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Investigations on the refolding of chromatin using scanning force microscopy have recently renewed interest in the three-dimensional organization of the higher order structure, so complementing several observations on the molecular functioning of the transcription machinery showing that the unfolding of heterochromatin represents an obligatory step in gene activation and vice versa. A few years ago we (Allera et al. J. Biol. Chem. 272, 10817, 1997) reported several striking observations on the status of chromatin of apoptotic thymocytes. All of the genome was found to be organized into a woven network of 11 nm fibers, arising from the close face to face packing of nucleosomes, a conformation strongly reminiscent of the organization of the columnar phases of core particles described by Levforestier and Livolant (Biophys. J. 73, 1771, 1997). In addition, the onset of apoptosis involved a significant (from 42 to 70%) increase in the unacetylated isoform of histone H4. In this report we show first that H4 acetylation does not directly modulate chromatin higher order structure. Indeed, apoptosis is induced quite efficiently in rat thymocytes by treatment with sodium butyrate, an inhibitor of histone deacetylases, as judged from the observation of the relevant hallmarks. Also the ultrastructure of chromatin was undistinguishable from that isolated from thymocytes treated with methylprednisolone sodium succinate; the percentage of the unacetylated isoform of H4 (32%) was, however, lower than that of control cells (42 %). Ultrastructural observation in combination with image analysis and Fourier filtering confirmed that the 11 nm fiber arises from the face to face packing of nucleosomes. When the fiber is restrained in the network, however, it experiences a considerable elastic stress. Indeed, a basic morphological motif is the presence of interwound regions giving rise to a characteristic figure of eight configuration. When the intercalating agent ethidium bromide (EB) is added to the chromatin solution at a molar ratio r of 0.028 (r=molecules of bound EB/molecules of DNA phosphate) we observe the complete relaxation of the superstructure but not the separation of the core particles, while at the same value or r the linker appears as a straight segment connecting well separated cores in control chromatin. A second relevant morphological feature is the origin of the crosslinking regions in the network. Image analysis clearly shows that these regions correspond to short stretches of the polynucleosomal chain in which the core particles interact edge to edge, and can therefore firmly associate with another stretch through face to face interactions. Our results demonstrate for the first time that the bulk of apoptotic chromatin is negatively supercoiled. A structural and thermadynamic investigation on the mechanism of heterochromatin formation from our laboratory has shown that this process is driven by an all or none phase transition inside the loop (Balbi et al., Biophys. J., 77, 2725, 1999). Since we have shown that apoptotic condensation occurs in the absence of DNA cleavage, it is plausible to assume that also the chromatin transitions in apoptosis are entirely determined by internucleosomal interactions inside a topologically isolated unit.

BACKGROUND. After the discovery that nuclear matrix (NM) directs the spatial organization of DNA transcription and replication, there has been an increasing interest in studying NM changes associated with malignant transformation and their potential usefulness in the clinical setting. METHODS. High-resolution two-dimensional gel electrophoresis was used to analyze the NM proteins (NMP) of specimens of prostate cancer tissue obtained from the prostates of 75 patients undergoing retropubic prostatectomy. RESULTS. Nine NMP with different molecular weights and isoelectric points have been identified. They were expressed differently by prostate cancer tissues. An increasing trend toward the expression of such proteins like NMP 6-8 was evident with increasing tumor stage and dedifferentiation. NMP 6-8 were also significantly correlated with the risk of biochemical progression. However, Gleason score was the only significant discriminant in this regard in multiparametric models. CONCLUSIONS. This study confirms that prostate cancer progression is related to profound changes in NMP expression patterns. However, due to the complexity of the methods required to define these latter, the clinical relevance of NMP appears to be still limited.

N-terminally truncated amyloid-beta (Abeta) peptides are present in early and diffuse plaques of individuals with Alzheimer's disease (AD), are overproduced in early onset familial AD and their amount seems to be directly correlated to the severity and the progression of the disease in AD and Down's syndrome (DS). The pyroglutamate-containing isoforms at position 3 [AbetaN3(pE)-40/42] represent the prominent form among the N- truncated species, and may account for more than 50% of Abeta accumulated in plaques. In this study, we compared the toxic properties, fibrillogenic capabilities, and in vitro degradation profile of Abeta1-40, Abeta1-42, AbetaN3(pE)-40 and AbetaN3(pE)-42. Our data show that fibre morphology of Abeta peptides is greatly influenced by the C-terminus while toxicity, interaction with cell membranes and degradation are influenced by the N- terminus. AbetaN3(pE)-40 induced significantly more cell loss than the other species both in neuronal and glial cell cultures. Aggregated AbetaN3 (pE) peptides were heavily distributed on plasma membrane and within the cytoplasm of treated cells. AbetaN3(pE)-40/42 peptides showed a significant resistance to degradation by cultured astrocytes, while full- length peptides resulted partially degraded. These findings suggest that formation of N-terminally modified peptides may enhance beta-amyloid aggregation and toxicity, likely worsening the onset and progression of the disease.

A light scattering method, together with complementary electron microscopy observations, was designed to investigate the self-assembly of fibrin. Calcium-free monomer was used, and clot reconstitution was carried out in solvents corresponding to limit interaction energies of the protein with the medium. The self-assembly process, under physiological conditions, conforms to the following sequence of events. 1) A fast polymerization step leading to linear aggregates. 2) Fiber growth; at this stage onset of the network occurs. 3) Gelation (clot formation). Bound calcium was found to be structurally required for gelation. Its removal results in the formation of thick fibers, which are unable to clot. Evidence is reported favouring our previous hypothesis (Conio et al., 1976) on the onset of the network: branching of linear aggregates is a prerequisite for clotting. The occurrence of a crystallization process, which overlaps to fiber growth, is demonstrated in this paper for the first time. Its dependence on solvent-protein interactions is analyzed. Our results suggest that fibrin monomer is to some extent a flexible molecule. Both flexibility and crystallization may play a functional role in the clotting process in vivo.

Different lines of evidence suggest that the nuclear matrix (NM), the protein scaffold of the nucleus, represents a functional unit playing a pivotal role in the spatial and temporal coordination of the events of gene activation. Any change in the gene expression pattern, which occurs during carcinogenesis, may partially depend on an impairment of the regulatory functions of the NM. Therefore, increasing interest has been addressed to the study of NM modifications associated with malignant transformations and to potential clinical applications. Here, recent results on the NM changes in prostate cancer are discussed. Tumor cells are characterized by a more complex NM protein pattern compared to normal tissue: the development of poorly-differentiated tumors is characterized by the expression of proteins that are not present in hyperplastic tissues or in more differentiated tumors. In addition, a few newly-expressed proteins are significantly correlated with the risk of biochemical progression. The potential application of these proteins at the diagnostic and prognostic levels calls for further studies.