[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.

[Cluster size polymorphism of active human ribosomal genes and simulation of the conditions of its stability through generations].

Based on selective silver nitrate staining of active ribosomal gene (AcRG) clusters in nucleolus organizer regions (NORs) of human metaphase chromosomes, a technique was developed earlier to estimate the AcRG dosage in individual genomes as a sum of arbitrary units (0-3) ascribed to the silver precipitate (AgNOR) on ten NORs. The AcRG dosage was considered to be an additive quantitative trait determined by five polymorphic autosomal loci (with for allelic forms for each locus). A database was created to contain the data on AcRG cluster variants for more than 1000 individual human genomes. In this study, the population frequencies of AcRG cluster variants were determined. The results agreed with the hypothesis that stabilizing selection acts at the zygotic and/or early embryogenetic stage to restrain the AcRG genomic dosage (copy number) within a range from 14.9 to 23.7 arbitrary units (the cell is unviable when the trait is beyond this range). The average zygotic losses due to selection were estimated at 9.1-9.9% for a real population. A computer model where the AcRG dosage of a progeny results from a random combination of the AgNORs of the five acrocentric chromosome pairs of the parents was developed and used to simulate the formation of a certain AcRG genomic dosage through generations in a human panmictic population with nonoverlapping generations. A combination of stabilizing selection by total AcRG copy number and a certain spontaneous mutation rate (the probability of changes in the cluster size of a NOR as a result of unequal crossingover in meiotic prophase) was shown to be a sufficient condition for the restrain of equilibrium population frequencies of AgNOR size variants in a human panmictic population. Using the model, the most probable spontaneous mutation frequency was predicted to be (2.1-2.3) x 10(-2) per NOR per generation for human AgNORs. The predicted frequency was within the 95% confidence interval of the experimental rate, which was determined by studying the inheritance of AgNOR variants in real families.

[CpG-DNA inhibits cell reactions accompanied with the development of the adaptive response in human lymphocytes after low-dose X-ray exposure].

The lymphocytes of peripheral blood of healthy donors were influenced by X-ray radiation (10 cGy) or a fragments of the transcribed region of rDNA (TRrDNA) transmitted to the incubation medium of non-irradiated cells. Both factors induced transposition of the loci 1q12 of homologous chromosomes from the membrane to the centre of the nucleus in lymphocytes; produced the activation of the genes TLR9 and MyD88 expression, the chromosomal nucleolus-forming regions, TNF-alpha and caspase-3; and also increased nuclease activity and synthesis RNA of the cells. However all the investigated reaction in the cells did not developed during the synergetic radiation and TRrDNA but the activity level of the cytokine TNF-alpha was increasing. The reactions of human lymphocytes on the induced influence are discussed herein.

[Extracellular DNA fragments from culture medium of low-dose irradiated human lymphocyte trigger instigating of the oxidative stress and the adaptive response in non-irradiated bystander lymphocytes].

We have previously shown that the induced by X-ray radiation (10 cGy) in human lymphocytes reactions of transposition of the loci of homologous chromosomes from the membrane to the centre of the nucleus, and activation of the chromosomal nucleolus-forming regions (NFR) are transmitted via DNA fragments to the nonirradiated cells--the so-called bystander effect (BE). In the present study, the blockade of the oxidative stress (OS) with alpha-tocopherol prior to irradiation or treatment with H2O2 induced no effects of either chromosomal loci transposition or activation of the NFR; neither in the presence of alpha-tocopherol were these reactions induced by the addition of the DNA fragments from the growth medium of the exposed (X-irradiated or H2O2-treated) lymphocytes to the bystander cells. Moreover, after inhibiting the activity of caspase 3 in the H2O2-treated/irradiated lymphocytes or suppression of the toll-like receptors (TLR9) in their bystander cells, we observed no transposition of the chromosomal loci. Based on the reported and previously obtained findings we suggest that the induced OS specifically modifies nuclear DNA, instigating the mechanisms of the adaptive response (AR) and apoptosis of the radiation-sensitive lymphocytes, while the interaction of the DNA fragments released therefrom with the TLR9 of the bystander cells leads to the development of the OS in last, to be followed by the AR (BE). Possibilities of such a pathway are discussed herein.

[The DNA fragments obtained from the culture media exposed to adaptive doses of the ionizing radiation as factors of stress signaling between lymphocytes and bystander cells].

At the initial stages of an adaptive response the transposition of the homologous chromosome loci from the peripheral parts of the nucleus and their approach happens. It is necessary for the repair of DNA double strand breaks in the process of the homologous recombination. Was shown that the chromosome loci transposition and accompanied by the nucleolus activities took place first in the irradiated (X-rays, 10 cGy) G0-lymphocytes, and then in the intact (bystander) cells incubated in the growth medium of irradiated lymphocytes. If there is a bystander effect the quantity of irradiated cells may be three order less than the bystander cells that affirms the great capacity of stress-signalization system. Moreover, the DNA fragments (the factors of stress signaling) were obtained from the growth medium supernatant of the irradiated and of the intact lymphocytes. In other independent experiments they were inoculated into the growth medium of recipient cells. Was demonstrated that there is loci transposition of homologous chromosomes loci and of nucleus activity after introducing the DNA fragments of irradiated cells. After introducing the DNA fragments of non-irradiated cells the both effects were not observed. In the work the characteristics of the obtained factors and the possible ways of stress signaling between the irradiated and the bystander lymphocytes were discussed.

[Stress signaling between human lymphocytes after induction of bystander effect by exposure to ionizing radiation in adaptive doses].

We previously reported that the consequence of human lymphocytes irradiation by the adaptive doses (X-rays, 10 cGy) was a transposition of the homologous chromosome loci in the cell nucleus (FISH method); this phenomenon was mediated by the increase of nucleolus activity. They both are transmited to non-irradiated cells by the bystander effect (BE). We shown that the reaction of stress signaling is induced by the DNA fragments of irradiated lymphocytes. The study shows that after the inhibition of caspase 3 activity in irradiating lymphocytes or the blockade TLR9 in bystander cells the transposition was not observed. A signaling way of BE from irradiated lymphocytes apoptosis to bystander cells receptors is discussing.

Stimulatory effect of fragments from transcribed region of ribosomal repeat on human peripheral blood lymphocytes.

Fragments from the transcribed region of the ribosomal repeat include considerable amounts of unmethylated CpG DNA motifs. These motifs activate immune cells via the interaction with Toll receptors. In vitro experiments confirmed the stimulatory effect of transcribed region of ribosomal repeat on human lymphocytes. Culturing of lymphocytes in a medium containing 2-20,000 ng/ml fragments from transcribed region of ribosomal repeat was accompanied by structural changes in the nucleus in a considerable number of cells. These changes manifested in translocation of pericentromeric heterochromatin from the membrane to the center of the nucleus and activation of the nucleolus and were accompanied by a significant increase in interleukin-6 production and slight stimulation of tumor necrosis factor-a synthesis. The transcribed region of the ribosomal repeat and E. coli DNA had various effects on quantitative parameters of lymphocytes. Our results suggest the existence of mechanisms of stimulation not mediated by the interaction of CpG DNA motifs with Toll receptors.

[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.

[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.

[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.

[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.

[Relation between the intensity of transcription and the rRNA gene content of individual nucleolar-forming regions of human chromosomes]. / Zavisimost' mezhdu intensivnost'iu transkriptsii i soderzhaniem genov rRNK v individual'nykh iadryshkoobrazuiushchikh raionakh khromosom cheloveka.

A comparison has been carried out of satellite strand lengths, Ag-staining intensity and labelling intensity in in situ hybridization of 3H-thymidine-labelled 28S-rRNA gene fragment in nucleolar-forming regions (NFR's) of individual acrocentric chromosomes from blood lymphocytes of 2 karyotypically normal individuals. To identify chromosomes modified R-staining was performed (5-bromdesoxy-pyridine + Höchst fluorochrome 33258 + Giemsa dye). The data obtained demonstrate, firstly, the variability between 10 acrocentric human chromosomes both in the content of ribosome genes and in silvering intensity and NFR satellite strand length and, secondly, a positive correlation between three studied characteristics of individual chromosomes. In one case an exception has been noted in one homologue of chromosome 21 of an individual A: high intensity of hybridization labelling was accompanied by weak Ag-staining and short satellite strand. It was concluded that the variability of transcription activity of individual NFR's detected by Ag-staining is, as a rule, based on the variability in the content of ribosome gene DNA in them and, in some cases, the presence of rRNA gene copies in inactive state.

[Population polymorphism of silver-stained nucleolar organizer chromosome regions in man]. / Populiatsionnyi polimorfizm okrashivaiushchiksia serebrom iadryshkoobrazuiushchikh raionov khromosom u cheloveka.

Activity of nucleolar organizer regions (NORs) of acrocentric chromosomes determined by Ag-staining was comparatively studied in 40 individuals of Bulgarian and 40 individuals of Russian populations. Chromosome 21 was found to be significantly more often stained in both populations. The other NORs did not differ significantly in staining from the means. No differences were noted between individual NORs, in respect of the intensity of Ag-staining in both populations, except chromosome 15 which showed markedly decreased staining capacity in Russians. The data obtained are compared with those published in literature concerning four other populations.

[Molecular and cytogenetic approach to the mapping of methylated polymorphic restriction fragments of human rRNA genes]. / Molekuliarno-tsitogeneticheskii podkhod k kartirovaniiu metilirovannykh polimorfnykh restriktsionnykh fragmentov genov rRNK

Combined restriction with Bam H I and Sal I (or Hpa II) has revealed Bam H I fragment on a non-transcribed spacer of rRNA genes in one out of four individuals under study. Using Ag-staining and hybridization in situ, chromosome 13p+ enriched by inactive rRNA gene copies was found in the given individual. Since Sal I does not restrict methylated sequences and rRNA genes are repressed by methylation, it is concluded that methylated Banm I-restricted rRNA gene fragments of non-transcribed spacer are localized in chromosome 13p+ of the individual in question.

[Sequential staining of the polymorphic regions of human acrocentric chromosomes using different cytochemical methods]. / Posledovatel'noe okrashivanie polimorfnykh raionov akrotsentricheskikh khromosom cheloveka s pomoshch'iu razlichnykh tsitokhimicheskikh metodov.

The chromosome preparations of 12 normal individuals were consequently stained with four different techniques (RV, Q, C and Ag). Correlations between morphological and staining peculiarities of specific polymorphic segments of the short arms of the same acrocentrics were investigated. It was determined that except correlations for the length of satellite stalks and the size of Ag-deposits, the polymorphic signs revealed with Q-, C- and Ag-techniques appear to be independent and not correlating with each other. These data allow to think that in spite of morphological similarities between acrocentric arms, the individual chromosomes can differ in structure and properties of the nucleoprotein material available in the examined regions, which may cause a non-identical response to cytochemical treatment.

[Comparative analysis of the population polymorphism of human silver-staining chromosomal nucleolus organizer regions]. / Sravnitel'nyi analiz populiatsionnogo polimorfizma okrashchivaiushchikhsia serebrom iadryshkoobrazuiushchikh raionov khromosom u cheloveka.

The population study of nucleus organizing regions (NOR) activity in individuals of Georgian nationality was carried out using the Ag-method. The results obtained were compared with those for other populations: Russian, Viena-Ulm, Estonian etc. The mean number of active NORs for Georgians is 9.0, which significantly exceeds analogous indexes for the rest of populations, except Viena-Ulm one. The population differences in the distribution of NOR activity and the ability to be stained was found for individual NORs. The lowest activity was registered in chromosome 14, the highest being observed for chromosomes 21 and 13. It is assumed that some mechanism exists that controls the level of general NOR activity in the cell.

Genetic determination of NOR activity in human lymphocytes from twins.

Activity of nucleolar organizer regions (NORs) was studied in cultured blood lymphocytes from 20 monozygotic (MZ) and 20 dizygotic (DZ) twin pairs. The number of Ag-stained NORs, the degree of staining, and the frequency of acrocentric associations were used as criteria of the NOR activity, the acrocentric chromosomes being identified by G-banding. Analysis of intrapair concordance as well as of intrapair variance showed the number of Ag + NORs and the size of Ag-deposits to be highly heritable traits. Intrapair differences in acorocentric association frequency were not significantly higher in DZ compared with MZ twins.

Polymorphisms of Ag-stained nucleolar organizer regions in man.

Polymorphisms of the NORs as tested by Ag-staining of metaphase G-banded chromosomes were investigated in cultured blood lymphocytes of karyotypically normal individuals from the Moscow population. The study of cell-to-cell variability in the number of Ag-stained NORs carried out on 14 monozygotic twin pairs showed the phenomenon to have some features of real intercellular variation. In 40 unrelated individuals the individual acrocentric chromosomes were compared by the number of Ag-stained NORs, their degree of staining, and their participation in acrocentric association. Chromosome 21 was found to be significantly more active than four others by all the criteria, and chromosome 15 was less active compared with the others by the size of the Ag deposits and the frequency of participation in NOR associations. The frequency distribution of homozygotes and heterozygotes for Ag-stained NORs in the same group of 40 individuals was in accordance with the Hardy-Weinberg law.