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Biomedical subjects

B F Vaniushin

Publications and source records attributed to B F Vaniushin.

At least 19 recordsLinked to original sources
PubMed1991

[Effect of sphingomyelin and its enzymatic hydrolysis products on heterologous methylation of calf thymus DNA by cytosine-DNA-methyltransferase EcoRII].

It was found that sphingomyelin and its enzymatic hydrolysis products, choline and sphingosine, influence the degree of DNA methylation in the reaction of heterologous methylation by methylase EcoRII in vitro. Sphingomyelin was found to be able to was activate (by 20%), sphingosine and choline inhibit methylation. Phosphatidylcholine had no effect on DNA methylation in an in vitro system. The role of lipids in the regulation of gene expression during enzymatic modification (methylation) of DNA is discussed.

Animals↗
PubMed1990

[The effect of preparations of Hirudo medicinalis leeches on DNA methylation in the rat liver].

The effect of the salivary gland secretion and dialysable part of the homogenate of the leeches Hirudo medicinalis on the methylation of DNA in the rat liver after the intraperitoneal injection and perfusion of isolated liver has been analysed. The maximum concentration of 5-methylcytosine is observed 1 h later the injection of preparations: for the salivary gland secretion the increase is 39%, for the dialysate of leech homogenate is 28%. The 5-methylcytosine content increases on 28% after the perfusion of isolated liver with the leech saliva and after the dialysate of the leech homogenate--on 20%. No other changes in DNA content is observed. It is suggested that the DNA-methylation of the liver cells is due to the penetration of biologically active substances produced by the medical leech into the cell-targets accompanied by the forming of corresponding ligand-receptor complexes.

5-Methylcytosine↗
PubMed1989

[Cytokinins do not noticeably change the methylation of adenine residues of DNA in cultured Escherichia coli B].

6-Benzylaminopurine (6-BAP) (1 mg/ml) does not influence the growth of E. coli B cell cultures or the number of [8-14C] labeled N6-methyladenine (m6A) residues in the total DNA [(100.m6A/(A x m6A) = 1.7]. The growth of bacterial cells in the presence of adenine or cytokinins (6-BAP, kinetin, zeatin) (1 mg/ml) was unaccompanied by significant changes in the intracellular content of plasmid pBR 322. The mode of restriction by endonuclease Cfu I hydrolyzing the Gm6ATC site of plasmids pBR 322 from E. coli B cells grown in the presence of adenine or one of the above-mentioned cytokinins is identical. These plasmids also have identical restriction products Mbo I or Sau 3AI. Thus, the cytokinins under study do not markedly affect the methylation of adenine residues in total DNA of E. coli B cell cultures and the GATC sequence in plasmids pBR 322 isolated from these cells.

Adenine↗
PubMed1987

[Properties and specificity of action of DNA methylases from blood lymphocytes of healthy cattle and cattle with chronic lymphoid leukemia].

Using cell extract fractionation with ammonium sulfate and subsequent chromatography on DEAE- and DNA-cellulose and Blue Sepharose, two cytosine DNA-methylases were isolated from blood lymphocytes of cows suffering from lympholeukosis; one cytosine DNA-methylase was isolated from blood lymphocytes of healthy animals. The DNA-methylases from normal lymphocytes was purified 383-fold; the enzyme has a specific activity of 2.3 u./mg, Mr of 114 000 Da and a pH optimum of 7.6. The molecular mass of DNA-methylases from leukemic lymphocytes is about 130,000 Da. The enzymes isolated from leukemic lymphocytes, i.e., DNA-methylase I (568-fold purification, specific activity 14.2 u./mg) and DNA-methylase II (524-fold purification, specific activity 13.1 u./mg) possess different action optima at pH 7.8 and 6.7, respectively. The total DNA-methylase activity of leukemic lymphocytes is about 4 times that of normal lymphocytes. All the DNA-methylases under study methylate in vitro bacterial and animal DNAs of different base composition; bacterial DNAs are the best (GC content is approximately 70%), while homologous DNAs- the worst acceptors of CH3-groups. Heat-denaturated DNAs are methylated more intensively than initial DNAs. The optimal NaCl concentration in the reaction mixture is approximately 50 mM; EDTA (greater than 10 mM) inhibits the reaction. DNA-methylase I of leukemic and normal lymphocytes show the same pH optimum and specificity of action. In vitro methylation of bacterial DNA by these DNA-methylases in the presence of [3H-methyl]S-adenosylmethionine results in the similar type of label distribution among pyrimidine isopliths in the DNA. DNA-methylase II from leukemic lymphocytes methylates about two times more Pu-C-Pu sequences in th same DNA than does DNA-methylase I from leukemic and normal lymphocytes and thus reveals a different specificity. The changes in the type of DNA methylation as well as the appearance of an additional DNA-methylase possessing a different specificity of action in leukemic lymphocytes may be responsible for cell transformation and transcriptional changes in chronic lympholeukosis.

Animals↗
PubMed1987

[DNA methylation and interaction with glucocorticoid receptor complexes in the rat liver].

DNA from the livers of 20-day-old rat embryos contains 1.8 mol.% of 5-methylcytosine (m5C), i.e., it is methylated approximately 2-fold as compared with adult (m5C = 1.0 mol.%) and aged (20-month-old; m5C = 0.9 mol.%) animals. 90 min after intraperitoneal injection of adult adrenalectomized rats with hydrocortisone (5 mg per 100 g of body weight), the m5C content in liver DNA becomes 0.7 mol.%, i.e., it decreases by about 30% against control values. It was assumed that hydrocortisone induces enzymatic demethylation of m5C residues in rat liver DNA. A comparative study of competition between the DNA under study and DNA-cellulose for the binding to highly purified (approximately 2000-fold) glucocorticoid-receptor complexes (GRC) of rat liver revealed that the highly methylated rat embryo liver DNA bind the activated [3H]triamcinolone-receptor complexes with a lower (approximately 1.8-2 times) efficiency than do the DNA from the livers of adult and aged rats, including the hydrocortisone-induced ones. An inverse correlation was observed between the m5C level in rat liver DNA and the ability of the latter to bind GRC. After in vitro methylation of liver DNA of adult rats in the presence of S-adenosylmethionine by DNA-methylase from blood lymphocytes of suffering from chronic lympholeukosis cows, the GRC-receptor ability of these DNA was practically lost. Hence, GRC of rat liver can recognize sequences that are recognizeable by animal DNA-methylase. The observed modulations in the RNA-methylation upon ageing and those induced by hydrocortisone can control the effectiveness of glucocorticoid hormone action on gene expression in ontogenesis and at various inducible functional states of the organism.

5-Methylcytosine↗
PubMed1985

[Determination of DNA methylase activity in animal tissue extracts].

An express method for measuring the level of in vitro DNA methylation in homogenates and nuclei from animal tissues as well as during initial steps of DNA methylase isolation and purification when methylase activity is low and hardly testable by other methods has been suggested. The method is based on the measuring the radioactivity incorporated in filter adsorbed DNA (acid-insoluble material) 3H-label from S-adenosile-L-methionine as a result of in vitro DNA methylation. The advantage of the method consists in the replacement of a long-duration repeated deproteinization procedure traditionally used by a relatively simple procedure (15 min incubation of the mixture at 80 degrees C with 10 volumes of the 8M urea, 5 mM EDTA, 5% n-butanol, 2% sodium dodecilsulfate, 1 M sodium chloride solution) and the absence of any loss of DNA. The method is fit for the fast serial assay of DNA methylase activity taking into consideration that about one third of the total acid-insoluble radioactivity is due to the radioactivity in 5-methylcytosine residues in DNA.

Adsorption↗
PubMed1984

[Isolation and properties of membrane-bound DNA of the chromatin peripheral layer in the blood of healthy cattle and cattle with chronic lympholeukemia].

The nuclei and the fraction of chromatin peripheral layer bound to the nuclear membrane were isolated from blood lymphocytes of healthy and chronic lympholeukosis (CLL)-stricken cows. The isolation procedure included treatment of nucleic with DNase I with subsequent extraction of chromatin by low ionic strength salt solutions and purification of nuclear membranes by sucrose density gradient centrifugation. The blood lymphocyte nuclei from cows suffering from CLL differ from nuclei of healthy animals by a higher degree of chromatin condensation and a lower RNA content. Amount of DNA in the peripheral chromatin layer in lymphocyte nuclei of cows suffering from CLL is two times as high as that in healthy animals. The GC-content in the peripheral chromatin layer DNA from lymphocyte nuclei of healthy and CLL-stricken animals does not differ from that of total nuclear DNA (about 45 mol.%). Both in healthy and CLL-stricken animals, the 5-methylcytosine content in the chromatin peripheral layer DNA is by about 20% less than in the total nuclear DNA of healthy cows. In the chromatin peripheral layer DNA of blood lymphocytes from CLL-stricken animals the methylation level is unaffected. Therefore, a certain decrease in the total nuclear DNA methylation in CLL-stricken animals observed is due to methylation changes in DNA which is not bound to the nuclear membrane.

Animals↗
PubMed1984

[Age-related decrease of the binding of isolated rat liver and brain nuclei with glucocorticoid-receptor complexes].

Isolated nuclei from liver and brain of rats of various age bind isolated and 1000-2000-fold purified glucocorticoid-receptor complexes (GRC) containing [3H]dexamethasone. The binding of GRC by the nuclei decreases with an increase in the NaCl concentration from 0 to 0.4 M in a medium. The maximal degree of GRC binding is observed in the liver and brain nuclei of newborn rats (1-day-old); the nuclei of adult animals aged 3 months bind GRC 1/2 times as much. in old (25-month-old) and grown-up animals, no significant differences in GRC binding by nuclei from respective tissues were revealed. Brain nuclei of newborn rats were shown to bind GRC to a greater extent than liver nuclei.

Aging↗
PubMed1983

[Comparative analysis of the interaction of highly purified glucocorticoid-receptor complexes from rat liver with homologous and heterologous cell nuclei].

The binding of glucocorticoid-receptor complexes (GRC) from rat liver purified 1000-2000-fold to homologous and heterologous cell nuclei was investigated. The purified GRC preparations were examined by gel filtration, ion-exchange chromatography on DEAE-cellulose and electrophoresis in polyacrylamide gel in the presence of Na-DS. The binding of GRC to the nuclei was characterized in terms of associate resistance to KCl. It was found that the isolated nuclei of the non-homologous tissue of the same species (rat brain) or of the heterologous tissues (liver and brain) of phylogenetically different species (pigeon, frog) can bind rat liver GRC in the same degree or even more than the homologous nuclei. Some tissue- and species-dependent peculiarities of GRC binding capacity of the nuclei were revealed. The GRC binding capacity of pigeon nuclei is somewhat higher than that of rat nuclei, while the corresponding ability of frog brain nuclei is higher than that of frog liver nuclei. The GRC-nuclei associates are more resistant to KCl than the GRC--pure DNA associates.

Animals↗
PubMed1983

[Mitochondrial nature of newly synthesized DNA in aging coleoptiles of etiolated wheat shoots].

In mature ageing coleoptiles as well as in cut-off etiolated wheat shoots exhausted by incubation in H2O nuclear DNA (nDNA) synthesis does not occur. However, the synthesis of another DNA is continued under these conditions. The parameters of this DNA (e. g. buoyant density, rho = 1.716 g/cm3, and composition - 56 mol.% GC) differs from those of nDNA (rho = 1.700 g/cm3, 44 mol.% GC). The newly synthesized DNA is non-methylated and metabolically stable. It is not sensitive to cycloheximide (20 micrograms/ml) but is markedly inhibited by ethidium bromide (2-10 micrograms/ml). The synthesis of this DNA is localized in the mitochondria, thus indicating that in ageing coleoptiles and exhausted wheat shoots all the newly synthesized DNA is of mitochondrial origin.

Aging↗
PubMed1983

[Synchronous synthesis of DNA in coleoptiles and the initial leaf of developing etiolated wheat shoots: the nature and correlation of nuclear and mitochondrial DNA syntheses].

In etiolated coleoptiles and initial leaf of developing wheat shoots the DNA synthesis is periodical and synchronous. In the initial leaf each step of DNA synthesis results in a stepwise increase of DNA content and is doubled at the first three steps. During the leaf plane formation the synthesis of nuclear DNA (nDNA) is decreased, while that of mitochondrial DNA (mitDNA) continues in synchronous cycles. This is the cause of relative stabilization of DNA content per unit of leaf plane length. The DNA increase in this organ occurs due to synchronous synthesis of nDNA and mitDNA in intercalary meristem cells. In coleoptiles a marked replication of nDNA is observed at the first three steps of the synthesis; in each cycle nDNA synthesis precedes that of mitDNA. With completion of coleoptile formation the nDNA synthesis in it practically ceases, whereas that of mitDNA continues in synchronous cycles. MitDNA is non-methylated and its composition (56 mol.% GC) differs significantly from that of the newly synthesized nDNA (44 mol.% GC; 100 X m5C/(C + m5C) = 16-17%). It may be concluded that in various organs of wheat shoots the composition and methylation of newly synthesized DNA depend on the age of the shoot and on the ratio of nDNA/mitDNA syntheses.

Aging↗
PubMed1983

[Methylation of newly formed DNA in developing wheat seedlings].

The base composition (45 divided by 56 mol.% GC) and methylation level (100 . m5C/(m5C + C) = = 20% divided by 0.4%) of pulse-labelled newly formed DNA in etiolated wheat seedlings and their individual organs are markedly changed with age. The base composition of total leaf and coleoptile DNA (45 mol.% GC) remains unchanged; the changes in the methylation level are not significant (1). In the course of development of the plant organs (e. g. leaf) the level of synthesis of DNA whose composition is identical to that of nuclear DNA, is decreased and the incorporation of labelled precursors into the GC-rich (56 mol.% GC) practically unmethylated DNA is increased. In ageing coleoptiles only the synthesis of unmethylated DNA of the GC-type takes place. It is assumed that this DNA may correspond to mitochondrial DNA.

Base Composition↗
PubMed1982

[Replicative methylation of DNA in L-cells: effect of S-isobutyladenosine and cycloheximide and possible existence of two DNA-methylases].

Methylation of DNA in cultured cells of mouse fibroblasts (L-cells) occurs at least in two steps, i. e. methylation of Okazaki fragments (up to 100 . m5C/C + m5C = 2.8-2.9) and methylation of linkage sites of DNA formed by ligation of the fragments (up to 6.0). The synthesized Okazaki fragments are not subjected to further methylation, since about one half of their methylation sites (CG) remains non-modified. The transmethylation inhibitor S-isobutyladenosine (SiBA) inhibits the methylation of the "linkage" sites of the newly synthesized DNA without affecting that of the Okazaki fragments. The repression of protein synthesis (including that of histones) by cycloheximide in the course of replication reveals some additional methylation sites. The level of methylation of the newly synthesized polymeric DNA reaches thereby 6.0, which corresponds to modification of all its CG-dinucleotides. A model for replicative methylation of DNA based on the existence of two DNA-methylases differing in their specificity is proposed. It is assumed that one of the DNA methylases is highly specific and functions within the complex with DNA-polymerase, while the other possesses a restricted specificity and functions in a free form (i. e. apart from the replicative complex).

Animals↗