[Oxidative stress, rRNA genes, and antioxidant enzymes in pathogenesis of schizophrenia and autism: modeling and clinical advices].

Ribosomal genes (RG), or genes for rRNA, are represented by multiple tandem repeats in eukaryotic genomes, and just a part of them is transcriptionally active. The quantity of active copies is a stable genome feature which determines the cell's capability for rapid synthesis of proteins, necessary to cope with stress conditions. Low number of active RG copies leads to reduced stress resistance and elevated risk of multifactorial disorders (MFD). Oxidative stress (OS) in the brain cells is believed to be involved in the pathogenesis of infantile autism (IA) and schizophrenia, i.e., MFDs with a manifested genetic predisposition. With autism, OS markers are found almost in every research, whilst with schizophrenia, the OS data are contradictory. Earlier, in a sample of patients with schizophrenia, we have found significantly higher quantity of active RG copies than at the average in healthy population. Here we have estimated the number of active RG copies in a sample of patients with IA (n = 51) and revealed significantly lower mean value than in healthy population. A novel mathematical model of the dynamic pattern of OS has been proposed. The model is realized as an ordinary differential equation system, supposing induction of antioxidant protection enzymes being mediated by reactive oxygen species (ROS), with the subsequent decrease of ROS content in a cell. The rate of synthesis of antioxidant protection enzymes is limited by the ribosome synthesis rate which depends on the number of active RG copies. Analysis of the model showed that the system always approaches a single stable equilibrium point along a damped oscillation trajectory, which in some degree resembles the dynamics of 'predator-prey' interaction in Lotka-Volterra model. The stationary ROS level inversely depends on the number of active RG copies. Our study explains the inconsistency of clinical data of OS in schizophrenia and suggests a novel criterion for discriminative cytogenetic diagnostics of schizophrenia and IA, as well as allows to assume that antioxidant therapy should be effective only for children with low number of active RG copies.

[Ribosomal genes in the human genome: identification of four fractions, their organization in the nucleolus and metaphase chromosomes].

Completion of human genome reading stimulated intense studies in the field of functional genomics and characterization of individual genomes. Of considerable importance is the study of the complex of multicopy ribosomal genes (RGs), but its thorough analysis was not a task of the "Human Genome" program. In this short review we present our data on the copy number of rRNA genes in individual human genomes and on their heterogeneity in the functional respect. Fractions of active and potentially active RGs as well as fractions of inactive and silent RGs intensively methylated in the transcribed region are characterized. Their location in the nucleolus structures and in metaphase chromosomes is discussed.

[Determination of genome dose of active ribosome genes and several quantitative parameters of extracellular dna in test-subjects of the experiment with 7-day immersion].

Genome dose of active ribosome genes (ARG), average of nucleolus argyrophil structures in lymphocyte nuclei, levels of extracellular DNA (DNA(e)) concentrations and ratio of antibodies to total DNA (AB(DNA)) and ribosomal DNA (AB(DNA-rib)), and nuclease activity were determined in peripheral blood of 8 volunteered subjects (21-26 y.o.) in the experiment with 7-d DI. Results of the investigation revealed a broad individual variability ensued from heterogeneity of the group of the test-subjects as to ARG values. There was an inverse negative relationship between ARG values and increment of the ribosome genes activity index. Part of the subjected exhibited increased DNA(e) levels on completion of the experiment, whereas the others decreased the parameter demonstrating individual character of body reaction. No correlation was established between DNA(e) content and nuclease activity in blood. Concentrations of AB(DNA) and DNA AB(DNA-rib) before and after immersion were essentially unchanged; however, they were higher as compared with the control group of blood-donors. Diversity of subjects' reactions was accounted to the broad range of ARG values. Therefore, selection of test-subjects for ground-based simulation experiments should be conducted with due consideration of the parameter.

[Dynamics of blood biochemical parameters in an experiment with 7-day immersion].

Blood samples were taken from 8 volunteers (21 to 26 years of age) for 7-day immersion 7 days prior to, on days 3 and 7 of the experiment and on days 1 and 8 of recovery. Serum was analyzed for 38 biochemical markers of the functional state of the myocardium, skeletal muscles, hepatobiliary system, kidney, pancreas, GI tract, prostate, and protein-nucleic, carbohydrate, electrolyte and mineral metabolism. Seven-day immersion was found to alter the biochemical parameters within the physiological norm boundaries. The observed changes included lower activities of enzymes associated with muscular and myocardial constellation, shifts in electrolytes (K, Na, Mg), and increases in the biliary function parameters. Increased concentrations of the lipid metabolism parameters suggest a higher risk of atherogenesis. Biochemical parameters of bone tissues and erythrocyte activity were essentially unchanged. Most of the parameters returned to pre-experimental values by day 8 of recovery.

[Ribosomal genes in inbred mouse strains: interstrain and intrastrain variations of copy number and extent of methylation].

Quantitative dot hybridization was used to estimate the rDNA copy number in brain tissues of five inbred mouse strains (AKR/JY, NZB/B1OrlY, CBA/CaLacY, 101/HY, and 129/JY), which were obtained from the collection of the Research Center of Biomedical Technologies (Y). In each strain, 9-12 mice aged 1-2 months were examined. The rDNA copy number per diploid genome in strains AKR (range 105-181, mean +/- SD 136 +/- 27) and NZB (129-169, 148 +/- 12) was significantly lower than in strains CBA (172-267, 209 +/- 31), 101 (179-270, 217 +/- 30), and 129 (215-310, 264 +/- 33). Mice of strain NZB were relatively homogeneous in this trait (CV = 8.1%). Strains AKR, CBA, 101, and 129 displayed significant between-group differences, CV varying from 12.5 to 19.9%. The same DNA specimens were digested with MspI or HpaII and used to estimate the extent of methylation of the 28S rDNA region. Regardless of the strain, all mice could be classed into two groups. One group (20 mice) had a methylated fraction accounting for less than 8% of rDNA and included all nine mice of strain NZB, seven out of nine mice of strain 101, and three out of ten mice of strain 129. In the other group (29 mice), the methylated fraction varied from 18 to 38%. A possible role of methylation and the genome dosage of ribosomal genes in phenotypic variation (quantitative trait variation) of inbred mouse strains is discussed.

[Early and late responses to oxidative stress in human dermal fibroblasts of healthy donors and rheumatoid arthritis patients. Relationship between the cell death rate and the genomic dosage of active ribosomal genes].

A study was made of the effect of the oxidizing agent potassium chromate (K2CrO4, PC) on cultured dermal fibroblasts of a healthy donor and three patients with rheumatoid arthritis (RA). Characteristics of the rRNA gene (RG) complex-RG copy number, active RG (ARG) dosage, and 18S rRNA content--were determined for each cell line. In cells of the healthy donor, oxidative stress caused by low doses of PC (2-4 microM, 1-4 h) induced an early response, including a 50-80% increase in total RNA and rRNA. An appreciable activation of the nucleolus was observed cytochemically, by silver staining and morphometry. The early response grew considerably lower with the increasing passage number and/or PC concentration. Exposure to 6-12 microM PC for 24 h led to a progressive cell death (late response). The existence and intensity of the early response correlated positively with the cell survival during further culturing. Cells of the RA patients displayed almost no early response even at early passages: total RNA did not increase, and rRNA increased by no more than 10%. Cell disruption (apoptosis) during further culturing was more intense than in the line originating from the healthy donor. The apoptosis intensity characterized by the increase in the content of DNA fragments in the culture medium and in the caspase 3 activity, was inversely proportional to the ARG dosage in the genome. The results provide the first quantitative characterization of the early and late responses of cells to PC-induced oxidative stress and suggest a role of the ARG dosage in cell survival in stress.

[Rate of DNA replication and size of replicons in human diploid cells]. / Skorost' replikatsii DNK i razmery replikonov vdiploidnykh kletakh cheloveka.

The rate of DNA replication and the distances between initiation sites (size of replicons) have been studied in human cultured fibroblasts. The modified Huberman and Riggs technique of DNA fiber autoradiography has been used: the pulse-labelled regions were analysed in DNA fibers preliminarily labelled along the whole length. This enabled us: a) to analyse the arrangement of replicons along the length of labelled DNA fibers with the lengths of 200-750 micron, reaching 2700 micron in some cases; b) to select only single DNA molecules for the analysis. This technique decreases the danger of a mistake when minor labelled regions belonging to different DNA molecules are referred to the same one. The rate of DNA replication varies from 0.2 to 1.2 micron/min, the average of 0.6 micron/min. This conforms with findings of other authors. The distances between initiation sites vary from 15 to 140 micron with the modal interval of 50-60 micron. This value is twice higher than those obtained by other authors. The possible reasons for such difference are discussed.

[Elements of structural organization of the transcribed area of the ribosomal repeat (rDNA) in human peripheral blood lymphocytes]. / Elementy strukturnoi organizatsii transkribiruemoi oblasti ribosomnogo povtora (rDNK) v limfotsitakh perifericheskoi krovi cheloveka.

The rDNA transcribed region (TR) was tested for accessibility to RsaI recognizing 15 TR sites, DNase I, and photoinducible arylazide (N-(4-azido-2-hydroxybenzoyl)-N,N'-diaminoheptane acetate) in isolated nuclei and, with arylazide, in intact cells. Arylazide entered cells well and did not appreciably affect the chromatin structure. Its photolysis products efficiently modified DNA in accessible sites. Single-strand breaks made by DNase I were not transformed in double-stranded in rDNA TR, suggesting the necessity of denaturing electrophoresis for such an analysis. About 70% of all rDNA copies proved poorly inaccessible to endonucleases and arylazide, the accessibility being higher in their 18S and 5.8S rRNA gene regions than in the regions of the external transcribed spacers (ETSs) and the 28S rRNA gene. Proteinase K disrupted this structure, and the corresponding copies were extracted from nuclei. This explained why in situ hybridization occasionally fails to reveal rDNA in the nucleolar fibrillar center (FC) on electron microscopic preparations. In other rDNA copies, TR (excluding 5'-ETS) was accessible to nucleases and arylazide. These copies were not extracted from nuclei treated with proteinase K. Some of their RsaI sites were protected by tightly bound proteins. Seven such regions were identified in TR. Possible association of the molecular structure, nucleolar location, and functional state of rDNA is discussed.

[Quantitative analysis of repetitive sequences in human genomic DNA and detection of an elevated ribosomal repeat copy number in patients with schizophrenia (the results of molecular and cytogenetic analysis)]. / Kolichestvennoe opredelenie povtoriaiushchikhsia posledovatel'nostei v genomnoi DNK cheloveka. Obnaruzhenie uvelichennogo kolichestva ribosomnykh povtorov v genomakh bol'nykh shizofreniei (rezul'taty molekuliarnogo i tsitogeneticheskogo analizov).

A modified version of quantitating repetitive sequences in genomic DNA was developed to allow comparisons for numerous individual genomes and simultaneous analysis of several sequences in each DNA specimen. The relative genomic content of ribosomal repeats (rDNA) was estimated for 75 individuals, including 33 healthy donors (HD) and 42 schizophrenic patients (SP). The rDNA copy number in HD was 427 +/- 18 (mean SE) per diploid nucleus, ranging 250-600. In SP, the rDNA copy number was 494 +/- 15 and ranged 280-670, being significantly higher than in HD. The two samples did not differ in contents of sequences hybridizing with probes directed to a subfraction of human satellite III or to the histone genes. Cytogenetic analysis (silver staining of metaphase chromosomes) showed that the content of active rRNA genes in nucleolus organizer regions is higher in SP compared with HD. The possible causes of the elevated rRNA gene dosage in SP were considered. The method employed was proposed for studying the polymorphism for genomic content of various repeats in higher organisms, including humans.

[The effect of methylnitronitrosoguanidine on DNA synthesis in human and animal cells. Inhibition of DNA synthesis in asynchronous and synchronous cultured cells]. / Deistvie metilnitronitrozoguanidina na sintez DNK v kletkakh cheloveka i zhivotnykh. Podavlenie sinteza DNK v asinkhronnykh i sinkhronnykh kul'turakh kletok.

Methylnitronitrosoguanidine (MNNG) is reported to inhibit DNA synthesis in intact human cells, in the cells from patients with ataxia telangiectasia (AT) or the cells from two rodent species. DNA synthesis in different cell lines exhibits varying sensitivity to MNNG inhibitory effect. 4-5-fold higher concentrations of MNNG are required for 50% inhibition of DNA synthesis in AT cells or in field vole cells as compared with the concentration required for human cells or Chinese hamster. The different compactness of two chromatin fractions might possibly result in lower sensitivity of DNA synthesis in heterochromatin to MNNG-induced inhibition as compared with the sensitivity of euchromatin. The genetic expression of AT defect on the cellular level is supposed to be connected with changes in supramolecular packaging of chromatin in interphase nuclei.

[Effect of methylnitronitrosoguanidine on DNA synthesis in animal cells: decrease in the rate of replication fork movement]. / Deistvie metilnitronitrozoguanidina na sintez DNK v kletkakh zhivotnykh: snizhenie skorosti dvizheniia vilki replikatsii DNK.

The effect of methylnitronitrosoguanidine (MNNG) on the rate of DNA replication fork (RF) progress has been studied by DNA fiber autoradiography in asynchronous Microtus agrestis and Chinese hamster cells. The rate of RF progress has been shown to be decreased by 14% and 36% at MNNG concentrations of 50 and 100 microM in M. agrestis cells; the rate of DNA synthesis being reduced by 50 and 75% respectively. In Chinese hamster cells the MNNG concentration of 5 microM does not affect the rate of RF and that of 10 microM decreases the latter by 11%, the respective fall in the synthesis of DNA rate being 13 and 57%. It has been concluded that the decreased RF rate contributes only partially to the overall DNA synthesis inhibition following the MNNG administration. Inhibition of DNA synthesis at the MNNG concentrations reducing the DNA synthesis by less then 40-50% is mainly caused by the inhibition of the initiation points and, possibly, by the stopping of operating RF. Further DNA synthesis inhibition (at the MNNG concentrations leading to DNA synthesis decrease exceeding 50%) is mostly due to the reduced RF progress rate.

[The effect of bleomycin on DNA synthesis in human cells: evidence for grouping of initiation of replicons in the S-period]. / Vlianie bleomitsina na sintez DNK v kletkakh cheloveka: dokazatel'stvo sgruppirovannosti aktov initsiatsii replikonov v S-periode.

The effect of bleomycin (Blm) on DNA synthesis has been studied in a synchronous culture of human embryonic lung cells. The data obtained suggest that in the Blm presence in a medium (20 micrograms/ml) DNA synthesis initiation in new replicons is suppressed. The Blm action at different S-phase intervals has been shown to inhibit DNA synthesis unequally. Four discrete time intervals have been singled out in the course of the 10-hr S-phase in which a grouped initiation of replicon portions can be supposed. Together with the data on DNA replication in large-size replicon units (50-500 microns), the obtained results account well for the uneven DNA synthesis in S-phase, manifested by 3 or 4 peaks of [3H]-thymidine incorporation in pulse-labelled cells.

[A new approach to cloning of tandem repetitive DNA sequences]. / Novyi podkhod k klonirovaniiu tandemno-organizovannykh povtoriaiushchikhsia posledovatel'nostei DNK.

A new approach to screening of the repeated human DNA sequences tandemly arranged in the genome is described. Efficiency of the developed approach for search of tandemly arranged DNA sequences is corroborated by the obtained experimental data.

[Activation of total and ribosomal RNA transcription under adapting doses of ionizing radiation inducing displacement of chromosome loci in human G0-lymphocyte]. / Aktivatsiia transkriptsii total'noi i ribosomnoi RNK pri vozdeistvii adaptiruiushchikh doz ioniziruiushchei radiatsii, indutsiruiushchikh izmenenie koordinat lokusov khromosom v iadrakh G0-limfotsitov cheloveka.

As we demonstrated earlier, the adapting X-ray doses (3 and 10 cGy) induced movement of chromosome centromeric loci in G0-lymphocyte nuclei. In the present study we investigated the influence of X-rays with 3 and 10 cGy doses on the content of total, 18S and 45S rRNA in human G0-lymphocytes because it is known that the transcription products participate in nucleus organization. It was shown that 3 h after irradiation the content of both total and 18S RNA was significantly increased. The 3 cGy dose induced higher level of the rRNA than 10 cGy dose did in cells of some individuals. At the same time, the 45S RNA content was not changed significantly. This result may suggest that process of rRNA transcription and primary transcript (45S rRNA) processing have been completed during 3 h after irradiation. The data about an activation of rRNA synthesis were confirmed by cytological observation. Under 3 and 10 cGy doses both nuclei diameter and area of the Ag-stained granules were increased, depending on dose. These data also may be connected with an initiation of rRNA transcription because of correlation of Ag-painting with nucleolus activity. Thus, adapting X-ray doses induce displacement of chromosome loci in lymphocyte nuclei and activation of rRNA transcription. Further investigations are required for understanding of these phenomena interconnection.

[Organization of mammalian genome replication: data on the high rate of DNA replication in replicons of structural heterochromatin]. / Organizatsiia replikatsii genoma mlekopitaiushchikh: dannye o vysokoo skorosti replikatsii DNK v replikonakh strukturnogo geterokhromatina.

By the autoradiography, the increase of the mean rate of replication fork movement in DNA was shown for human skin fibroblasts during S-period induced by reseeding the stationary culture; the portion of high-rate replicons growing parallel to the increase in numbers of cells, in which the constitutive heterochromatin was replicating. Furthermore, the heterogeneity of replicons was investigated in regard to the rate of DNA chain growth in asynchronous cell populations of human and three rodent species (Microtus agrestis, Tscherskia triton, Cricetulus griseus). A positive correlation was found between the portion of replicons, with a relatively high rate of replication (more than 60 micron/h), and the relative amounts of C-heterochromatin in genomes of these species. On the basis of these studies it has been assumed, that the replicons with the high rate of DNA chain growth belong to the constitutive heterochromatin.

[Cytogenetic of nucleolar organizer regions (NOR) of human chromosomes: identification of four morphofunctional variants of NOR, their inter-individual and inter-chromosomal distribution]. / Tsitogenetika iadryshkoobrazuiushchikh rainov (IaOR) khromosom cheloveka: vydelenie chetyrekh morfofunktsional'nykh variantov IaOR, ikh mezhindividual'noe i mezhkhromosomnoe rasprelenie.

Cytogenetic characters of the nucleolus organizer regions (NORs) located on the short arms of five acrocentric chromosomes were studied in chromosome preparations obtained from cultured blood cells of 17 donors. In situ hybridization to a 3H-labeled probe was used to estimate the relative copy number of ribosomal genes (RGs) in all NORs of chromosomes identified by G-banding in each sample. The relative amount of potentially active RGs (0 to 4 arbitrary units) in each NOR was estimated from the size of AgNOR selectively stained with silver nitrate. Linear regression analysis revealed clusters of silent RGs (CSRGs) in 24 out of 170 NORs (14%). Based on the presence or absence of active and inactive RG clusters, NORs of human chromosomes were classified into four morphological functional variants (MFVs): (1) Ag-/CSRG-, (2) Ag-/CSRG+, (3) Ag+/CSRG-, and (4) Ag+/CSRG+. These variants were observed in 7.65%, 2.35%, 11.8%, and 78.2% of 170 analyzed NORs, respectively. NORs with CSRGs (MFV 2 and 3) were absent in 5 out of 17 donors. One, two, and three NORs with CSRGs were observed in four donors each. Analysis of the chromosome distribution of NOR MFVs showed that their frequencies remained almost the same in group-D and group-G acrocentric chromosomes. Although the tested samples were small (34 chromosomes for each pair), two observations were made with regard to individual chromosomes. First, almost half MFV-1 NORs (6 out of 13) were detected on chromosome 15. Second, the frequency of CSRGs was higher in chromosome 21 (29%) than in the other chromosomes (10%).

[Interindividual and intercellular differences in the total activity of ribosomal genes detectable by the Ag staining of nucleolus organizer regions in human acrocentric chromosomes]. / Mezhindividual'nye i mezhkletochnye razlichiia summarnoi aktivnosti ribosomnykh genov, vyiavliaemye AG-okraskoi iadryshkoobrazuiushchikh raionov akrotsentricheskikh khromosom cheloveka.

Selectively Ag-stained nucleolar organizing regions (NORs) of human chromosomes were analysed using four size categories: 0, 1, 2 or 3 grades. A criterion of NORs' total activity has been proposed as a sum of grades (sigma (+]. On this basis, interindividual polymorphism was defined in 60 healthy individuals with normal karyotypes. The reaction norm of sigma (+) was determined (from 16 to 22 grades). In the cells of the patients with two nucleolar organizing chromosomes involved in Robertsonian translocations the sigma (+) was within the reaction norm (16-19). The total NORs activity was determined in a patient having both normal karyotype cells and two cell clones with one or two small bisatellited chromosomes: sigma (+) in three cell clones amounted to 20.5, 23.0 and 26.3. In the clones with additional NORs, the silver staining intensity for 10 NORs of the main set did not change, which leads to a suggestion that no compensatory change in the number of rRNA gene copies working takes place in man. The data obtained allow to suppose that zygotic selection operates in man, which ensures maintainance of the number of the ribosomal gene's copies necessary for viability of an individual.

[Analysis of chromosome aberrations and nucleolar organizer regions of chromosomes in workers producing pyromellitic dianhydride: the possibility of the adaptive role of Ag-NOR variants]. / Analiz khromosomnykh aberatsii i iadryshkoobrazuiushchikh raionov khromosom u rabochikh proizvodstva piromellitovogo diangidrida: O vozmozhnoi adaptivnoi roli variantov Ag-IaOR.

To assess the mutagenic danger of pyromellitic dianhydride (PMDA) production, the frequency of chromosome aberrations in peripheral blood lymphocytes from 56 workers and 37 control donors from Ufa was determined. A significant increase in the frequency of metaphases with aberrant chromosomes was found in the industrial group (5.3%) as compared with the control (2.9%). The effect of toxic factors on the functional state of acrocentric NORs was analyzed. No significant differences between PMDA-exposed workers and the control group in cumulative functional activity of 10 NORs (silver staining) was revealed. At the same time, a decreased proportion of cells with associations was found (76% in workers and 82% in donors), which may be a consequence of changes in immune status and in compensatory activation of cell proliferation, which leads to accumulation of young circulating lymphocytes with low associative capacity of acrocentrics. In addition, a significantly increased proportion of individuals carrying extreme Ag-NOR variants (grades 3.5-4.0) was observed in the industrial group (up to 37% vs 6% in the control), which may be due either to genotype selection at the number of active rRNA gene copies or to compensatory activation of repressed copies of ribosomal genes in some NORs.

[Interconnection of the functional activity of nucleolar organizer regions of chromosomes with human reproductive pathologies]. / Vzaimosviaz' funktsional'noi aktivnosti iadryshkoobrazuiushchikh raionov khromosom s reproduktivnoi patologiei cheloveka.

Comparative analysis of transcriptional activity of human nucleolus organizer regions (NOR) of the chromosomes in the group of the control phenotypically healthy individuals (I) and in the spouses with repeated spontaneous abortions (II) was conducted in an attempt to verify the hypothesis: whether elimination of zygotes having received a very large or very small number of the copies of active rRNA genes may serve as a factor decreasing the fecundity of some spouses? It has been shown that the groups I and II have no differences in total activity of 10 NOR (Ag staining, rating estimation). At the same time, the II group is characterized by higher, in comparison with the I, heterogeneity of Ag-NOR variants in homologues of 5 nucleolus-organizing chromosomes. As a result, in the individuals of the group II the gametes are formed which are more heterogenous than in the group I for the Ag-NOR pattern variants. The imitation computer experiments revealed that in the group II elimination of zygotes as a consequence of inherited Ag-NOR variants combination should occur more frequently (in 22.2% cases) than in the group I (15.9%), p < 0.05. Thus, the hypothesis under test was substantiated in the present study.

[Localization of the CD4 receptor gene in the chromosomes of clone cells of the monocytoid line U-937 characterized by different sensitivities to HIV]. / Lokalizatsiia gena retseptora CD4 v khromosomakh kletok klonov monotsitoidnoi linii U-937, kharakterizuiushchikhsia razlichnoi chuvstvitel'nost'iu k VICh.

Karyotypes of two clones of U-937 line, with high and low sensitivity to HIV-1, were studied. The CD4-receptor gene-cellular receptor of HIV-1 was mapped. CD4-receptor gene was located according to in situ hybridization method, in locus 12 p11-p12, both in cells of high-sensitive clone U-937/16, and in cells of low-sensitive clone U-937/4. It is determined that in both the clones chromosomes 12 are presented in two copies and are not affected by rearrangements. That allows to conclude that the sensitivity of cells U-937 to HIV-1 does not depend on the dose of this gene, or on its transference in chromosomes.