N6-(5-Phenylpentan-1-yl)adenine-A New Non-competitive Receptor-Specific Anti-cytokinin.

For the first time, N6-(5-phenylpentan-1-yl)adenine, a synthetic adenine derivative with a receptor-specific anticytokinin effect, was obtained. This compound exhibits a pronounced anticytokinin effect, reducing cytokinin-induced expression of the GUS reporter gene when interacting with the cytokinin receptor CRE1/AHK4 of the model plant Arabidopsis thaliana. This effect manifests itself much weaker with the related AHK2 receptor and is not observed at all with the AHK3 receptor. We showed that N6-(5-phenylpentan-1-yl)adenine does not bind to the ligand-binding sites of the Arabidopsis cytokinin receptors, which does not allow it to be classified as a true cytokinin antagonist. Despite the currently unknown mechanism of action, this compound may find its use as a component of plant growth regulators. Like true anticytokinins, it enhances root growth of Arabidopsis seedlings, apparently suppressing the action of endogenous cytokinins on the "root" receptor CRE1/AHK4.

Comparative Analysis of the Biosynthesis of Isoprenoid and Aromatic Cytokinins.

To compare the biosynthesis pathways of aromatic and isoprenoid cytokinins, a series of nucleoside derivatives of natural cytokinins was synthesized and their cytokinin activity was determined in a test system based on the model plant Arabidopsis thaliana. Cytokinin nucleosides are known to lack the hormonal activity until cleaving the ribose moiety at the position 9. Our experiments have shown that both ribo- and 5'-deoxyribo derivatives of N6-isopentenyladenine were able to turn into active cytokinins in planta exhibiting cytokinin activity. By contrast, 5'-deoxy nucleosides of aromatic cytokinins did not show similar activity. Since 5'-deoxy nucleosides cannot phosphorylate in vivo, the direct pathway of active cytokinin formation by cleavage of nucleotides is blocked here. The detected activity in 5'-deoxy nucleosides of isoprenoid cytokinins and the lack of the activity in 5'-deoxy nucleosides of aromatic cytokinins indicates the difference in the biosynthesis of these compounds.

[Epigenetic mutagenesis as program of age-related protein dysfunction and aging].

DNA methylation plays an important polyfunctional role in ontogenesis of human and mammals. A steep rise in probability of mutational substitution of CpG dinucleotide on TpG dinucleotide in the genome is one of the consequences of DNA methylation. All spectrum (17) of possible DNA and protein mutations caused by CpG-dinucleotide methylation in DNA were characterized, and the three most dangerous mutations (able to result in protein inactivation) were isolated. The computer program that allows one to predict all most probable mutations in the analyzed gene and encoded protein was created. On the example of genes from humans and various mammals, it was demonstrated that the amount of potentially dangerous sites of epigenetic mutagenesis in exons was drastically decreased as a result of genome evolution. But, at the same time, unforced preservation of such sites and their persistence were established, indicating the occurrence of age-related protein dysfunction built into the genome epigenetic program, resulting in apoptosis and aging; this program is based on the set and position of methylated codons in exonic gene regions. It is assumed that the program of epigenetic mutagenesis limits the lifetime of an individual, accelerating the deliverance of the population from long-lived individuals that completed the reproductive period.

Molecular structure of phospholipase D and regulatory mechanisms of its activity in plant and animal cells.

Phospholipase D (PLD) catalyzes hydrolysis of phospholipids with production of phosphatidic acid, which often acts as secondary messenger of transduction of intracellular signals. This review summarizes data of leading laboratories on specific features of organization and regulation of PLD activity in plant and animal cells. The main structural domains of PLD (C2, PX, PH), the active site, and other functionally important parts of the enzyme are discussed. Regulatory mechanisms of PLD activity are characterized in detail. Studies associated with molecular design, analysis, and synthesis of new nontoxic substances capable of inhibiting different PLD isoenzymes in vivo are shown to be promising for biotechnology and medicine.

[Identification of genetically modified vegetable sources in food and feed using hydrogel oligonucleotide microchip].

A method of multiplex polymerase chain reaction (PCR) followed by the hybridization on a hydrogel oligonucleotide biochip was developed for simultaneous identification of ten different transgenic elements of plant DNA in feed and food products. The biochip contained 22 immobilized probes intended for (i) detection of plant DNA; (ii) plant species determination (soybean, maize, potato, rice); (iii) identification of transgenic elements, including 35S CaMV, 35S FMV, rice actine gene promoters, nos, 35S CaMV, ocs, pea rbcS1 gene terminators, and bar, gus, nptII marker genes. The limit of detection was 0.5% of genetically modified (GM) soybean and maize in analyzed samples. Identification of transgenic DNA in food and feed products using either the developed approach or real-time PCR led to virtually identical results. The assay can be used for selection of GM samples by screening food and feed products for subsequent quantitative determination of the GM component based on the identified transgene.

Core features of the hormonal status in in vitro grown potato plants.

Some time ago, potato transformants expressing Agrobacterium-derived auxin synthesis gene tms1 were generated. These tms1-transgenic plants, showing enhanced productivity, were studied for their hormonal status, turnover and responses in comparison with control plants. For this purpose, contents of phytohormones belonging to six different classes (auxins, cytokinins, gibberellins, abscisic, jasmonic and salicylic acids) were determined by a sensitive UPLC-MS/MS method in tubers and shoots of in vitro grown plants. To date, this study represents the most comprehensive analysis of the potato hormonal system. On the basis of obtained results, several new generalizations concerning potato hormonal status were drawn. Overall, these data can serve as a framework for forthcoming integrative studies of the hormonal system in potato plants.

Methylation of reiterated sequences in mammalian DNAs. Effects of the tissue type, age, malignancy and hormonal induction.

The content of 5-methylcytosine in sequences of various repetition degree obtained from some mammalian (rat, mouse, cow) DNAs has been studied. The minimal 5-methylcytosine content - about 0.8 mol% - is characteristic of unique DNA sequences of all DNAs studied; the maximal 5-methylcytosine content, usually exceeding 2 mol%, is found in most highly repeated sequences. The 5-methylcytosine content in low repetitive (10--1000-fold) sequences, which are known to contain genes for rRNA, tRNA and histones, is markedly higher than in unique sequences. In total DNAs of vertebrates, as well as in mammalian DNA fractions, 5-methylcytosine occurs at almost the same frequency as does dinucleotide (5')-CG-(3'). This suggests that a greater part (if not all) of 5-methylcytosine in mammalian DNAs is a product of DNA methylases which recognize the (5')-CG-(3') DNA sequence. In cows the 5-methylcytosine content in reiterated DNA sequences decreases in thymus and heart with age and in lymphocytes on chronic lympholeukosis. The methylation degree of reiterated sequences increases in rat liver DNA after administration of hydrocortisone. On the contrary, in all cases the extent of methylation of unique sequences hardly ever changes, which seems to result from nearly complete methylation of cytosine residues in sequence (5')-CG-(3'). Specific changes observed in tissue on methylation of reiterated DNA sequences on aging, leukosis and hormone treatment support the idea that DNA methylation is associated with cellular differentiation or transformation and may be one of the possible mechanisms for regulation of transcription and replication in eukaryotes.

Cytosol induces apparent selectivity of glucocorticoid receptor binding to nucleic acids of different secondary structure.

Unpurified rat liver glucocorticoid-receptor complexes within cytosol show a distinct binding preference for double-stranded DNA over single-stranded DNA; the binding to Escherichia coli rRNA is negligible. Extensive purification of the receptor abolishes its ability to distinguish among DNAs of different secondary structure and the affinity of the purified receptor toward RNA is greatly enhanced, reaching 30-50% of that of DNA. The purification effect is reversible: after cytosol addition to purified receptor preparation the binding preference restores. NaCl does not mimic the effect of cytosol. The flow-through fraction of a phosphocellulose column retains the ability of crude cytosol to produce selective decrease in the receptor binding to single-stranded DNA. This effect may also be observed by using two types of DNA-cellulose bearing double-stranded or denatured DNA, pretreated with crude cytosol. Additionally, pretreatment of immobilized DNA with even low cytosol concentrations has been shown to markedly enhance receptor binding, although this enhancement was lacking specificity with respect to DNA secondary structure. The nature of cytosolic active principle and some possible regulatory implications are discussed.

Purified glucocorticoid-receptor complexes from rat liver cytosol preferentially bind in vitro to a homologous DNA fraction whose transcription is activated by cortisol.

The in vitro binding of 2000-fold purified rat liver glucocorticoid-receptor complexes to functionally differing homologous DNA fractions has been studied. The effectiveness of receptor binding to the transcriptionally active DNA, isolated from rat liver 4 h after cortisol administration, increased by 1.6-1.8-fold with simultaneous reduction in receptor affinity for the hardly extractable DNA fraction. The analogous DNA fractions from control animals did not differ in ability to bind the receptor. This suggests that sites of high affinity for the receptor are directly involved in the in vivo process of hormonal transcription activation.

Morphology and tuber formation of in-vitro-grown potato plants harboring the yeast invertase gene and/or the rolC gene.

Growth and tuber formation of transgenic potato plants (Solanum tuberosum cv. Désirée) harboring the yeast invertase gene and the rolC gene individually or in combination under the transcriptional control of the patatin promoter were investigated under different conditions in vitro. Plants expressing only the invertase gene were morphologically similar to control plants. rolC transgenic plants had an increased tiller number, improved root growth, and a higher total biomass. Tuber formation and growth were altered by the introduced transgenes. The sucrose requirement to induce tubers was shifted to lower or higher concentrations for invertase- or rolC-expressing clones, respectively. In addition, rolC plants formed tubers of altered morphology. A comparison with soil-grown plants showed that morphological parameters can be predicted to some extent from in vitro studies, while for reliable prescreening of parameters concerning tuber formation and growth, an optimization of currently used protocols is necessary.

[Possible relation between terminal modification of pre-mRNA and mRNA and discrete organization of genes]. / O vozmozhnoi sviazi kontsevykh modifikatsii pre-mRNK i mRNK s preryvistoi organizatsiei genov.

A suggestion of the importance of end modifications of pre-mRNA and RNA for selective defence of RNA coding sequences in the course of processing allows postulating the relationship between RNA modifications and presence of introns in genes. This conclusion was verified by comparing the presence of introns with RNA end modifications in different types of organisms, subcellular organelles and some genes; in the whole an obvious correlation of these phenomena was shown.

[Comparative analysis of the kinetics of renaturation of newly synthetized and total DNA of eukaryotes as a means of demonstrating selective genome synthesis]. / Sravnitel'nyi analiz kinetiki renaturatsii vnov' sintezirovannoi i summarnoi DNK éukariotov kak sposob vyiavleniia izbiratel'nogo sinteza genoma.

A simple and sensitive method for determining the selective eucaryotic DNA synthesis (amplification, ordered replication) amounting up to 10(-6)% of genome has been proposed. The essence of the method is measuring of the combined renaturation of total and in vivo labelled newly synthesized DNA. By means of a special computer program a theoretical investigation of amplification (from 50 to 2-fold) was accomplished, based on the experimentally found renaturation curve of rat liver DNA. A new method of graphical analysis of experimental results is also proposed based on the changes in the specific activities against Cot values. Some advantages of the new graphical method, i. e. greater sensitivity and reliability, simplicity of calculation of the amplification level and of the proportion of radioactivity in the amplified sequences are shown. The major patterns of the changes in the specific activities in different examples of amplification were revealed. Evidence is presented that the amplification necessarily brings about essential changes in the specific activities of denatured and renatured DNA fraction at high Cot values. On the basis of the method developed, the mode of liver DNA synthesis in normal and hormone-induced rats was established. Both in the normal and hydrocortison-induced rat livers no selectivity of DNA synthesis was found in the sense that hormonal induction of RNA synthesis is not a consequence of additional synthesis of DNA templates.

[Intragenomic specificity of DNA methylation in animals. Qualitative differences in tissues and changes in methylation of repeating sequences during aging, carcinogenesis and hormonal induction]. / Vnutrigenomnaia spetsifichnost' metilirovaniia DNK u zhivotnykh. Tkanevaia raznokachestvennost' i izmenenie metilirovaniia povtoriaiushchikhsia posledovatel'nostei pri starenii, kantserogeneze i gormonal'noi induktsii.

5-methylcytosine (m5C) is nonrandomly distributed in mammalian genome. The unique sequences in DNA of all species studied (mouse, rat, cow) methylated to a similar extent and are characterized by a minimal m5C content (0.8--0.9 mole%). The very renaturating sequences (including satellite DNA) possess a maximal m5C amount. The rarely repeated (10 to 1000 folds) sequences which are known to contain genes for rRNA and tRNA as well as for histones are characterized by an elevated level of methylation. It is established that tissue differences in m5C content in DNA as well as decrease in the DNA methylation level with age and on spontaneous lympholeukosis in cattle and, on the contrary, increase in DNA methylation degree in rat liver as a result of induction with hydrocortisone, are due to tissue specific changes in the methylation level of repeated but not unique sequences. The methylation level of sequences differing in the reiteration degree seems to be mainly due to CG-dublet frequencies. In the unique sequences this particular dinucleotide is almost completely methylated. Nevertheless, the multiplicity of DNA methylases in the animal nucleus is not ruled out. Methylation of the CG sequence is supposed to protect animal DNA's, especially structural genes, against restriction endonucleases. The decrease in DNA methylation in animals with age and on lympholeukosis detected by us is considered to be one of the possible reasons for chromosome lesions and for distortions in DNA replication and transcription.

[Glucocorticoid-receptor complexes of rat liver. II. Interaction with natural and synthetic polynucleotides]. / Gliukokortikoid-retseptornye kompleksy pecheni krys. II. Vzaimodeistvie s estestvennymi i sinteticheskimi polinukleotidami.

The relative affinities of [3H]dexamethasone-labelled glucocorticoid-receptor complexes (GRC) from rat liver to various natural and synthetic polynucleotides differing in base composition and secondary structure have been determined. The modified competition assay procedure with use of DNA-cellulose was employed. The interaction of 150-2000 fold purified GRC with polyribo- and polydeoxyribonucleotides greatly depends on their base composition and nucleotide sequences. This interaction is hardly affected by the structure (ribo- or deoxyribo-) of the sugar-phosphate backbone or its size and is in fact independent of the secondary structure (single- or double-stranded forms) of polynucleotides. Mixtures of mononucleotides or apurinic DNA do not compete with DNA-cellulose for GRC binding. In the presence of cytosol, GRC bind double-stranded rather than single-stranded DNA, the fact possibly reflecting different effects of cytoplasmic proteins on the accessibility to GRC of polynucleotides of dissimilar secondary structure. The apparent equilibrium dissociation constants (Kd) of the ternary complexes of GRC with some synthetic polynucleotides were calculated. In case of double-stranded polydeoxyribonucleotide poly(dA) . poly(dT) Kd is equal to (1.2 +/- 0.6) x 10(-4) M; this value may characterize a nonspecific GRC--DNA interaction. It is supposed that GRC interact with high affinity (Kd approximately 10(-7)--10(-8) M) with short (5-10 nucleotides long) DNA sequences in which certain purine and pyrimidine residues are interspersed. Such DNA sequences may represent sites of primary action of GRC on the cellular genome in vivo.

[Glucocorticoid-receptor complexes of rat liver. I. Kinetic and equilibrium parameters of their interaction with DNA: ionic strength and temperature dependence]. / Gliukokortikoid-retseptornye kompleksy pecheni krys. I. Kineticheskie i ravnovesnye parametry vzaimodeistviia s DNK: vliianie ionnoi sily i temperatury.

The interaction of about 2000-fold purified rat liver glucocorticoid-receptor complexes (RGC) with DNA immobilized on cellulose was investigated. The sedimentation coefficient of GRC before or after purification is about 4S in 0.3 M NaCl. The parameters of GRC-DNA interaction were quantitatively determined using arbitrary constants expressed in M of DNA nucleotide residues. The GRC oligomeric forms which are predominant in low ionic strength media are supposed to have a higher DNA-binding affinity as compared to the monomeric forms which are predominant in high salt solutions. The interaction of GRC with DNA-cellulose qualitatively differs from the interaction of GRC with phosphocellulose. This suggests that electrostatic forces do not determine the formation of GRC-DNA complexes. So, in addition to nonspecific interaction, some specific "recognition" in GRC-DNA complexes may occur. Calculations made on the basis of GRC-DNA binding parameters derived for the conditions close to physiological (0.15 M NaCl, 20-30 degrees C, pH 7.4) demonstrate that rapid and nearly complete in vivo translocation of GRC form cytoplasm to nuclei may be a consequence of direct interaction of GRC with cellular DNA.

[The morphometric and immunohistochemical indices of the gastric and duodenal mucosa in the dynamic treatment of erosive gastroduodenitis in those who worked in the cleanup of the aftermath of the accident at the Chernobyl Atomic Electric Power Station in the follow-up period]. / Morfometricheskie i immunogistokhimicheskie pokazateli slizistoi obolochki zheludka i dvenadtsatiperstnoi kishki v dinamike lecheniia érozivnogo gastroduodenita u likvidatorov posledstvii avarii na Chernobyl'skoi AES v otdalennom periode.

62 Chernobyl wreckers with erosive gastroduodenitis (50 cases) and ulcer (12 cases) were admitted to hospital where they received standard drugs and local He-Ne laser treatment of erosions and ulcer located in the antral stomach and duodenal bulb. Histological, bacterioscopic, morphometric and immunohistochemical examinations have identified chronic inflammation and disturbance of local immunity. After treatment epithelialization of erosions was not accompanied by normalization of morphometric and immunohistochemical parameters. This suggests a high risk of recurrences.

[Role of current large-film fluorography in the improved diagnosis of gastric polyps in mass screening of the population]. / Rol' sovremennoi krupnokadrovoi fliuorografii v uluchshenii diagnostiki polipov zheludka pri provedenii dispanserizatsii naseleniia.

The paper is concerned with a study of present-day potentialities for improving the diagnosis of stomach polyps. As the main diagnostic method the authors proposed gastric LFF with subsequent (as per indications) endoscopic examination of persons at high risk of development of the above pathology. Proceeding from an analysis of 110 cases of stomach polyps diagnosed at examination of over 11000 persons at high risk of developing stomach tumors, the authors emphasized a high efficacy of the proposed methods to find a solution to the problem of improving polyp diagnosis and issued their recommendations for their introduction into practice.

[The characteristics of gastroduodenal pathology in the workers of a plant for plastic construction materials]. / Osobennosti gastroduodenal'noi patologii u rabochikh zavoda stroitel'nykh plastikov.

Out of 162 workers exposed to chemicals in the manufacture of construction plastics an outpatient and inpatient examination revealed gastrointestinal problems in 84.3%, vegetative nervous disorders in 62.6% of the examinees. In a random sample of 47 workers high acid production occurred in 63.3% and erosions, ulcers, cicatrices in the stomach and duodenum in 45.2% of them. No HLA genetic predisposition to duodenal ulcer was reported. Histological findings on gastroduodenal mucosa biopsies were indicative of the dystrophy in the chief and lining cells of the gastric glands, epithelium and stroma with formation of the dark cells. The authors insist on consideration of the above facts in examination and treatment of workers exposed to chemicals at construction plastics plants.

[Endoscopic assay and surgery at chronic pancreatitis]. / Endoskopicheskie issledovaniia i operatsii pri khronicheskom pankreatite.

OBJECTIVE: To assess the possibilities of endoscopic surgeries at the pathology of major duodenal papilla in patients with the painful form of chronic pancreatitis. The study involved 35 patients with the painful form of chronic pancreatitis including 19 subjects with cholecystectomy as a result of cholelithiasis, 8 subjects with alcohol-induced pancreatitis (4 of them had external pancreatic fistulas) and 8 patients with idiopathic pancreatitis. As many as 60 different X-ray and endoscopic procedures were carried out altogether: ERCPG, EPST and dissection of pancreatitis duct entrances as well as nasopancreatic drainage and stenting. ERCPG discovered the pathology of major duodenal papilla in the form of stenosis of the common bile duct entrance and/or main pancreatic duct entrance in 25 (71%) patients. At the same time, the ultrasonic examination discovered the pathology only in 7 patients (20%). EPST was performed in 20 patients of 25. The dissection of the major pancreatitis duct entrance was carried out in 16 of them; the dissection of the additional pancreatic duct was performed in 1 patient; the external pancreatic duct drainage was conducted in 5 patients; the additional pancreatic duct stenting was carried out in 1 patient. Fifteen patients (75%) felt better immediately upon the surgery. Ten patients were followed-up from 2 months to 3 years; steady amelioration was observed in 7 of them. There were complications in the form of acute pancreatitis in 3 patients after ERCPG and in 2 patients after endoscopic surgeries. There were no other complications or fatalities. For patients with the painful form of chronic pancreatitis, ERCPG is an informative and relatively safe technique enabling to discover the stenosis of the common bile duct entrance and/or main pancreatic duct entrance in 71% of cases. Endoscopic surgeries make it possible to produce an immediate positive result in 75% of cases.

[Removal of foreign bodies from the upper segment of the digestive tract using an endoscope]. / Udalenie inorodnykh tel iz verkhnikh otdelov pishchevaritel'nogo trakta s pomoshch'iu éndoskopa.

The work presents data of the endoscopic diagnostics and treatment of 73 patients with foreign bodies in the upper part of the digestive tract (93 objects). High efficiency and little traumatism of removals of the foreign bodies from the upper parts of the digestive tract was achieved due to using a rational tactic of curative endoscopy consisting in using different models of modern fibroendoscopes and instrumental devices.