Changes in the molecular packing of fibrillin microfibrils during extension indicate intrafibrillar and interfibrillar reorganization in elastic response.

Fibrillin-rich microfibrils are the major structural components of the extracellular matrix that provide elasticity in a majority of connective tissues. The basis of elastic properties lies in the organization of fibrillin molecules, which, unfortunately, is still poorly understood. An X-ray diffraction study of hydrated fibrillin-rich microfibrils from zonular filaments has been conducted to give an insight into the molecular structure of microfibrils in intact tissue. A series of measurements was taken during controlled tissue extension to observe alterations in the lateral packing of microfibrils. Computer-generated simulated patterns were used to fit the experimental X-ray scattering data and to obtain the fibril diameter and lateral distance between the fibrils. The results suggest a nonlinear correlation between external strain and decrease in fibril diameter and lateral spacing. This was accompanied by a nonlinear increase in axial periodicity and a structure with a 160-nm periodicity, which is reported here for the first time using X-ray diffraction. These changes may reflect the unraveling of fibrillin from the complex folded arrangement into a linear structure. This finding supports a pleating model where fibrillin molecules are highly folded within the microfibrils; more importantly, the connection is made between the interaction of individual microfibrils and the change in their suprafibrillar coherent organization during extension. We suggest that the intermediate states observed in our study reflect sequential unfolding of fibrillin and can explain the process of its reversible unraveling.

DNA damage and repair in human peripheral blood lymphocytes following treatment with microcystin-LR.

The purpose of this study was to find a possible explanation of the inconsistency of data regarding the genotoxicity of microcystin-LR (MC-LR). We compared the results of the comet assay with the results of the analysis of chromosome aberrations and apoptosis. In order to investigate the influence of MC-LR on DNA damage in human lymphocytes, cells were treated with MC-LR at different concentrations (1, 10 and 25 microg/ml) for 6, 12, 18 and 24 h. Analyses of Olive Tail Moment (OTM) as an indicator of DNA damage showed that MC-LR treatment induced DNA damage in a time-dependent manner, reaching its maximum after 18 h. The lowest values of OTM were observed after 24 h. MC-LR had no effect on the frequency of chromosome aberrations in lymphocytes. Since some data available in the literature indicate that apoptosis may lead to overestimated or false positive results regarding the genotoxicity of mutagens in the comet assay, we measured the frequency of late apoptotic cells by use of the comet assay and the frequency of early apoptotic cells with the TUNEL method. The comet assay results revealed that the highest level of apoptosis was observed after 24 h and the lowest after 18 h. The comparison of the frequency of apoptotic cells determined by the comet assay with DNA damage (OTM) examined by the comet assay revealed a statistically significant, negative correlation. The TUNEL results showed that the frequency of apoptotic cells progressively increased in a dose- and time-dependent manner. The comparison of the frequency of apoptotic cells determined by TUNEL method with DNA damage (OTM) examined by the comet assay showed a significant positive correlation for lymphocytes treated with MC-LR for 6, 12 and 18 h. Therefore, our findings indicate that microcystin-LR-induced DNA damage observed in the comet assay may be related to the early stages of apoptosis due to cytotoxicity but not genotoxicity. In addition, we examined the DNA repair kinetics in lymphocytes following treatment with microcystin-LR and ionizing radiation. Our results indicate that MC-LR has an inhibiting effect on the repair of radiation-induced damage.