and patients with mammary tumors.

The concentrations of extracellular DNA and RNA were measured in the plasma of donors and patients with fibroadenoma and breast cancer. The content of extracellular DNA surpassed the normal in 80% plasma samples from patients with mammary tumors. Extracellular RNA was detected in 30% plasma samples from donors and patients with breast tumors. No correlations were found between plasma concentration of extracellular DNA and size and stage of tumor growth. Hence, measurement of extracellular DNA in the plasma of patients can be used only as an accessory test for tumor diagnosis.

Breast Neoplasms↗
PubMed2003

Interaction of keratin K1 with nucleic acids on the cell surface.

The interaction of surface proteins from A431 cells and cellular extracts with nucleic acids was investigated using affinity modification with 32P-labeled reactive oligonucleotide derivatives. Proteins with molecular weights of 68, 46, 38, and 28 kD as well as several low molecular weight proteins capable of binding to nucleic acids were found on the surface of intact cells. It was demonstrated that a protein with molecular weight of 68 kD is exposed at the cell surface, since the treatment of cells with trypsin results in the cleavage of this protein. Disruption of the integrity of the cell membrane (scrapping, treatment with trypsin, or permeabilization of the cell membrane with streptolysin O or saponin) disrupts the interaction of the reactive oligonucleotides with the cell surface proteins. Affinity modification of the cytosolic and membrane-cytosolic cell fractions with labeled oligonucleotides results in the modification of a large number of proteins, where proteins with molecular weights of 68, 46, 38, and 28 kD can be found as minor components. Surface oligonucleotide-binding proteins with molecular weight of ~68 kD were isolated by affinity chromatography after the modification of intact A431 cells with a reactive oligonucleotide derivative. The isolated surface oligonucleotide-binding proteins from A431 cells were sequenced, and one of the proteins was identified as keratin K1.

Animals↗
PubMed2001

The Rossmann fold of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a nuclear docking site for antisense oligonucleotides containing a TAAAT motif.

The subcellular localisation of oligodeoxynucleotides (ODN) is a major limitation for their use against nuclear targets. In this study we demonstrate that an antisense ODN directed against cytosolic phospholipase A(2) (cPLA2) mRNA is efficiently taken up and accumulates in the nuclei of endothelial cells (HUVEC), human monocytes and HeLa cells. Gel shift experiments and incubation of cells with oligonucleotide derivatives show that the anti-cPLA2 oligo binds a 37 kDa protein in nuclear extracts. The TAAAT sequence was identified as the major binding motif for the nuclear protein in competition experiments with mutated ODNs. Modification of the AAA triplet resulted in an ODN which failed to localise in the nucleus. Moreover, inserting a TAAAT motif into an ODN localising in the cytosol did not modify its localisation. The 37 kDa protein was purified and identified after peptide sequencing as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). It was shown by confocal microscopy that GAPDH co-localises with anti-cPLA2 ODN in the nucleus and commercial GAPDH effectively binds the oligo. Competition experiments with increasing concentration of NAD(+) co-factor indicate that the GAPDH Rossmann fold is a docking site for antisense oligonucleotides containing a TAAAT motif.

Amino Acid Sequence↗
PubMed1999

Characterisation of membrane oligonucleotide-binding proteins and oligonucleotide uptake in keratinocytes.

Inadequate cellular compartmentalisation of plasmid DNA and antisense oligodeoxynucleotides (ODNs) is generally considered as a major limitation in their use. In this study, an approach combining in situ visual-isation of rhodamine-labelled ODNs and affinity modification of proteins by radiolabelled-alkylating ODN derivatives has been used to investigate the uptake of ODNs into keratinocytes. We confirm here that unmodified ODNs are efficiently taken up and accumulate in cell nuclei in primary keratinocytes as well as in HaCaT and A431 keratinocyte cell lines. Uptake is fast, irreversible, saturable and not significantly altered by incubation at low temperature. Affinity modification studies in keratinocyte cell lines has revealed two high-affinity, cell-specific interactions between ODNs and proteins of 61-63 kDa and 35 kDa. Trypsin pre-treatment of A431 cells and pre-incubation with polyanions, or with unlabelled nucleic acid competitors, inhibited the accumulation of rhodamine-labelled ODNs in nuclei as well as the affinity labelling of the 61-63 kDa doublet and 35 kDa ODN-binding proteins by reactive ODN derivatives. Finally, cell fractionation studies indicated that these ODN-binding proteins were essentially localised in the plasma membrane. Our results suggest that these ODN-binding proteins might be involved in the recognition and transport of ODNs into keratinocytes.

Cells, Cultured↗
PubMed1999

Activation of spleen lymphocytes by plasmid DNA.

Plasmid pUC19 DNA was shown to stimulate in vitro proliferation of CBA mouse splenocytes in a dose-dependent manner. Simultaneous treatment of the cells with the plasmid DNA and Con A or LPS produced an additive effect, while PMA acted synergistically with DNA. Monovalent Fab fragments of rabbit anti-mouse Ig (RAMIg) antibodies significantly inhibited plasmid DNA-induced polyclonal lymphocyte activation suggesting the involvement of Ig receptors in this process. Affinity modification of lymphocytes membrane-cytosole proteins with a 32P-labeled alkylating oligonucleotide derivative resulted in labeling of 67-82 and 23 kDa polypeptides corresponding to IgD and IgM heavy and light chains respectively. The immunoglobulin nature of the 82 and 23 kDa oligonucleotide-binding polypeptides was confirmed by immunoprecipitation with RAMIg antibodies.

Animals↗
PubMed1997

Interaction of oligonucleotides with barrier fluid proteins.

Affinity modification of proteins was used to study their interaction with oligonucleotides barrier fluids. Several proteins of saliva and tears were shown to undergo affinity modification during incubation with an alkylating derivative of deoxyribooligonucleotides. In tears, such proteins were lactoferrin, immunoglobin G and lysozyme; in saliva, immunoglobulin A and lysozyme. The data showed that the affinity for oligonucleotides decrease in the order lactoferrin > lysozyme > immunoglobulin A > immunoglobulin G. The binding of reactive oligonucleotide derivatives with the proteins was competitively inhibited by polyanions, such as oligonucleotides of various nucleotide compositions, single-stranded and double-stranded DNA, heparin, and dextran sulfate. Interactions between oligonucleotides and proteins can strongly affect the metabolism of oligonucleotides and their ability to permeate biological barriers.

Adenosine Triphosphate↗
PubMed1988

[Immune electron microscope localization of RNA-polymerase on the Escherichia coli chromosome using monoclonal antibodies against the beta-subunit].

Possibility of the immunoelectron microscopic visualization of RNA polymerase on the Escherichia coli chromosome with monoclonal antibodies against the beta-subunit labelled by [protein A.gold] complex was demonstrated. Using this method RNA polymerase molecules were revealed within nucleoid as well as on the membrane-free chromosome.

Antibodies, Monoclonal↗
PubMed1986

[A simple method of determination of antigenic determinants in proteins with known primary structure].

A simple and rapid method is proposed for the localization of antigenic determinants in proteins of known primary structure exemplified by human myoglobin. The polypeptide chain of myoglobin was cleaved with BrCN (at Met residues) or with bromosuccinimide (at Trp and Tyr residues) under conditions which on average gave less than one scission per myoglobin molecule. The "single-hit" cleavage products were separated by gel electrophoresis and transferred to nitrocellulose by electroblotting. The peptides containing intact antigenic determinants were vizualized by immuno-peroxidase staining with four monoclonal anti-myoglobin antibodies. Comparison of the lengths of the immuno-reactive peptides with the known positions of methionine, tryptophan and tyrosine residues suggested that the four monoclonal antibodies were bound by myoglobin over the region Trp-14 to Met-55. As compared with other methods of localization, the method proposed is much faster and takes much lesser amount of protein.

Amino Acid Sequence↗
PubMed1966

[Anal pruritus].

Explore the source record for details and available documents.

Nursing↗
PubMedDate not supplied

Glyceraldehyde-3-phosphate dehydrogenase is responsible for intranuclear localization of some oligonucleotides.

Nuclear accumulation of ODNs has been associated with their binding to a series of nuclear proteins. These interactions could be responsible for the sequence-independent effects of ODNs as well as for their sequence-specific interactions and their intracellular distribution. Investigation of interaction of ODNs with these proteins may shed light on the mechanisms of cellular uptake and nuclear accumulation of oligonucleotides.

Binding, Competitive↗
PubMedDate not supplied

Cell surface oligonucleotide-binding proteins of human squamous carcinoma A431 cells.

Affinity modified with Flu-DAP-p(N)16degU oligonucleotide-binding proteins were isolated by affinity chromatography using Ultrogel A2-anti fluorescein antibodies. After separation by SDS-PAGE the proteins with molecular masses about 68 kDa were MS/MS sequenced and identified as keratin K1, keratin K10, keratin K2e and albumin.

Binding Sites↗
PubMedDate not supplied

[Development of a two-site immunoassay using polyclonal antibodies for determination of lactoferrin in human blood].

The two-site enzyme-linked immunosorbent assay (ELISA) for lactoferrin using polyclonal antibodies to spatially distant epitopes has been developed. The assay sensitivity defined as minimal detectable lactoferrin concentration for p = 0.05 is 0.5 ng/ml. Accuracy of the assay (variance coefficient) is 7% within the clinical range of antigen concentrations. Human albumin, hemoglobin, and transferrin in concentrations up to 5 mg/ml practically do not interfere with the measurement. Sera of healthy donors and viral hepatitis patients were investigated using the two-site ELISA. The lactoferrin content in 44 donors' sera was 130 +/- 40 ng/ml (medium +/- standard deviation). A study of the serum specimens of 95 patients with hepatitis A, B, and C showed significant increase in serum lactoferrin concentration: 850 +/- 420, 780 +/- 580, and 680 +/- 500 ng/ml respectively. The assay showed good characteristics and may be recommended for lactoferrin measurement in patients' sera.

Animals↗