Biomedical subjects
Publications and source records attributed to L A Iakubov.
Using transplantable Ehrlich ascites tumor, hepatoma HA-1 and Lewis carcinoma it was shown, that preparations of fragmented genomic DNA can more or less effectively inhibit such tumors as well as the growth of their metastases. Such effects were produced by DNA preparations derived from tissues of mice, both syngeneic or allogeneic to tumor-bearer, as well as from human tissues.
The cytostatic drug cyclophosphamide (CPA) in high dosage suppressed hemopoiesis by causing multiple double-strand breaks to occur in hemopoietic cell DNA and leading to mutation, chromosomal abberations and finally cell death. We tested fragmented DNA drugs for an ability of CPA to protect murine leukopoiesis on an assumption that once exogenous fragmented DNA had infiltrated into a cell, it might integrate with chromosomal DNA through homologous recombinations thus repairing damaged segments. DNA drugs did promote repair of leukocyte count in murine peripheral blood with leukopoiesis being suppressed by CPA administration. The levels of DNA derived from murine organs and human placenta were higher than those from salmon roe. Tumor growth was significantly inhibited following injection of placental DNA into mice bearing intramuscularly transplanted lymphosarcoma. Antitumor effect of combined CPA and DNA treatment was much higher than after CPA alone.
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Using the 32P-labelled alkylating oligonucleotide derivative (pT)16, the oligonucleotide interaction with mammalian cells has been studied. The majority of fibroblastoid cell lines tested in this study (COS-1, vero, L-671, Ag 17-1, CHO, B7) as well as mouse hepatocytes were found to contain proteins specifically interacting with oligonucleotides. Cells of Ag 17-1 and COS-1, apart from the 79 kDa protein specific for all fibroblast lines, contained also a protein with a molecular mass of 83 kDa. In BALB/c mouse hepatocytes the 83 kDa protein is the major oligonucleotide binding protein. Analysis of concentration dependencies of specific modification of receptor proteins in liver cells has made it possible to determine the values of constants for the oligonucleotide derivatives binding to the protein. The binding constant for the alkylating oligonucleotide derivative pT16 and the corresponding phosphothioate oligonucleotide has been found to be equal to 5 x 10(6) M-1.
The interaction of alkylating derivatives of deoxyribonucleotides whose 5'-terminal phosphate group contains a 4-[(N-2-chloroethyl-N-methyl)amino]benzylamine residue with serum proteins has been studied. Incubation of whole human sera with various concentrations of the alkylating derivative, p(T)16, resulted in affinity modification of several proteins, among which albumin as well as IgM and IgG were the most readily detectable ones. The type of dependence of the degree of modification of these proteins on oligonucleotide concentration suggests that the oligonucleotides display a higher affinity for IgM than for IgG and albumin. Binding of reactive oligomer derivatives to serum proteins was inhibited by polyanions of different oligomeric composition, two-chain DNA and heparin, the latter being the strongest inhibitor of the immunoglobulins. These data point to a role of nonspecific ion-to-ion interactions in the IgG-oligonucleotide complex formation. Oligonucleotide interaction with murine monoclonal antibodies GI was inhibited by a specific antigen which suggests that oligonucleotides may interact with immunoglobulin either at or near a site where the antigen is recognized by the antibody.
Distribution and stability of benzyl-[5'-32P] phosphoramides of oligonucleotides and their phosphorothioate analogs were investigated. Oligonucleotide derivatives are distributed among all murine organs, the maximal concentration being observed in the liver and kidney, and minimal in the brain. Intravenous and intraperitoneal injections resulted in a faster distribution of oligonucleotides among the tissues than subcutaneous injections. Hydrolysis rate varied from tissue to tissue, and in 30 min after injection 5 to 50% of intact oligonucleotides were found. Hydrolysis did not depend on the injected dose in the range of 0.15-150 nmol per mouse. The 3'-terminal cholesterol groups protected the oligonucleotides considerably. In the blood stream and pancreas phosphorothioate oligonucleotides showed much higher stability than their phosphodiester counterparts. A protein interacting specifically with the oligonucleotides was discovered in the liver, kidney and pancreas.
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A procedure for isolation of neuraminidase from influenza virus using the nonionic detergent Triton x-100 was developed. To achieve further purification, the protein mixture was passed through a Sepharose column packed with immobilized antibodies against hemagglutinin. The neuraminidase preparation thus obtained fully retained its enzymatic and antigenic properties and during electrophoretic separation under denaturating conditions gave one protein band.
Interaction of secretory immunoglobulins A of a varying degree of purity with oligonucleotides and ATP has been studied by the method of affinity modification. For this aim we used reactive derivatives of 32P-labeled deoxyoligonucleotide (ClRCH2NHp(T)14) and [gamma-32P]ATP (ClR-32PppA) or ATP (ClR-pppA) bearing a 4-[(N-2-chloroethyl-N-methyl)amino]benzylamine residue. Preparations of sIgA were obtained from human milk by sequential chromatography on protein A-sepharose (P1), DEAE-fractogel (P2) and by gel-filtration in 50 mM NaOH (P3). It was revealed, that the H- and L-chains of sIgA P1; H-, L-chains and secretory component (SC) in sIgA P2 and only SC in sIgA P3 were exposed to modification after incubation with ClRCH2NHp(T)14. LPS, DNA, tRNA, heparin, sufficiently inhibited the modification of chains of sIgA P1. These competitors did not influence the modification of H- and L-chains of sIgA P2, but DNA, tRNA, heparin, inhibited binding of SC with the modifier. Suppressing affect of binding of ClRCH2NHp(T)14 with secretory component of sIgA P3 by d(T)14 has been observed as well. The research of ClR-32PppA interaction with sIgA P3 has shown that H- and L-chains of sIgA are exposed to modification. ATP inhibited the reaction. Study of the influence of modification on the protein kinase activity of sIgA P3 has revealed, that the preliminary incubation of sIgA P3 with ClR-pppA leads to inhibition of protein kinase activity. We suggest that sIgA, possessing the protein kinase activity (sIgA-abzymes) has an ATP-binding center (catalytic center) and has an oligonucleotide-binding center as well.
A reaction of native Drosophila proteins with an alkylating oligonucleotide derivative bearing 4-[(N-2-chlorethyl-N-methyl)amino]benzylamine at the 5' terminal phosphate has been investigated. It was found, that the reagent alkylates a few proteins (90, 50, 44, 39, 32 kDa). The modification was organ specific. The labeled 39 kDa protein is present in the ovaries only, while the modified 32 kDa protein is found only in the bulbus.
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