RESUMO
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
RESUMO
Chromatin structure is a major regulator of transcription and gene expression. Herein we explore the use of osmotic modulation to modify the chromatin structure and reprogram gene expression. In this study we use the extracellular osmotic pressure as a chromatin structure and transcriptional modulator. Hyposmotic modulation promotes chromatin loosening and induces changes in RNA polymerase II (Pol II) activity. The chromatin decondensation opens space for higher amounts of DNA engaged RNA Pol II. Hyposmotic modulation constitutes an alternative route to manipulate cell fate decisions. This technology was tested in model protocols of induced pluripotency and transdifferentiation in cells growing in suspension and adherent to substrates, CD34+ umbilical-cord-blood (UCB), fibroblasts and B-cells. The efficiency and kinetics of these cell fate modulation processes were improved by transient hyposmotic modulation of the cell environment.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Transdiferenciação Celular/efeitos dos fármacos , Cromatina/química , Meios de Cultura/farmacologia , Pressão Osmótica , Células-Tronco/efeitos dos fármacos , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Linfócitos B/ultraestrutura , Células Cultivadas , Cromatina/ultraestrutura , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Meios de Cultura/química , DNA/genética , DNA/metabolismo , Sangue Fetal , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Humanos , Células K562 , Cinética , Osmose , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Células-Tronco/metabolismo , Células-Tronco/ultraestrutura , Transcrição Gênica/efeitos dos fármacosRESUMO
OBJECTIVE: To improve knowledge of the mechanisms of cellular differentiation and proliferation during retinal development, by studying cellular and molecular damage in a rat model of prenatal ethanol exposure. METHODS: Female, juvenile Wistar rats (200g body weight) and their offspring were divided into two groups, which were fed a liquid diet: 1) ethanol-exposed group (5% ethanol weight/vol as 35% of daily total calories) and 2) isocaloric control group (maltose/dextrin as 35% of daily total calories). Eyeballs were obtained at 21 days of gestation, embedded in paraffin, and immunodetection procedures performed on apoptotic (TUNEL) and mitotic profiles, which were observed and photographed using a confocal microscope. RESULTS: Analysis of the microphotographs revealed a statistically significant increase of apoptotic profiles and a decrease in mitotic profiles in the ethanol exposed group compared to controls (p<0.05). Ganglion cells and photoreceptors showed more changes than other retinal cell phenotypes. These findings suggest that abnormalities in the differentiation and proliferation processes of the retina were caused by the alcohol exposure. CONCLUSIONS: Alcohol abuse during pregnancy alters development of the visual system by inducing developmental changes in the mitotic and apoptotic processes of the retina. These latter changes may be the result of changes in the expression of regulatory genes as well as the result of alteration in signalling pathways for both differentiation-proliferation and apoptotic events.
Assuntos
Apoptose/efeitos dos fármacos , Etanol/toxicidade , Olho/efeitos dos fármacos , Olho/patologia , Mitose/efeitos dos fármacos , Animais , Feminino , Gravidez , Ratos , Ratos Wistar , SíndromeRESUMO
It is widely assumed that the vital processes of transcription and translation are spatially separated in eukaryotes and that no translation occurs in nuclei. We localized translation sites by incubating permeabilized mammalian cells with [3H]lysine or lysyl-transfer RNA tagged with biotin or BODIPY; although most nascent polypeptides were cytoplasmic, some were found in discrete nuclear sites known as transcription "factories." Some of this nuclear translation also depends on concurrent transcription by RNA polymerase II. This coupling is simply explained if nuclear ribosomes translate nascent transcripts as those transcripts emerge from still-engaged RNA polymerases, much as they do in bacteria.
Assuntos
Núcleo Celular/genética , Biossíntese de Proteínas , Transcrição Gênica , Animais , Autorradiografia , Biotina/metabolismo , Compostos de Boro/metabolismo , Células COS , Fracionamento Celular , Permeabilidade da Membrana Celular , Núcleo Celular/metabolismo , Cicloeximida/farmacologia , Citoplasma/metabolismo , Fluorescência , Células HeLa , Humanos , Imuno-Histoquímica , Mitocôndrias/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Transporte Proteico , Proteínas/metabolismo , RNA Polimerase II/metabolismo , Aminoacil-RNA de Transferência/metabolismo , Ribossomos/metabolismo , Células Tumorais CultivadasRESUMO
Recent evidence suggests that active RNA polymerases are concentrated in discrete 'factories' where they work together on many different templates. The evidence that such factories specialize in the transcription of particular groups of genes is reviewed.
Assuntos
Núcleo Celular/metabolismo , Transcrição Gênica/fisiologia , Nucléolo Celular/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Células HeLa , Humanos , Fatores de Transcrição/metabolismoRESUMO
Epidemiological and experimental studies suggest the involvement of lipid peroxidation (LPO) in retinal diseases. Clinicians usually prescribe antioxidants to help in the treatment of proliferative diabetic vitreoretinopathy and age-related macular degeneration. In spite of this, these processes inexorably induce visual impairment and may progress towards blindness. In addition to other pathogenic mechanisms not fully understood, it may be that peroxidic aldehydes from LPO occurring in the eyes, acting as cytotoxic chemicals, mediate in these chronic disorders. To test the mechanisms of removing peroxidic aldehydes from retinal cells and in an attempt to understand long-lasting changes induced by LPO, the distribution and activity of aldehyde dehydrogenases (ALDH) in the rat retina were studied and compared with the LPO sites induced by iron/nicotine adenine dinucleotide phosphate. Histochemical and immunocytochemical assays revealed the colocalization of LPO and ALDH, mainly in the photoreceptors and inner retinal layers. This suggests the involvement of ALDH in detoxifying peroxidic aldehydes from the retina. Any change in ALDH retinal expression and distribution might be of crucial importance in assessing the paths of LPO-mediated vitreoretinopathies. Further research is needed to evaluate these findings and their application to new ophthalmic therapy.
Assuntos
Aldeído Desidrogenase/metabolismo , Ferro/farmacologia , Peroxidação de Lipídeos , Peróxidos Lipídicos/metabolismo , NADP/farmacologia , Retina/enzimologia , Animais , Técnica Indireta de Fluorescência para Anticorpo , Imuno-Histoquímica , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Ratos , Retina/ultraestruturaRESUMO
The path that RNA takes through nuclear pores was mapped using two high-resolution techniques. Unexpectedly, no RNA in HL60 cells was detected by immunogold labelling in the central axis of the pore complex on its way to the transporter at the nuclear membrane; instead, it was distributed around the sides, apparently entering just before the membrane. In rat liver nuclei, poly(A)(+) RNA, hnRNPs A1 and C, mrnp 41, ASF, and a phosphorylated subset of SR proteins were also distributed like mRNA, as were various transport factors and their cargoes (NTF2, Ran, RCC1, karyopherin (beta), Rch1, transportin (alpha), m(2,2,7)-trimethylG). Many pores were associated with particular transport factors/cargoes to the exclusion of others; some were associated with poly(A)(+) RNA or phosphorylated SR proteins (but not NTF2), others with NTF2 (but not poly(A)(+) RNA or the SR proteins). Electron spectroscopic imaging confirmed these results. Some pores contained phosphorus-rich RNA apparently entering from the sides; others lacked any phosphorus, and were surrounded by a ribosome-free zone in the cytoplasm. The results also suggest that pores have different functional zones where SR proteins are dephosphorylated, and where hnRNP C is removed from messages.
Assuntos
Membrana Nuclear/metabolismo , Proteínas de Transporte Nucleocitoplasmático , RNA/metabolismo , Animais , Transporte Biológico Ativo , Proteínas de Transporte/metabolismo , Células HL-60 , Humanos , Hibridização In Situ , Fígado/metabolismo , Fígado/ultraestrutura , Microscopia Eletrônica , Membrana Nuclear/ultraestrutura , Proteínas Nucleares/metabolismo , Fósforo/metabolismo , RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos WistarRESUMO
Some SR proteins are associated with eukaryotic transcripts as they move from synthetic sites (transcription "factories"), through downstream sites, to nuclear pores. Downstream sites can also be isolated as large nuclear ribonucleoprotein particles of approximately 200 S (diameter approximately 50 nm). In ultrathin sections of HeLa nuclei, indirect immunogold labeling with a specific antibody gives many small clusters of approximately 10 gold particles (diameter 50-80 nm). We gauged errors in estimating the diameter of underlying structures marked by immunogold probes (lengths approximately 20 nm). We examined systematically how probe dimensions affected cluster diameter. Probes contained one to three immunoglobulin molecules, sometimes a protein A molecule, and a gold particle of 5-15 nm. We found that (a) immunolabeling particles were tightly packed, (b) reducing particle size by 5 nm reduced cluster diameter by 10 nm, (c) reducing the number of immunoglobulins in the immunolabeling sandwich from three to two reduced cluster diameter by approximately 4 nm, (d) replacing the last immunoglobulin in a sandwich with protein A increased diameter by approximately 7 nm and led to a peripheral concentration of particles, and (e) increasing the number of layers in the sandwich increased sensitivity. Assuming that underlying structures had diameters of 50 nm, we find that errors ranged from -20% to +50%.
Assuntos
Imuno-Histoquímica , Proteínas Nucleares/química , Fosfoproteínas/química , Células HeLa/química , Humanos , Microscopia Imunoeletrônica , Proteínas de Ligação a RNA/química , Sensibilidade e Especificidade , Fatores de Processamento de Serina-ArgininaRESUMO
The route taken by transcripts from synthetic sites in the nucleus to the cytoplasm has been under scrutiny for years, but details of the pathway remain obscure. A new high-resolution method for mapping the pathway is described; HeLa cells are grown in Br-U so that the analogue is incorporated into RNA and exported to the cytoplasm, before Br-RNA is localized by immuno-electron microscopy. After exposure to low concentrations of Br-U for short periods, cells grow normally. Br-RNA is first found in several thousand extra-nucleolar transcription sites or factories (diameter 50-80 nm), before appearing in several hundred new downstream sites (diameter 50-80 nm) each minute; subsequently, progressively more downstream sites become labelled. These sites can be isolated on sucrose gradients as large nuclear ribonucleoprotein particles of approximately 200 S. Later, Br-RNA is seen docked approximately 200 nm away from approximately 20% nuclear pores, before exiting to the cytoplasm. Individual downstream sites are unlikely to contain individual transcripts; rather, results are consistent with groups of transcripts being shipped together from synthetic sites to pores. A subset of SR proteins are excellent markers of this pathway; this subset is concentrated in tens of thousands of sites, which include transcription, downstream and docking sites. Growth in high concentrations of Br-U for long periods is toxic, and Br-RNA accumulates just inside nuclear pores.
Assuntos
Microscopia Imunoeletrônica/métodos , Membrana Nuclear/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/análise , Bromouracila/análogos & derivados , Nucléolo Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Células HeLa , Humanos , Imuno-Histoquímica , Membrana Nuclear/química , Cloreto de Sódio , Transcrição Gênica , Uridina/análogos & derivadosRESUMO
Using HeLa cells, we have developed methods to determine 1) the number of RNA polymerases that are active at any moment, 2) the number of transcription sites, and 3) the number of polymerases associated with one transcription unit. To count engaged polymerases, cells were encapsulated in agarose, permeabilized, treated with ribonuclease, and the now-truncated transcripts extended in [32P]uridine triphosphate; then, the number of growing transcripts was calculated from the total number of nucleotides incorporated and the average increment in length of the transcripts. Approximately 15, 000 transcripts were elongated by polymerase I, and approximately 75,000 were elongated by polymerases II and III. Transcription sites were detected after the cells were grown in bromouridine for <2.5 min, after which the resulting bromo-RNA was labeled with gold particles; electron microscopy showed that most extranucleolar transcripts were concentrated in approximately 2400 sites with diameters of approximately 80 nm. The number of polymerases associated with a transcription unit was counted after templates were spread over a large area; most extranucleolar units were associated with one elongating complex. These results suggest that many templates are attached in a "cloud" of loops around a site; each site, or transcription "factory," would contain approximately 30 active polymerases and associated transcripts.
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Transcrição Gênica , Bromouracila/análogos & derivados , Nucléolo Celular , Núcleo Celular , Citidina Trifosfato/metabolismo , Ativação Enzimática , Células HeLa , Humanos , RNA Polimerase I/metabolismo , RNA Polimerase II/metabolismo , Uridina/análogos & derivadosRESUMO
Current models for RNA synthesis involve an RNA polymerase that tracks along a static template. However, research on chromatin loops suggests that the template slides past a polymerase immobilized in a large transcription factory. The evidence for immobilized polymerases is reviewed, and a model for transcription by such fixed enzymes is presented. According to the model, gene activation would involve reducing gene-factory distance and increasing the affinity of a promoter for a factory. Locus controlling regions and enhancers would attach to a factory and increase the chances that a promoter could bind to a polymerase; after transcriptional termination, the gene would detach from the factory. As some RNA processing occurs cotranscriptionally, processing sites are also likely to be associated with the factory.
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Modelos Genéticos , Transcrição Gênica , DNA/genética , DNA/metabolismo , Enzimas Imobilizadas , Regulação da Expressão Gênica , Regiões Promotoras Genéticas , RNA/biossíntese , RNA/genética , Processamento Pós-Transcricional do RNA , Ativação TranscricionalRESUMO
Nascent transcripts in permeabilized HeLa cells were elongated by approximately 30-2,000 nucleotides in Br-UTP or biotin-14-CTP, before incorporation sites were immunolabelled either pre- or post-embedding, and visualized by light or electron microscopy. Analogues were concentrated in approximately 2,100 (range 2,000-2,700) discrete sites attached to a nucleoskeleton and surrounded by chromatin. A typical site contained a cluster (diameter 71 nm) of at least 4, and probably about 20, engaged polymerases, plus associated transcripts that partially overlapped a zone of RNA polymerase II, ribonucleoproteins, and proteins rich in thiols and acidic groups. As each site probably contains many transcription units, these results suggest that active polymerases are confined to these sites, which we call transcription 'factories'. Results are consistent with transcription occurring as templates slide past attached polymerases, as nascent RNA is extruded into the factories.
Assuntos
Núcleo Celular/enzimologia , RNA Polimerases Dirigidas por DNA/metabolismo , Sítios de Ligação , Biotina/metabolismo , Compartimento Celular , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Citidina Trifosfato/metabolismo , Células HeLa , Humanos , Microscopia Imunoeletrônica , RNA/genética , RNA/metabolismo , Transcrição Gênica , Uridina Trifosfato/análogos & derivados , Uridina Trifosfato/metabolismoRESUMO
It is well known that light emission is related to lipid peroxidation in biological material, and that this process occurs spontaneously in the brain. tert-Butyl hydroperoxide (tBHP) is an organic peroxide widely used as initiator of free radical production in several biological systems. However, the prooxidant capacity of this compound remins unclear. To clarify its role in brain spontaneous autooxidation, rat brain homogenates were incubated with and without tBHP. Light emission and lipid peroxidation were measured by luminometry and the TBARs test, respectively. Several inhibitors of free radical-induced lipid peroxidation were also used. These inhibitors included ascorbate, EDTA, and desferrioxamine. Our results indicate that the pattern of light emission spontaneously produced in brain was different from that observed after the addition of tBHP to the homogenates, and that these differences depended on the tBHP concentration. The main difference was that tBHP caused a rapid light emission that reached its maximum more quickly than in the case of spontaneous emission. Addition of ascorbate resulted in an increase in chemiluminescence in presence of tBHP. In contrast, EDTA and desferrioxamine inhibited light emission in homogenates both with and without tBHP. The results of MDA determination were similar to those described, including the effect of inhibitors. A common feature in MDA and luminometric determinations was the dispersion of data. In conclusion, these results suggest that tBHP, under specific conditions, modify the kinetic pattern of brain spontaneous autooxidation.
Assuntos
Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Medições Luminescentes , Peróxidos/farmacologia , Animais , Antioxidantes/farmacologia , Ácido Ascórbico/farmacologia , Desferroxamina/farmacologia , Ácido Edético/farmacologia , Feminino , Histocitoquímica , Ferro/farmacologia , Malondialdeído/metabolismo , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Ratos , Ratos Wistar , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , terc-Butil HidroperóxidoRESUMO
We report here that a 62 kDa calmodulin-binding protein (p62), recently identified in the nucleus of rat hepatocytes, neurons and glial cells, consists of four polypeptides showing pI values between 5.9 and 6.1. By using a DNA-binding overlay assay we found that the two most basic of the p62 polypeptides bind both single- and double-stranded DNA. The intranuclear distribution of calmodulin and p62 was analysed in hepatocytes and astrocyte precursor cells, and in proliferating and differentiated astrocytes in primary cultures by immunogold-labeling methods. In non-dividing cells nuclear calmodulin was mostly localized in heterochromatin although it was also present in euchromatin and nucleoli. A similar pattern was observed for p62, with the difference that it was not located in nucleoli. p62/calmodulin complexes, mainly located over heterochromatin domains were also observed in interphasic cells. These complexes remained associated with the nuclear matrix after in situ sequential extraction with nucleases and high-salt containing buffers. In dividing cells, both calmodulin and p62 were found distributed over all the mitotic chromosomes but the p62/calmodulin aggregates were disrupted. These results suggest a role for calmodulin and p62 in the condensation of the chromatin.
Assuntos
Proteínas de Ligação a Calmodulina/metabolismo , Calmodulina/metabolismo , Ciclo Celular/fisiologia , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Astrócitos/ultraestrutura , Nucléolo Celular/química , Células Cultivadas , Cromatina/química , DNA/metabolismo , DNA de Cadeia Simples/metabolismo , Interfase/fisiologia , Fígado/citologia , Fígado/metabolismo , Fígado/ultraestrutura , Microscopia Imunoeletrônica , Mitose/fisiologia , Ligação Proteica , RatosRESUMO
The endocytic-lysosomal system of proliferating and differentiated astrocytes in primary culture was investigated using a combination of cytochemical, immunocytochemical and biochemical procedures. These included impregnation with osmium tetroxide and potassium iodide, phosphotungstic acid staining, cytochemical demonstration of acid phosphatase and thiamine pyrophosphatase activities and incorporation of cationized ferritin. The acid phosphatase activity was also analyzed using biochemical techniques. Our results indicate that while all astrocytes in primary culture have a developed endocytic-lysosomal system, this system is different in proliferating cells from that in differentiated astrocytes. Whereas in proliferating astrocytes it appears to be composed mainly of a variety of vacuoles and vesicles displaying a heterogeneous osmium tetroxide staining pattern, differentiated cells are characterized by the presence of small size vesicles showing an intense reaction. Both types of astrocyte showed abundant lysosomes, including multivesicular bodies, which presented an intense phosphatase acid activity. Biochemical analyses demonstrated that this activity increase during the proliferation period, reaching a maximum at 15 days of culture. Incorporation of cationized ferritin revealed that lysosomes and endosomes constitute separate systems. Finally, we have also found that the activity of thiamine pyrophosphatase, a marker for the Golgi complex, increases throughout the culture period. These results indicate that astrocytes could play an important role in regulating the macromolecular composition of the extracellular space.
Assuntos
Astrócitos/ultraestrutura , Endocitose , Lisossomos/ultraestrutura , Vacúolos/ultraestrutura , Fosfatase Ácida/análise , Animais , Astrócitos/fisiologia , Encéfalo/citologia , Diferenciação Celular , Divisão Celular , Células Cultivadas , Ferritinas/metabolismo , Tetróxido de Ósmio , Ácido Fosfotúngstico , Ratos , Coloração e RotulagemRESUMO
Alcohol dehydrogenase (ADH), the major enzyme catalyzing the biological oxidation of ethanol in mammals, includes four classes with very different capacities for ethanol oxidation. Class III ADH is present in all the tissues and is well conserved throughout evolution. This enzyme has a low activity with ethanol, is specific for the glutathione-dependent oxidation of formaldehyde, and is therefore a formaldehyde dehydrogenase (FALDH). Until now there have been few and conflicting studies concerning its intracellular distribution, which is important for the understanding of its role in cell function. In the present work we used biochemical and immunocytochemical methods to assess the distribution of FALDH in rat hepatocytes and astroglial cells. With the glutathione-dependent formaldehyde dehydrogenase assay, we found the highest activity in the cytosol of hepatocytes and brain cells (12 and 2.6 mU/mg protein, respectively), but nuclei also exhibited significant activity (1.16 and 2.1 mU/mg protein, respectively). The immunocytochemical results showed the presence of FALDH binding sites in both the cytoplasm and the nucleus of the different cell types studied. Whereas no specific gold particle labeling was seen associated with any cytoplasmic component, in the nucleus the particles were found mainly over condensed chromatin and interchromatin regions. Finally, the gold particle density over both the nucleus and cytoplasm was greater in differentiated than in proliferating astrocytes in primary culture. In contrast, class I ADH, primarily responsible for ethanol metabolism, was found only in the cytoplasm of hepatocytes. We propose that one of the functions of FALDH is to protect cell structures, including DNA, from the toxic effects of endogenous formaldehyde, which is an intermediate in many metabolic process.
Assuntos
Aldeído Oxirredutases/análise , Núcleo Celular/enzimologia , Aldeído Oxirredutases/imunologia , Aldeído Oxirredutases/isolamento & purificação , Animais , Anticorpos/imunologia , Especificidade de Anticorpos , Astrócitos/enzimologia , Astrócitos/ultraestrutura , Encéfalo/citologia , Encéfalo/enzimologia , Encéfalo/ultraestrutura , Fracionamento Celular , Núcleo Celular/ultraestrutura , Células Cultivadas , Cromatina/química , Imuno-Histoquímica/métodos , Fígado/citologia , Fígado/enzimologia , Fígado/ultraestrutura , Microscopia Eletrônica , RatosRESUMO
This study has investigated the effect of prenatal alcohol exposure on the qualitative and quantitative ultrastructure of proliferating and differentiated astrocytes in primary cultures as well as on the cytochemical activity of several subcellular phosphatase markers, including acid phosphatase, uridine diphosphatase, thiamine pyrophosphatase, 5'-nucleotidase and glucose-6-phosphatase. The astrocytes were obtained from 21-day-fetuses of both control and alcohol-fed rats. Our results show that several cell components, such as mitochondria, rough endoplasmic reticulum and lysosomes, exhibit qualitative and/or quantitative ultrastructural changes during the process of astrocyte maturation. In some cases these morphological changes are accompanied by variations in the cytochemical activity of enzymes located in these and other cell components, suggesting that these enzymes, and therefore the functional state of these organelles, are modulated during astrocyte development. When prenatally exposed to ethanol, both proliferating and differentiated astrocytes showed striking ultrastructural alterations compared with controls, including an increment of lysosomes as well as a decrease in the values of stereological parameters relative to mitochondria, rough endoplasmic reticulum and Golgi apparatus. Cytochemical analysis of these cells indicates that prenatal exposure to ethanol decreased the activities of all the enzymes tested, except for acid phosphatase, which was increased in both groups of treated astrocytes. These results suggest that prenatal exposure to ethanol could affect astrocytes during development in two different but probably complementary ways: a) by causing a delay in astrocyte maturation and, b) by inducing a direct toxic effect on these cells.