Biomedical subjects

B F Vanyushin

Publications and source records attributed to B F Vanyushin.

At least 19 recordsLinked to original sources

PubMed2006

DNA methylation in plants.

DNA in plants is highly methylated, containing 5-methylcytosine (m5C) and N6-methyladenine (m6A); m5C is located mainly in symmetrical CG and CNG sequences but it may occur also in other non-symmetrical contexts. m6A but not m5C was found in plant mitochondrial DNA. DNA methylation in plants is species-, tissue-, organelle- and age-specific. It is controlled by phytohormones and changes on seed germination, flowering and under the influence of various pathogens (viral, bacterial, fungal). DNA methylation controls plant growth and development, with particular involvement in regulation of gene expression and DNA replication. DNA replication is accompanied by the appearance of under-methylated, newly formed DNA strands including Okazaki fragments; asymmetry of strand DNA methylation disappears until the end of the cell cycle. A model for regulation of DNA replication by methylation is suggested. Cytosine DNA methylation in plants is more rich and diverse compared with animals. It is carried out by the families of specific enzymes that belong to at least three classes of DNA methyltransferases. Open reading frames (ORF) for adenine DNA methyltransferases are found in plant and animal genomes, and a first eukaryotic (plant) adenine DNA methyltransferase (wadmtase) is described; the enzyme seems to be involved in regulation of the mitochondria replication. Like in animals, DNA methylation in plants is closely associated with histone modifications and it affects binding of specific proteins to DNA and formation of respective transcription complexes in chromatin. The same gene (DRM2) in Arabidopsis thaliana is methylated both at cytosine and adenine residues; thus, at least two different, and probably interdependent, systems of DNA modification are present in plants. Plants seem to have a restriction-modification (R-M) system. RNA-directed DNA methylation has been observed in plants; it involves de novo methylation of almost all cytosine residues in a region of siRNA-DNA sequence identity; therefore, it is mainly associated with CNG and non-symmetrical methylations (rare in animals) in coding and promoter regions of silenced genes. Cytoplasmic viral RNA can affect methylation of homologous nuclear sequences and it maybe one of the feedback mechanisms between the cytoplasm and the nucleus to control gene expression.

Adenine↗

PubMed2005

Enzymatic DNA methylation is an epigenetic control for genetic functions of the cell.

In eukaryotic cells nuclear DNA is subjected to enzymatic methylation resulting in formation of 5-methylcytosine residues mainly in CG and CNG sequences. In plants and animals, this DNA methylation is species-, tissue-, and organelle-specific. It changes (diminishes) with age and is regulated by hormones. On the other hand, genome methylation can control hormonal signal. There are replicative and post-replicative DNA methylations. They are served by multiple DNA-methyltransferases with different site specificity. Replication is accompanied by appearance of hemi-methylated sites in DNA; pronounced asymmetry of DNA chain methylation disappears at the end of the cell cycle; a model of regulation of replication by DNA methylation is suggested. DNA methylation controls all genetic processes in the cell (replication, transcription, DNA repair, recombination, gene transposition) and it is a mechanism of cell differentiation, gene discrimination, and silencing. Prohibition of DNA methylation stops development (embryogenesis), switches on apoptosis, and is usually lethal. Distortions in DNA methylations result in cancerous cell transformation, and the DNA methylation pattern is one of the safe cancer diagnostics at early stages of carcinogenesis. The malignant cell has a different DNA methylation pattern and a set of DNA-methyltransferase activities expressed as compared with normal cells. Inhibition of DNA methylation in plants is accompanied by induction of genes of seed storage proteins and flowering. In eukaryotes one and the same gene can be methylated both on cytosine and adenine residues; thus, there are, at least, two different and probably interdependent systems of DNA methylation in the cell. First higher eukaryotic adenine DNA-methyltransferase was isolated from plants; this enzyme methylates DNA with formation of N6-methyladenine residues in the sequence TGATCA --> TGm6ATCA. Plants have AdoMet-dependent endonucleases sensitive to DNA methylation status; therefore, like microorganisms, plants seem to have a restriction-modification (R-S) system. Revelation of an essential role of DNA methylation in the regulation of genetic processes has laid a foundation for and materialized epigenetics and epigenomics.

Animals↗

PubMed2004

Apoptosis in plants: specific features of plant apoptotic cells and effect of various factors and agents.

Apoptosis is an integral part of plant ontogenesis; it is controlled by cellular oxidative status, phytohormones, and DNA methylation. In wheat plants apoptosis appears at early stages of development in coleoptile and initial leaf of 5- to 6-day-old seedlings. Distinct ultrastructural features of apoptosis observed are (1). compaction and vacuolization of cytoplasm in the apoptotic cell, (2). specific fragmentation of cytoplasm and appearance in the vacuole of unique single-membrane vesicles containing active organelles, (3). cessation of nuclear DNA synthesis, (4). condensation and margination of chromatin in the nucleus, (5). internucleosomal fragmentation of nuclear DNA, and (6). intensive synthesis of mitochondrial DNA in vacuolar vesicles. Peroxides, abscisic acid, ethylene releaser ethrel, and DNA methylation inhibitor 5-azacytidine induce and stimulate apoptosis. Modulation of the reactive oxygen species (ROS) level in seedling by antioxidants and peroxides results in tissue-specific changes in the target date for the appearance and the intensity of apoptosis. Antioxidant butylated hydroxytoluene (BHT) reduces the amount of ROS and prevents apoptosis in etiolated seedlings, prolongs coleoptile life span, and prevents the appearance of all apoptotic features mentioned. Besides, BHT induces large structural changes in the organization of all cellular organelles and the formation of new unusual membrane structures in the cytoplasm. BHT distorts mitosis and this results in the appearance of multiblade polyploid nuclei and multinuclear cells. In roots of etiolated wheat seedlings, BHT induces differentiation of plastids with the formation of chloro(chromo)plasts. Therefore, ROS controlled by BHT seems to regulate mitosis, trigger apoptosis, and control plastid differentiation and the organization of various cellular structures formed by endocytoplasmic reticulum.

Antioxidants↗

PubMed2004

Apoptosis in wheat seedlings grown under normal daylight.

Apoptosis was observed in the coleoptile and initial leaf in 5-8-day-old wheat seedlings grown under normal daylight. Apoptosis is an obligatory event in early wheat plant ontogenesis, and it is characterized by cytoplasmic structural reorganization and fragmentation, in particular, with the appearance in vacuoles of specific vesicles containing intact organelles, chromatin condensation and margination in the nucleus, and internucleosomal fragmentation of nuclear DNA. The earliest signs of programmed cell death (PCD) were observed in the cytoplasm, but the elements of apoptotic degradation in the nucleus appeared later. Nuclear DNA fragmentation was detected after chromatin condensation and the appearance in vacuoles of specific vesicles containing mitochondria. Two PCD varieties were observed in the initial leaf of 5-day-old seedlings grown under normal daylight: a proper apoptosis and vacuolar collapse. On the contrary, PCD in coleoptiles under various growing (light) conditions and in the initial leaf of etiolated seedlings is only a classical plant apoptosis. Therefore, various tissue-specific and light-dependent PCD forms do exist in plants. Amounts of O2*- and H2O2 evolved by seedlings grown under normal daylight are less than that evolved by etiolated seedlings. The amount of H2O2 formed in the presence of sodium salicylate or azide by seedlings grown under normal daylight was increased. Contrary to etiolated seedlings, the antioxidant BHT (ionol) did not inhibit O2*- formation and apoptosis and it had no influence on ontogenesis in the seedlings grown under normal daylight. Thus, in plants grown under the normal light regime the powerful system controlling the balance between formation and inactivation of reactive oxygen species (ROS) does exist and it effectively functions. This system is responsible for maintenance of cell homeostasis, and it regulates the crucial ROS level controlling plant growth and development. In etiolated plants, this system seems to be absent, or it is much less effective.

Antioxidants↗

PubMed2004

H1 histone in developing and aging coleoptiles of etiolated wheat seedlings.

It has been established that the DNA and H1 histone contents in aged coleoptile of 8-day-old etiolated wheat seedling are about 40 and 30%, respectively, lower than those in young seedlings. H1 histone in wheat seedlings is represented as six electrophoretically different subfractions. The ratios of H1 histone subfractions in wheat coleoptile and initial leaf are similar. In contrast to some animal cells, apoptosis in wheat coleoptile is not accompanied by changes in the set and ratios of H1 histone subfractions. Aging of coleoptiles is associated with a progressive diminution of the H1 histone and DNA contents. H1 histone/DNA ratio in aged coleoptile is 1.5-2-fold higher than that in the young organs. Therefore, the content of H1 histone in chromatin of coleoptile decreases with age more slowly than DNA content.

Apoptosis↗

PubMed2003

Effect of ethylene producer ethrel and antioxidant ionol (BHT) on the proteolytic apparatus in coleoptiles of wheat seedlings during apoptosis.

It was established that total proteolytic activity in etiolated wheat seedlings changes in ontogenesis in cycles: peaks of proteolytic activity correspond to the 3rd, 5th, and 8th days of seedling growth, respectively. The maximum of proteolytic activity preceded the maximum of nuclease activity, which may be due to activation of nucleases by proteolytic enzymes. According to inhibitory analysis the cysteine and serine proteases play the main role in apoptosis in wheat coleoptiles. Growing of seedlings in the presence of ethrel stimulated apoptosis in the coleoptile, and it increased (almost 6-fold) the proteolytic activity in its cells. On the other hand, the antioxidant ionol (BHT) suppressed the induction of proteases, particularly at the second stage of coleoptile development, and it slowed down the increase in the nuclease activity after 6th day of the seedling life. It is suggested that phytohormones and antioxidants participate in regulation of apoptosis in the ageing coleoptile, directly or indirectly effecting the proteolytic apparatus in the coleoptile cells.

Antioxidants↗

PubMed2002

Apoptosis in the initial leaf of etiolated wheat seedlings: influence of the antioxidant ionol (BHT) and peroxides.

Apoptosis was observed in the initial leaf of 5-8-day-old etiolated wheat seedlings. A condensation of cytoplasm in apoptotic cells, formation of myelin-like structures, specific fragmentation of cytoplasm, appearance in vacuoles of specific vesicles containing subcellular organelles, condensation and margination of chromatin in the nucleus, and internucleosomal fragmentation of nuclear DNA are ultrastructural features of apoptosis in the initial wheat leaf. Single-membrane vesicles detected in vacuoles of the leaf cells resemble in appearance the vacuolar vesicles in the coleoptile apoptotic cells described earlier (Bakeeva, L. E., et al. (1999) FEBS Lett., 457, 122-125); they contain preferentially plastids but not mitochondria as was observed in coleoptile. The vacuolar vesicles are specific for the apoptotic plant cells. Thus, apoptosis in various tissues is an obligatory element of plant (wheat) growth and development even in the early stages of ontogenesis. Contrary to strong geroprotecting action in coleoptile, the known antioxidant BHT (ionol, 2.27 x 10(-4) M) does not prevent in the leaf cells the apoptotic internucleosomal DNA fragmentation and appearance of specific vacuolar vesicles containing subcellular organelles. Therefore, the antioxidant action on apoptosis in plants is tissue specific. Peroxides (H2O2, cumene hydroperoxide) stimulated apoptosis (internucleosomal DNA fragmentation) in coleoptile and induced it in an initial leaf when apoptosis in a control seedling leaf was not yet detected. Thus, apoptosis that is programmed in plant ontogenesis and controlled by reactive oxygen species (ROS) can be modulated by anti- and prooxidants.

Antioxidants↗

PubMed2002

Ionol (BHT) produces superoxide anion.

In aqueous medium etiolated wheat seedlings release superoxide anion (O2*-). Interaction of a synthetic antioxidant, butylated hydroxytoluene (BHT, ionol), with oxygen in the aqueous medium is accompanied by O2*- formation. This suggests that under certain conditions BHT behaves as a prooxidant. A natural antioxidant, superoxide dismutase (SOD), and also a wound healing preparation, emulsified denatured placenta (EDP), do not exhibit the prooxidant properties. In contrast to BHT, they reduce O2*- production by the etiolated wheat seedling system.

Antioxidants↗

PubMed2001

Putative DNA-(amino)methyltransferases in eucaryotes.

By computer analysis of the known data bases, we have established that the open reading frames (ORF) coding for proteins that possess high degree of homology with procaryotic DNA-(amino)methyltransferases are present in the genomes of Leishmania major, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Arabidopsis thaliana, Drosophila melanogaster, Caenorhabditis elegans, and Homo sapiens. Conservative motifs typical for bacterial DNA-(amino)methyltransferases are detected in the amino acid sequences of these putative proteins. The ORF of all putative eucaryotic DNA-(amino)methyltransferases found are encoded in nuclear DNA. In mitochondrial genomes including a few fully sequenced higher plant mtDNA, nucleotide sequences significantly homologous to genes of procaryotic DNA-(amino)methyltransferases are not found. Thus, ORF homologous to bacterial adenine DNA-methyltransferases are present in nuclei of protozoa, yeasts, insects, nematodes, vertebrates, higher plants, and other eucaryotes. A special search for corresponding proteins and, in particular, adenine DNA-methyltransferases in these organisms and a study of their functions are quite promising.

Adenine↗

PubMed2001

Effect of the antioxidant ionol (BHT) on growth and development of etiolated wheat seedlings: control of apoptosis, cell division, organelle ultrastructure, and plastid differentiation.

Ionol (BHT), a compound having antioxidant activity, at concentrations in the range 1-50 mg/liter (0.45 x 10(-5)-2.27 x 10(-4) M), inhibits growth of etiolated wheat seedlings, changes the morphology of their organs, prolongs the coleoptile life span, and prevents the appearance of specific features of aging and apoptosis in plants. In particular, BHT prevents the age-dependent decrease in total DNA content, apoptotic internucleosomal fragmentation of nuclear DNA, appearance in the cell vacuole of specific vesicles with active mitochondria intensively producing mtDNA, and formation of heavy mitochondrial DNA rho = 1.718 g/cm3) in coleoptiles of etiolated wheat seedlings. BHT induces large structural changes in the organization of all cellular organelles (nucleus, mitochondria, plastids, Golgi apparatus, endocytoplasmic reticulum) and the formation of new unusual membrane structures in the cytoplasm. BHT distorts the division of nuclei and cells, and this results in the appearance of multi-bladed polyploid nuclei and multinuclear cells. In roots of etiolated wheat seedlings, BHT induces intensive synthesis of pigments, presumably carotenoids, and the differentiation of plastids with formation of chloro- or chromoplasts. The observed multiple effects of BHT are due to its antioxidative properties (the structural BHT analog 3,5-di-tert-butyltoluene is physiologically inert; it has no effect similar to that of BHT). Therefore, the reactive oxygen species (ROS) controlled by BHT seem to trigger apoptosis and the structural reorganization of the cytoplasm in the apoptotic cell with formation of specific vacuolar vesicles that contain active mitochondria intensively producing mtDNA. Thus, the inactivation of ROS by BHT may be responsible for the observed changes in the structure of all the mentioned cellular organelles. This corresponds to the idea that ROS control apoptosis and mitosis including formation of cell wall, and they are powerful secondary messengers that regulate differentiation of plastids and the Golgi apparatus in plants.

Antioxidants↗

PubMed2000

Necessity of superoxide production for development of etiolated wheat seedlings.

It was found that production of superoxide (O(2)(*-)) is crucial for normal morphogenesis of etiolated wheat seedlings in the early stages of plant development. The development of etiolated wheat seedlings was shown to be accompanied with cyclic changes in the rate of O(2)(*-) production both in the entire intact seedling and in its separated organs (leaf, coleoptile). First increase in the rate of O(2)(*-) production was clearly observed in the period from two to four days of seedling development, then the rate of O(2)(*-) production decreased to the initial level, and then it increased again for two days to a new maximum. An increase in O(2)(*-) production in the period of the first four days of seedling development correlates with an increase in DNA and protein contents in the coleoptile. The second peak of increased rate of O(2)(*-) production observed on the sixth or seventh day of seedling development coincides with a decrease in DNA and protein contents and apoptotic internucleosomal nuclear DNA fragmentation in the coleoptile. Incubation of seedlings in the presence of the antioxidant BHT (ionol) strongly affects their development but it does not influence the increase in DNA and protein contents for the initial four days of seedling life, and it slows down the subsequent age-dependent decrease in protein content and fully prevents the age-dependent decrease in DNA content in the coleoptile. A decrease in the O(2)(*-) amount induced by BHT distorts the seedling development. BHT retards seedling growth, presumably by suppression of cell elongation, and it increases the life span of the coleoptile. It seems that O(2)(*-) controls plant growth by cell elongation at the early stages of seedling development but later O(2)(*-) controls (induces) apoptotic DNA fragmentation and protein disintegration.

Butylated Hydroxytoluene↗

PubMed1999

Subcellular reorganization of mitochondria producing heavy DNA in aging wheat coleoptiles.

Unusual closed membrane vesicles containing one or more mitochondria were isolated from homogenates of aging wheat coleoptiles. Very similar (or the same) bodies were shown to exist in situ in vacuoles of undividing cells in the apical part of intact senescent coleoptiles. Vesicles isolated from coleoptile homogenate free of nuclei by 10 min centrifugation at 1700 x g and traditional mitochondria (sedimented at between 4300 x g and 17,400 x g) are similar in respiration rate, composition and content of cytochromes and sensitivity to respiration inhibitors. However, vesicles contain about 2-fold more Ca2+ ions than free mitochondria do. The specific feature of vesicles containing mitochondria in aging coleoptiles is an intensive synthesis of heavy (rho = 1.718 g/cm3) mitochondrial DNA (H-mtDNA). Thus, aging in plants is accompanied by an increased selective H-mtDNA production and change in subcellular organization of mitochondria.

Apoptosis↗

PubMed1998

Butylated hydroxytoluene modulates DNA methylation in rats.

The major observation of this investigation is that a single intraperitoneal injection of butylated hydroxytoluene (BHT, 60 mg/kg body mass) results within a few hours in a strong increase in nuclear DNA(cytosine-5)-methyl transferase (methyl transferase) activity in the liver, kidneys, heart, spleen, brain and lungs of male rats. In most organs, the rise in methyl transferase activity is observed as early as 4 h after BHT injection, it reaches a maximum at 8 h and then, except for lungs and brain, gradually decreases to its initial level at 16 h. At the maximum induction times, the methyl transferase activity in liver, kidney and spleen increases by about 16-, 3- and 5-fold, respectively. A second BHT injection at 96 h results in a secondary rise in hepatic methyl transferase activity. Isoelectric focusing electrophoresis of control rat liver nuclear extracts showed methyl transferase activity in the pI 4.7 and 7.4 protein fractions. Both fractions methylate calf thymus DNA better than they do Drosophila melanogaster DNA. In similar extracts from BHT-treated rats, the methyl transferase activity is found in three protein fractions with pI values equal to 4.0, 6.2 and 9.5, respectively. Most of the methyl transferase fractions from the livers of BHT-treated rats methylate the completely unmethylated D. melanogaster DNA better than they do calf thymus DNA. Thus, BHT induces methyl transferase activity that preferably provides de novo DNA methylation. BHT injection had no significant effect on the hepatic contents of S-adenosylmethionine (AdoMet), S-adenosylhomocysteine (AdoHcy) and AdoMet/AdoHcy ratios. While BHT injection did not alter the 5-methyldeoxycytidine content in liver DNA, it did appear to alter such content in other organs. BHT appears to cause the reversible changes in the methylation status of an internal cytosine residue in some CCGG sites of the rat liver cytosine DNA-methyl transferase gene. BHT induces also hypomethylation of the renal methyl transferase gene and the hepatic c-Ha-ras gene. While BHT also increases the hepatic mRNA transcripts for the S-adenosylmethionine synthetase and the p53 genes, it had no detectable effects on the corresponding mRNA transcripts for methyl transferase homologous to murine methyl transferase. Thus, BHT induces tissue-specific reversible changes in methyl transferase activity and methylation of total DNA and various genes in rats. A strong increase in methyl transferase activity in rat liver is accompanied with BHT-induced change in the methyl transferase set observed in this organ.

Animals↗

PubMed1998

DNA methylation by wheat cytosine DNA methyltransferase: modulation by protease inhibitor E-64.

Cytosine DNA methyltransferase isolated from wheat seedlings and purified in the presence of metalloprotease and serine protease inhibitors has molecular mass and specific activity equal to about 85 kDa and 250 units/mg protein, respectively. Apparent K(m) for AdoMet and [I]50 for AdoHcy values are about 6 microM and 12 microM, respectively. The enzyme is active in wide pH range (pH 5.5-8.5) and is inhibited by NaCl. The enzyme rapidly loses its methyltransferase activity in the absence of substrates. Using the cysteine protease inhibitor E-64 it has been shown that rapid enzyme inactivation is caused by disappearance of essential enzyme SH-groups but is not due to proteolytic enzyme cleavage.

Cysteine Proteinase Inhibitors↗

PubMed1998

Elevated expression and altered pattern of activity of DNA methyltransferase in liver tumors of rats fed methyl-deficient diets.

DNA methyltransferase (MTase) activity in nuclear extracts from neoplastic and preneoplastic livers of rats fed a methyl-deficient diet (MDD) is elevated compared with that seen in the livers of control rats. Nuclear proteins were prepared in the presence of protease inhibitors including trans-epoxy succinyl-L-leucylamido-(4-guanido)butane and were fractionated by isoelectric focusing. In normal, control liver, two distinct MTase fractions were observed. In MDD-induced malignant liver, a third fraction, in addition to the previous two, was also seen. Both the DNA substrate and the cytosine site specificities of the third MTase fraction differ from those of the other two fractions. The distinct MTase activity in liver tumor has significantly more de novo MTase activity than do the MTase fractions of normal, control liver. Thus, normal and neoplastic rat livers differ in DNA MTase fractionation patterns and site specificities. The altered DNA MTase activity observed in rat liver tumors caused by MDDs may be one of the critical factors contributing to cancer formation through abnormal DNA methylation.

Animals↗

PubMed1998

RNA-polymerase, DNA-polymerase, DNA-methyltransferase and sphingomyelinase activities in liver nuclei of rats of different Age.

It has been established that activities of RNA-polymerase, DNA-polymerase, DNA-methyltransferase, and sphingomyelinase in the liver nuclei isolated from newborn (2-day-old) rats are by 18, 25, 27, and 610%, respectively, higher than those in mature (6-month-old) rats. It has been also shown that the number of single-stranded stretches in rat liver nuclear DNA increases with age: it is about 2-fold higher in mature rats than in newborn rats. Thus, the postnatal period of ontogenesis is accompanied by both significant changes in DNA structure and decrease in activities of enzymes serving for the most important genetic processes in cells of animals.

Aging↗

PubMed1996

Drosophila melanogaster genomic DNA sequence homologous to mammalian cytosine DNA-methyltransferase gene.

Using Southern blotting procedure we have shown that Drosophila genomic DNA hybridizes with 4423-bp C-terminal fragment of murine cytosine DNA-methyltransferase gene. Thus, the Drosophila genome has a sequence homologous to the mammalian cytosine DNA-methyltransferase gene. We assume that DNA methylation most likely responsible for strong CpG suppression in the Drosophila genome mainly was catalyzed by a cytosine DNA-methyltransferase that has since been lost.

Animals↗

PubMed1996

The methylation pattern of a cytosine DNA-methyltransferase gene in Arabidopsis thaliana plants.

Using a PCR-amplified 5'-end proximal 600-bp fragment and two cDNA clones of cytosine DNA-methyltransferase gene of Arabidopsis thaliana as specific probes in hybridization with plant DNA samples, hydrolyzed by methylation-sensitive restriction endonucleases HpaII and MspI, it has been established that CCGG sites located in the 5'-end proximal part of cytosine DNA-methyltransferase gene are highly methylated at internal C and less but detectably methylated at external C residues. On the contrary, all CCGG sites in 3'-terminal half of the coding region were found to be unmethylated at both external and internal C residues. No significant differences between methylation patterns of cytosine DNA-methyltransferase gene in various organs (leaf, stem, flower) of the Arabidopsis thaliana plant were detected.

Arabidopsis↗