RESUMO
Changes in the macromolecular orientation and metachromasy of glycosaminoglycans (GAG) in newly synthesized and assembled collagen fibers in rat Achilles tendon after tendon excision were investigated in toluidine blue (TB)-stained preparations, based in the selective absorption of polarized light (= linear dichroism, LD) and of absorption of unpolarized light in situ. Extrinsic LD was observed microspectrophotometrically from the early phases of tendon repair onwards, although the absorption peaks in both parallel and perpendicular directions with respect to the plane of polarized light and the long axis of the collagen fibers occurred at the same wavelength, and thus differed from the pattern situation in normal adult controls. Compared to normal adult tendons, the pattern of LD in newly synthesized and assembled fibers was still not fully attained 110 days after surgical tendon removal. This incomplete recovery possibly reflected the influence of aging during the repair process. There was no correlation between LD and metachromasy. The highest absorption values for metachromatic staining occurred on the 7th day after tendon removal, at a time when LD was not intense. Treatment with hyaluronidase showed that the LD in the early stages of tendon repair was mostly due to hyaluronate whereas the LD in the later stages was due to chondroitin sulfates. The changes in LD during Achilles tendon repair were attributed to gradual modifications in the composition and macromolecular orientation of GAGs relative to the long axis of the collagen fibers.
Assuntos
Colágeno/metabolismo , Glicosaminoglicanos/metabolismo , Traumatismos dos Tendões/terapia , Tendão do Calcâneo/lesões , Animais , Ratos , Ratos Wistar , Fatores de Tempo , Cicatrização/fisiologiaRESUMO
The present study was undertaken to test the reproducible formation of the extended chromatin fibres (ECF), beads and superbeads, and detect molecular order and crystallinity by optical anisotropies in those structures. Chicken erythrocyte smears and mouse liver cell imprints were treated with 2.0-3.0 m NaCl solution in 1% Triton-X100 vertically prior to staining with 0.025% toluidine blue at pH 4. Detection of birefringence and colours of abnormal dispersion of birefringence (ADB) following toluidine blue binding to DNA revealed that the DNA molecular order and crystallinity in decondensed and condensed chromatin are preserved after ECF formation. The tests for Con-A binding to mannose/glucose residues of glycoproteins was confirmed within nuclei, and in the ECF, beads and superbeads. ECF formation was not regular. Clumped cells did not show ECF, although chromatin mobility was observed within the nuclei. Electron microscopy demonstrated that after treatment of nuclei with 0.77 m NaCl ECF always spread from the nuclei, in clumped nuclei the fibres aggregated instead of spreading.
Assuntos
Núcleo Celular/ultraestrutura , Cromatina/ultraestrutura , Eritrócitos/ultraestrutura , Fígado/ultraestrutura , Animais , Núcleo Celular/metabolismo , Galinhas , Cromatina/metabolismo , Concanavalina A/metabolismo , Cristalização , Eritrócitos/metabolismo , Fígado/metabolismo , CamundongosRESUMO
When transfected to benzo[a]pyrene (BP)-transformed MCF-10F human breast epithelial cells (BP1 cell line) the c-Ha-ras oncogene has proven to enhance the neoplastic changes initiated by exposure to BP, giving rise to an aggressive tumorigenic cell line, BP1-Tras. We have previously demonstrated by image analysis that BP affects the DNA content and the chromatin supraorganization of MCF-10F cells. Here Feulgen-stained BP1-Tras cells were studied by image analysis in order to evaluate possible additional changes in DNA content and chromatin texture induced by insertion of the ras oncogene. A high variability in DNA content also including polyploidy or near-polyploidy, and an increase in the packing states of the chromatin which became still condensed in BP1 cells were found in BP1-Tras cells. The results differed from those reported for the BP1-E1 cell line which is also an aggressive tumorigenic cell line, but was attained through progressive passages of BP-transformed cells. It was demonstrated that different patterns of changes in DNA content and chromatin organization may be involved in equally aggressive tumorigenic BP-transformed cell lines originated from the same cell line by different mechanisms.
Assuntos
Benzo(a)pireno/farmacologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Cromatina/genética , DNA de Neoplasias/análise , Genes ras/genética , Corantes de Rosanilina , Linhagem Celular Transformada , Corantes , Células Epiteliais/citologia , Feminino , Humanos , Citometria por ImagemRESUMO
DNA content and chromatin supraorganization defined in terms of patterns of chromatin texture were studied by image analysis in Feulgen-stained human breast MCF-10F epithelial cells expressing different stages of tumorigenic progression after treatment with benzo[a]pyrene (BP). Nontransformed MCF-10F, nontumorigenic transformed BP1, and tumorigenic BP1-E1 cell lines were analyzed. A relationship between changes in DNA content and chromatin texture and the expression of different stages of tumorigenesis were researched in interphase nuclei. A significant decrease in Feulgen-DNA amounts and absorbing areas suggesting aneuploidy, and first detected in nontumorigenic transformed BP1 cells, became more relevant in the tumorigenic cell line BP1-E1. Simultaneously, an increase in optical densities and a change in related parameters, demonstrating increased chromatin higher order packing states, occurred in transformed BP1 cells and extended over larger chromatin areas in tumorigenic BP1-E1 cells. The results indicate loss of DNA and a change in chromatin higher order packing states accompanying the expression of different stages of the in vitro tumorigenesis process in BP-transformed human breast epithelial cells. It is suspected that the gradually acquired chromatin "condensation" supraorganization pattern is associated with activated transcription activities previously suggested for these cells.
Assuntos
Benzo(a)pireno , Mama/metabolismo , Cromatina/metabolismo , DNA de Neoplasias/metabolismo , Células Epiteliais/metabolismo , Processamento de Imagem Assistida por Computador , Mama/patologia , Linhagem Celular Transformada , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Cromatina/patologia , DNA de Neoplasias/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Feminino , Humanos , MicroespectrofotometriaRESUMO
Aspects of the molecular organization of the antennal sensilla trichodea of Triatoma infestans have been investigated with the use of both polarization and scanning electron microscopy. The sensilla have a smooth surface with minute bulbs on the tapered end. They showed strong positive birefringence, irrespective of the refractive index of the imbibing medium. The plotting of a form birefringence (FB) curve for native material showed slight increases in the retardation values with increases of the refractive index and at least 2 inflection points. On the other hand, a FB curve constructed for alkali-treated structures reveals higher values for the form birefringence and slight decreases in retardation values with raising refractive index. These results demonstrate that chitin fibrils are preferentially aligned with the sensilla long axis. Interestingly, the alkali treatment introduced no alterations in the retardation values measured at n = 1,435, which corresponds to the intrinsic birefringence of chitin. It is suggested that components removed by alkali-treatment have electronic transitions disposed perpendicularly to the chitin filaments and that the ill definition of the form birefringence curve of chitin is associated with the incomplete removal of chitin-associated components, which keep chitin fibrils apart from but strongly bound to each other. In addition, it is apparent from the results that cross-linking in a system with a parallel array of the chitin fibrils occurs predominantly perpendicular to the chitin fibrils.