RESUMO
CytoDiff reagent was developed to optimize the performance of a WBC differential evaluation. It determines the main population of white blood cells by the expression of linear markers. One of the additional possibilities of using CytoDiff is the differential diagnosis of reactive and tumor lymphocytosis. We studied peripheral blood samples of 76 patients of the All-Russian Central Research Center A.M. Nikiforova EMERCOM of Russia with absolute lymphocytosis of more than 3,0x109/l. The control group included 26 practically healthy people. All performed a clinical blood test on a 5Diff hematology analyzer with smear microscopy, and determination of leukocyte populations by phenotype using CytoDiff. Reference intervals were determined for subpopulations of lymphocytes using CytoDiff. An algorithm has been developed for evaluating the results obtained when determining leukocyte populations by phenotype using CytoDiff for differential diagnosis of reactive and tumor lymphoproliferation. To detect B-cell lymphoproliferative diseases, the use of a cut-off value of 13% or more of the number of leukocytes is optimal. At low values of the relative number of B-lymphocytes, it is important to take into account the results of microscopy of blood smears. If atypical mononuclear cells are absent in smears, then additional clinical and laboratory studies are necessary to establish the cause of lymphocytosis, including phenotyping of peripheral blood lymphocytes to exclude T-cell lymphoproliferative diseases. The expediency of using the CytoDiff reagent for the differential diagnosis of the reactive and tumor nature of lymphocytosis is shown. Already at the stage of primary screening studies, the use of CytoDiff makes it possible to efficiently collect blood samples from patients with possible lymphoproliferative diseases, which significantly reduces the time required for a diagnostic search.
Assuntos
Linfocitose , Neoplasias , Diagnóstico Diferencial , Citometria de Fluxo , Humanos , Linfocitose/diagnóstico , Neoplasias/complicações , Federação RussaRESUMO
The number of active ribosomal genes (AcRG) was evaluated in 172 carriers of chromosomal abnormalities (CA) such as Down's syndrome (DS), Robertsonian translocations (RT), Klinefelter's and Turner's syndromes, trisomy Ð¥, disomy Y, and various structural CA. In controls (n=318), AcRG dosage varied from 119 to 190 copies with a mean of 151 copies per diploid genome. In CA carriers, except for DS newborns, AcRG dosage was not beyond these limits. As shown previously, only within these limits cellular homeostasis and organism's viability can be supported, while genomes beyond these limits are eliminated by embryonic loss. About 10% of embryos with DS and 50% of embryos with RT die/are aborted exclusively due to a surplus (DS) or a shortage (RT) of AcRG. AcRG dosage also affects the CA carrier's viability after birth, as demonstrated by comparing newborn and aged (10-40 y.o.) DS patients. Sampling range of AcRG dosage becomes considerably narrower with age: DS newborns ranged from 139 to 194 RG copies (σ2=3.59), while aged DS patients varied from 152 to 190 copies (σ2=1.55) with the same mean. Each CA group showed peculiarities in AcRG dosage distribution. We found that carriers of numerical abnormalities of gonosomes (sex chromosomes) concentrate within the area of medium, most adaptive dosages, whilst carriers of structural CA can only survive with relatively high AcRG number. Our article is the first ever to report an association of CA viability with the genomic number of AcRG.
Assuntos
Aberrações Cromossômicas , Transtornos Cromossômicos/genética , Variações do Número de Cópias de DNA , Dosagem de Genes , RNA Ribossômico/genética , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Síndrome de Down/genética , Feminino , Humanos , Lactente , Recém-Nascido , Cariótipo , Masculino , Pessoa de Meia-Idade , Trissomia/genética , Síndrome de Turner/genética , Adulto JovemRESUMO
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.
Assuntos
Antioxidantes/metabolismo , Transtorno Autístico , Genes de RNAr , Modelos Biológicos , Estresse Oxidativo , Esquizofrenia , Adolescente , Transtorno Autístico/enzimologia , Transtorno Autístico/etiologia , Transtorno Autístico/genética , Criança , Pré-Escolar , Diagnóstico Diferencial , Humanos , Estresse Oxidativo/genética , Guias de Prática Clínica como Assunto , Esquizofrenia/enzimologia , Esquizofrenia/etiologia , Esquizofrenia/genéticaRESUMO
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.
Assuntos
Cromossomos Humanos/genética , Genes de RNAr/fisiologia , Instabilidade Genômica/fisiologia , Metáfase/fisiologia , Região Organizadora do Nucléolo/genética , Polimorfismo Genético/fisiologia , Células Cultivadas , Cromossomos Humanos/metabolismo , Dosagem de Genes/fisiologia , Loci Gênicos/fisiologia , Humanos , Mutação , Região Organizadora do Nucléolo/metabolismoRESUMO
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.
Assuntos
Doadores de Sangue , DNA Ribossômico/análise , Líquido Extracelular/química , Dosagem de Genes/genética , Componentes Genômicos/genética , Imersão , Ribossomos/genética , Adulto , Transfusão de Sangue , Humanos , Masculino , Ribonucleases/sangue , Adulto JovemRESUMO
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.
Assuntos
Aterosclerose/sangue , Biomarcadores/sangue , Imersão/efeitos adversos , Adulto , Aterosclerose/etiologia , Seguimentos , Humanos , Lipídeos/sangue , Masculino , Recuperação de Função Fisiológica/fisiologia , Valores de Referência , Adulto JovemRESUMO
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.
Assuntos
Artrite Reumatoide/metabolismo , Morte Celular , Dosagem de Genes , Estresse Oxidativo , RNA Ribossômico 18S/genética , Pele/metabolismo , Artrite Reumatoide/patologia , Células Cultivadas , Fibroblastos/metabolismo , Humanos , Pele/citologiaRESUMO
Most human somatic cells have no telomerase activity. This leads to terminal underreplication of chromosomes and, hence, proliferative ageing of cells. We studied the consequences of introduction of the gene of the catalytic component of human telomerase hTERT in the normal fibroblasts of adult human skin. The expression of this gene led to the appearance of telomerase activity in the fibroblasts, elongation of telomeres (to the size characteristic of the embryonic cells), and immortalization. The cells retained their normal karyotype. The activity of ribosomal genes remained unchanged: the degree of their methylation, abundance, and transcriptional activity (two clones were studied). The cells did not undergo significant changes after transition over the Hayflick's limit, retained the constant rate of proliferation (one of the clones was followed to the level of 200 duplications of the population), and resembled, in appearance, young diploid human fibroblasts. The initial cells and cells transfected by an empty vector could pass through no more than 68 duplications, their proliferation slowed down and they acquired the morphology characteristic for the ageing cells. The telomerized cells retained the normal capacity of entering the proliferative rest as a result of serum starvation. Telomerization did not eliminate the contact inhibition of proliferation but led to an increased saturating density of cells, which reached the levels characteristic for the early embryonic cells. The long-term suppression of the telomerase function by azidothymidine led to a shortening of telomeres and significantly slowed down cell proliferation. The cells that did not divided for a long time were enlarged, preserved their viability, and resembled, in appearance, the ageing cells. In the test on heterokaryons (index of telomerase activity on the chromosomes inside the cell), the telomerized cells behaved as other immortal cells. All these data suggest that the telomerized cells preserved the normal mechanisms of regulation of cell proliferation.
Assuntos
Linhagem Celular Transformada/citologia , Linhagem Celular Transformada/fisiologia , Telomerase/metabolismo , Telômero/fisiologia , Técnicas de Cultura de Células/métodos , Divisão Celular/efeitos dos fármacos , Divisão Celular/genética , Senescência Celular/fisiologia , Meios de Cultura Livres de Soro/farmacologia , Proteínas de Ligação a DNA , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Genes de RNAr , Humanos , Cariotipagem , Telomerase/genética , TransfecçãoAssuntos
Metilação de DNA , DNA Ribossômico/genética , DNA Ribossômico/metabolismo , Fibroblastos/fisiologia , Telômero/genética , Divisão Celular/genética , Transformação Celular Neoplásica/genética , Senescência Celular/genética , Sondas de DNA/genética , Proteínas de Ligação a DNA , Fibroblastos/citologia , Dosagem de Genes , Humanos , Sequências Repetitivas de Ácido Nucleico , Telomerase/genética , Telomerase/metabolismo , Telômero/metabolismo , TransfecçãoRESUMO
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%).
Assuntos
Variação Genética , Região Organizadora do Nucléolo/genética , Adulto , Células Sanguíneas , Mapeamento Cromossômico , Marcadores Genéticos , Humanos , Hibridização In Situ , Individualidade , Cariotipagem , Modelos Lineares , Pessoa de Meia-Idade , Região Organizadora do Nucléolo/ultraestruturaAssuntos
Alcoolismo/genética , Centrômero/ultraestrutura , Aberrações Cromossômicas , Metáfase/genética , Transtornos Relacionados ao Uso de Substâncias/genética , Adolescente , Adulto , Alcoolismo/sangue , Alcoolismo/tratamento farmacológico , Humanos , Cariotipagem , Linfócitos/ultraestrutura , Masculino , Transtornos Relacionados ao Uso de Substâncias/sangue , Transtornos Relacionados ao Uso de Substâncias/tratamento farmacológico , TemperançaRESUMO
Frequencies of extreme C segments of chromosomes 1, 9, and 16 were studied in patients with isolated gonadotropin deficiency (I), gonadotropin resistant ovary (I), and polycystic ovarian disease (III). Increased chromosome 9 heterochromatin was revealed for all syndromes, and increased chromosome 1 heterochromatin was revealed for groups II and III. Identical changes in heterochromatic parts of chromosomes, linked with increased heterochromatin, probably suggest a lack of specificity for the syndromes studied.
Assuntos
Cromossomos Humanos Par 1 , Cromossomos Humanos Par 9 , Gonadotropinas/deficiência , Doenças Ovarianas/genética , Polimorfismo Genético , Feminino , Gonadotropinas/fisiologia , Heterocromatina , Humanos , Doenças Ovarianas/fisiopatologiaRESUMO
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.
Assuntos
Antígenos CD4/genética , Cromossomos Humanos Par 12/genética , HIV-1/patogenicidade , Linhagem Celular , Células Clonais , DNA/genética , Sondas de DNA , Humanos , Cariotipagem , Hibridização de Ácido NucleicoRESUMO
We have obtained the alphoid DNA clones, pK1 and pK2, from the extrachromosomal DNA of Hela cells treated by cycloheximide (30 micrograms/ml). Nucleotide sequences of the clones were aligned. The sides of the pK1 and pK2 are 390 and 184 bp, respectively. The marked RELP for the clones was not observed. The results of in situ hybridization have shown an approximately equal distribution of Ag-grains over major part of human chromosomes, with a slight preference for chromosomes 1, 5 and 19 (the 1-st group of alpha-satellite DNA). Therefore, the obtained alphoid sequences seem to be rather conservative and non-chromosome-specific. We suppose that increase of the alphoid DNA content in the fraction of the extrachromosomal DNA under the cycloheximide treatment is a result of the sporadic statistical processes rather then consequence of the specific excision.
Assuntos
Sequência de Bases , DNA Circular , DNA Satélite , Cromossomos Humanos Par 1 , Cromossomos Humanos Par 19 , Cromossomos Humanos Par 5 , Clonagem Molecular , Cicloeximida/farmacologia , DNA Circular/genética , Herança Extracromossômica , Células HeLa , Humanos , Hibridização de Ácido Nucleico , Polimorfismo de Fragmento de RestriçãoRESUMO
Data are presented on the distribution of centromeric heterochromatin of the human X-chromosome in the interphase nucleus of a moving cell. The in situ hybridization made it possible to obtain some results leading to the following conclusions: in moving fibroblasts centromeric heterochromatin of the X-chromosome is located in end regions of the interphase nucleus; there was no preferential localization noted of the centromeric region of the X-chromosome in the front or back areas of the nucleus as to the direction of the movement.
Assuntos
Núcleo Celular/ultraestrutura , Cromossomo X , Movimento Celular , Centrômero , DNA Satélite , Heterocromatina/análise , Humanos , Interfase , Hibridização de Ácido NucleicoRESUMO
In this work a molecular-cytogenetic characteristic has been given to the alpha-satellite DNA of chromosome 6. Using different restrictases made it possible to evaluate structural heterogeneity of the obtained subgroup of the alphoid DNA. Polymorphic restriction fragments of DNA have been found which can be used in determining linkage groups.
Assuntos
Cromossomos Humanos Par 6 , DNA Satélite/isolamento & purificação , Cromatografia em Gel , Bandeamento Cromossômico , DNA Satélite/análise , DNA Satélite/genética , Humanos , Immunoblotting , Hibridização de Ácido Nucleico , Polimorfismo de Fragmento de Restrição , Mapeamento por RestriçãoRESUMO
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.
Assuntos
Clonagem Molecular/métodos , DNA Satélite/genética , Sequências Repetitivas de Ácido Nucleico , Sondas de DNA , Humanos , Cariotipagem , Hibridização de Ácido NucleicoRESUMO
Human rRNA genes contain variable regions, one of which is located in nontranscribed spacers (NTSs) closely downstream from the 3'-end of the transcribed region. This polymorphism may be detected by means of blot hybridization analysis as a set of distinct restriction fragments corresponding to this part of the rRNA genes. We have analyzed DNA of 51 individuals and found eight structural NTS variants of this region; two of these were common to all individuals analyzed, and six others were found in different combinations and with different frequencies. The copy number of each variant also differed but was not less than 10-20 copies per cell. The analysis of DNA isolated from leukocytes of the members of 11 families indicated that some of the structural variants (of the NTS region) are inherited as a single Mendelian locus. We propose that rRNA genes that belong to one particular structural variant form clusters on separate chromosomes. To test this proposition, we developed a combined method, including AgNO3-staining of chromosomes, in situ hybridization, and DNA analysis with methylation-sensitive restrictases, and used it for study of persons who had methylated rRNA genes located on AgNO3-negative nucleolar organizers. It was found that in three of four cases methylated genes really belonged to one structural variant. This approach may be used for detailed localization of separate classes of NTS structural variants of human rRNA genes.