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Biomedical subjects

Ivonne Pasquali Ronchetti

Publications and source records attributed to Ivonne Pasquali Ronchetti.

9 recordsLinked to original sources
PubMed2005

Identification of mineralized elastic fibers on wet samples by SEM.

A method is described that could be of potential use for the rapid ultrastructural identification of abnormal and fragmented elastic fibers in very small wet samples of dermal biopsies from patients affected by Pseudoxanthoma elasticum (PXE). Moreover, the method, which consists of the use of sealed capsules transparent to electrons, allows the rapid and accurate localization and detection of mineralized areas in PXE patients and of their ion composition by X-ray microanalysis. This methodology could be of great help in any tissue disorder, especially in connective tissue disorders, characterized by structural alterations associated with ion precipitation.

Biopsy↗
PubMed2005

Dissection of human tropoelastin: supramolecular organization of polypeptide sequences coded by particular exons.

Polypeptide sequences encoded by some exons of the human tropoelastin gene (EDP, elastin-derived peptide) have been analysed for their ability to coacervate and to self-assembly. The great majority of them were shown to form organized structures, but only a few were indeed able to coacervate. Negative staining and rotary shadowing transmission electron microscopy showed the polypeptides to adopt a variety of supramolecular organization, from filaments, as those typical of tropoelastin, to amyloid-like fibers. The results obtained gave significant insight to the possible roles played by specific polypeptide sequences of tropoelastin.

Amyloid↗
PubMed2005

Heparan sulphate interacts with tropoelastin, with some tropoelastin peptides and is present in human dermis elastic fibers.

A number of reports point to the presence of proteoglycans and/or glycosaminoglycans within elastic fibers in normal and in pathological conditions. We present data that heparan sulphate (HS)-containing proteoglycans are associated with normal elastic fibers in human dermis and that isolated HS chains interact in vitro with recombinant tropoelastin and with peptides encoded by distinct exons of the human tropoelastin gene (EDPs). By immunocytochemistry, HS chains were identified as associated with the amorphous elastin component in the human dermis and remained associated with the residual elastin in the partially degenerated fibers of old subjects. HS appeared particularly concentrated in the mineralization front of elastic fibers in the dermis of patients affected by pseudoxanthoma elasticum (PXE). In in vitro experiments, HS induced substantial changes in the coacervation temperature and in the aggregation properties of recombinant tropoelastin and of synthetic peptides (EDPs) corresponding to sequences encoded by exons 18, 20, 24 and 30 of the human tropoelastin gene. In particular, HS modified the coacervation temperature and favoured the aggregation into ordered structures of tropoelastin molecules and of EDPs 18, 20 and 24, but not of EDP30. These data strongly indicate that HS-elastin interactions may play a role in tissue elastin fibrogenesis as well as modulating elastin stability with time and in diseases.

Adult↗
PubMed2004

Supramolecular amyloid-like assembly of the polypeptide sequence coded by exon 30 of human tropoelastin.

Elastin is known to self-aggregate in twisted-rope filaments. However, an ultrastructural organization different from the fibrils typical of elastin, but rather similar to those shown by amyloid networks, is shown by the polypeptide sequence encoded by exon 30 of human tropoelastin. To better understand the molecular properties of this sequence to give amyloid fibers, we used CD, NMR, and FTIR (Fourier transform infrared spectroscopy) to identify the structural characteristics of the peptide. In this study, we have demonstrated, by FTIR, that antiparallel beta-sheet conformation is predominant in the exon 30 fibers. These physical-chemical studies were combined with transmission electron microscopy and atomic force microscopy to analyze the supramolecular structure of the self-assembled aggregate. These studies show the presence of fibrils that interact side-by-side probably originating from an extensive self-interaction of elemental cross beta-structures. Similar sequences, of the general type XGGZG(X, Z = V, L, A, I), are widely found in many proteins such as collagens IV and XVII, major prion protein precursor, amyloid beta A4 precursor protein-binding family, etc., thus suggesting that this sequence could be involved in contributing to the self-assembly of amyloid fibers even in other proteins.

Amino Acid Sequence↗
PubMed2004

The effect on rat thymocytes of the simultaneous in vivo exposure to 50-Hz electric and magnetic field and to continuous light.

Thymus plays an important role in the immune system and can be modulated by numerous environmental factors, including electromagnetic fields (EMF). The present study has been undertaken with the aim to investigate the role of long-term exposure to extremely low frequency electric and magnetic fields (ELF-EMF) on thymocytes of rats housed in a regular dark/light cycle or under continuous light. Male Sprague-Dawley rats, 2 months old, were exposed or sham exposed for 8 months to 50-Hz sinusoidal EMF at two levels of field strength (1 kV/m, 5 microT and 5 kV/m, 100 microT, respectively). Thymus from adult animals exhibits signs of gradual atrophy mainly due to collagen deposition and fat substitution. This physiological involution may be accelerated by continuous light exposure that induces a massive death of thymocytes. The concurrent exposure to continuous light and to ELF-EMF did not change significantly the rate of mitoses compared to sham-exposed rats, whereas the amount of cell death was significantly increased, also in comparison with animals exposed to EMF in a 12-h dark-light cycle. In conclusion, long-term exposure to ELF-EMF, in animals housed under continuous light, may reinforce the alterations due to a photic stress, suggesting that, in vivo, stress and ELF-EMF exposure can act in synergy determining a more rapid involution of the thymus and might be responsible for an increased susceptibility to the potentially hazardous effects of ELF-EMF.

Animals↗
PubMed2004

ABCC6 mutations in Italian families affected by pseudoxanthoma elasticum (PXE).

Pseudoxanthoma elasticum (PXE) is a genetic disorder, characterized by cutaneous, ocular and cardiovascular clinical symptoms, caused by mutations in a gene (ABCC6) that encodes for MRP6 (Multidrug Resistance associated Protein 6), an ATP-binding cassette membrane transporter. The ABCC6 gene was sequenced in 38 unrelated PXE Italian families. The mutation detection rate was 82.9%. Mutant alleles occurred in homozygous, compound heterozygous and heterozygous forms, however the great majority of patients were compound heterozygotes. Twenty-three different mutations were identified, among which 11 were new. Fourteen were missense (61%); five were nonsense (22%); two were frameshift (8.5%) and two were putative splice site mutations (8.5%). The great majority of mutations were located from exon 24 to 30, exon 24 being the most affected. Among the others, exons 9 and 12 were particularly involved. Almost all mutations were located in the intracellular site of MRP6. A positive correlation was observed between patient's age and severity of the disorder, especially for eye alterations. The relevant heterogeneity in clinical manifestations between patients with identical ABCC6 mutations, even within the same family, seems to indicate that, apart from PXE causative mutations, other genes and/or metabolic pathways might influence the clinical expression of the disorder.

Adult↗
PubMed2003

Normal human dermal fibroblasts: proteomic analysis of cell layer and culture medium.

Proteins present within the cell layer and those released in the cell medium from in vitro cultured normal human dermal fibroblasts were separated and characterized in terms of their isoelectric point and molecular weight, by two-dimensional (2-D) gel electrophoresis. All spots in the synthetic gel were firstly analyzed by the Melanie 3 software and compared with those of breast cancer cells, colorectal epithelial cells, HL60, lymphoma cells, and platelets, already available on-line. From the identification of 144 spots from both the cell layer and the medium, we were able to recognize 89 different proteins, since a certain number of spots represented different isoforms of the same molecule. Identifications were performed by matching with on-line 2-D databases, and by matrix assisted laser-desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS), in order to confirm the identification by matching, or to identify new proteins. The procedure we used allows (i) to design a highly reproducible reference map of the proteome of adult human normal fibroblasts in culture, (ii) to evaluate protein species produced in the cell layer as well as those released in the culture medium, and (iii) to compare data from gel matching with those obtained by MS. This work represents an essential step for a better knowledge of mesenchymal cells, given the widespread use of this cell type in both clinical and experimental investigations.

Adult↗
PubMedDate not supplied

Extracutaneous ultrastructural alterations in pseudoxanthoma elasticum.

Pseudoxanthoma elasticum (PXE) is caused by mutations in the ABCC6 gene, encoding for the membrane transporter MRP6, whose physiological role is still unknown. PXE is characterized by skin, eye, and cardiovascular alterations mainly due to mineralization of elastic fibers. The ultrastructural alterations of a large number of tissues obtained at autopsy from 2 PXE patients were analyzed and compared to clarify the involvement of the various organs in PXE and to identify cell types responsible for clinical manifestations. Ultrastructural alterations typical of PXE were present in all organs examined and consisted mostly of fragmentation and mineralization of a number of elastic fibers, abnormalities of collagen fibril shape and size, and, less frequently, deposition of aggregates of matrix constituents in the extracellular space. The severity of alterations was more pronounced in the organs affected by the clinical manifestations of PXE. Interestingly, veins and arteries were similarly damaged, the adventitia and the perivascular connective tissue being the most affected areas. Therefore, alterations in PXE are systemic and affect all soft connective tissues, even in the absence of specific clinical manifestations. The localization of alterations suggests that fibroblasts and/or smooth muscle cells are very likely involved in the pathogenesis of the disorder. These findings may help in the diagnosis of PXE when clinical manifestations affect internal organs.

Adult↗