RESUMO
A model based method for the accurate quantification of the 3D structure of fluorescently labelled cellular objects similar in size to the optical resolution limit is presented. This method is applied to both simulated confocal images of chromatin structures and to real confocal data obtained on a Fluorescence in situ Hybridization (FISH) labelled gene domain. The model assumes that the object is composed of a small number of discrete points which are convolved with the microscope point spread function to give the image. Fitting this model to image data results in a method to assess object structure which is accurate, shows a low bias, and does not require user intervention or the potentially subjective setting of a threshold.
Assuntos
Cromatina/ultraestrutura , Simulação por Computador , Modelos Biológicos , Proteínas Centrais de snRNP , Núcleo Celular/ultraestrutura , Sondas de DNA , Corantes Fluorescentes , Genes , Humanos , Hibridização in Situ Fluorescente/métodos , Linfócitos/ultraestrutura , Microscopia Confocal/métodos , Conformação Molecular , Síndrome de Prader-Willi/genética , Sensibilidade e Especificidade , Proteínas Centrais de snRNP/genética , Proteínas Centrais de snRNP/ultraestruturaRESUMO
The hypothesis that distinct chromatin domains expand and are remodelled differently when they undergo transcription, replication or cell cycle processes is well accepted. The condensation changes by which chromosomes are transformed at the metaphase-interphase transition are especially interesting and therefore extensively studied by light microscopy; however, quantitative information of the size on specific small chromatin domains during the cell cycle is scarce. In this respect, a serious problem is the determination of structural features close to the resolution limit. In this report we use a novel approach to quantify the lateral extent of the 8q24/c-myc gene domain and the centromeric region of chromosome 8 in doubly labelled normal human foreskin fibroblasts using confocal laser scanning microscopy (CLSM). The domains were analysed in both metaphase spreads and interphase nuclei. These high precision measurements revealed a somewhat smaller (few 10s of nm) lateral extension of the centromere region in metaphase compared to interphase. Surprisingly, within the same cells the lateral extension of the 8q24/c-myc region was significantly smaller in interphase than in metaphase. For comparison the centromere size was more condensed in metaphase than in interphase. This implies a different folding behaviour for specific chromatin domains with opposite condensation behaviour.
Assuntos
Centrômero/genética , Genes myc/genética , Metáfase , Ciclo Celular/genética , Núcleo Celular/genética , Cromatina , Cromossomos/genética , Cromossomos Humanos Par 8/genética , Fibroblastos , Humanos , Interfase/genética , Microscopia Confocal , MitoseRESUMO
Recently we described a cutaneous T-cell lymphoma expressing the gamma/delta T-cell receptor [5]. The patient suffering from this lymphoma showed low numbers of myeloid and T cells in peripheral blood, while B and NK cells were relatively increased. In vitro culture of the patient's bone marrow (BM) cells revealed a significant suppression of myeloid/monocyte colony formation (GM-CFU) compared with normal controls. This was not due to infiltration of the BM with lymphoma cells. We speculated that a soluble factor either secreted or induced by the lymphoma cells might be responsible for the marked suppression of hematopoiesis in this patient. From a skin biopsy with infiltrating gamma/delta T-lymphoma cells we established T-cell clones bearing the gamma/delta T-cell receptor and resembling the phenotype of the lymphoma cells. The supernatant (SN) of these gamma/delta T-cell clones reduced the number of colonies in a CFU-GM assay (using normal control BM) in comparison to SN of alpha/beta T-cell clones established from the same biopsy. This suppression was seen mainly on day 7 of culture and was not neutralized by the addition of placenta-CM. The main mediator of this suppression seems to be IFN-gamma, since it was detectable in high amounts in the SN of these gamma/delta T-cell tumor clones as well as in the serum of the patient. In addition, anti-IFN-gamma antibodies can reverse the T-cell SN-mediated suppression of CFU-GM. We conclude that high serum levels of interferon-gamma, which is secreted in high amounts from gamma/delta T-cells grown from a biopsy of a cutaneous lymphoma, can suppress hematopoiesis.
