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7 results on '"Rusciano, Giulia"'

6. Flexibility of the Prograamme of Spore Coat Formation in Bacillus subtilis: Bypass of CotE Requirement by Over-Production of CotH.

Author

Isticato, Rachele, Sirec, Teja, Giglio, Rosa, Baccigalupi, Loredana, Rusciano, Giulia, Pesce, Giuseppe, Zito, Gianluigi, Sasso, Antonio, De Felice, Maurilio, and Ricca, Ezio

Subjects
BACILLUS subtilis, BACTERIAL spores, BACTERIAL proteins, LYSOZYMES, PROMOTERS (Genetics), BACTERIAL sporulation, FLUORESCENCE microscopy
Abstract

Bacterial spores are surrounded by the coat, a multilayered shell that contributes in protecting the genome during stress conditions. In Bacillus subtilis, the model organism for spore formers, the coat is composed by about seventy different proteins, organized into four layers by the action of several regulatory proteins. A major component of this regulatory network, CotE, is needed to assemble the outer coat and develop spores fully resistant to lysozyme and able to germinate efficiently. Another regulator, CotH, is controlled by CotE and is present in low amounts both during sporulation and in mature spores. In spite of this CotH controls the assembly of at least nine outer coat proteins and cooperates with CotE in producing fully resistant and efficiently germinating spores. In order to improve our understanding of CotH role in spore formation, we over-produced CotH by placing its coding region under the control of a promoter stronger than its own promoter but with a similar timing of activity during sporulation. Over-production of CotH in an otherwise wild type strain did not cause any major effect, whereas in a cotE null background a partial recovery of the phenotypes associated to the cotE null mutation was observed. Western blot, fluorescence microscopy and Surface-Enhanced Raman Scattering spectroscopy data indicate that, in the absence of CotE, over-production of CotH allowed the formation of spores overall resembling wild type spores and carrying in their coat some CotE−/CotH-dependant proteins. Our results suggest that the B. subtilis spore differentiation programme is flexible, and that an increase in the amount of a regulatory protein can replace a missing partner and partially substitute its function in the assembly of the spore coat. [ABSTRACT FROM AUTHOR]

Published
2013
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7. Raman-microscopy investigation of vitrification-induced structural damages in mature bovine oocytes

Author

Giulia Rusciano, Rosa Carotenuto, Carolina De Canditiis, Gianluigi Zito, Bianca Gasparrini, María Roca, Antonio Sasso, Marcello Rubessa, Rusciano, Giulia, De Canditiis, Carolina, Zito, Gianluigi, Rubessa, Marcello, Roca, Maria Serena, Carotenuto, Rosa, Sasso, Antonio, and Gasparrini, Bianca

Subjects
0301 basic medicine, Cytoplasm, Embryology, lcsh:Medicine, Reproductive technology, Spectrum Analysis, Raman, Biochemistry, Oogenesis, Protein Structure, Secondary, Cryopreservation, Oxidative Damage, 0302 clinical medicine, Animal Cells, Macromolecular Structure Analysis, Vitrification, Birth Rate, Zona pellucida, lcsh:Science, Principal Component Analysis, 030219 obstetrics & reproductive medicine, Multidisciplinary, Fertility Preservation, Lipids, Cell biology, medicine.anatomical_structure, OVA, bovine oocytes, vitrification, Raman-microscopy, Female, Structural Proteins, Cellular Types, Cellular Structures and Organelles, Research Article, Protein Structure, Cell Survival, Specimen Preservation, Biology, Research and Analysis Methods, Cryobiology, 03 medical and health sciences, Botany, medicine, Animals, Blastocyst, Molecular Biology, Zona Pellucida, lcsh:R, Biology and Life Sciences, Proteins, Cell Biology, Oocyte cryopreservation, Lipid Metabolism, Oocyte, Germ Cells, 030104 developmental biology, Specimen Preparation and Treatment, Oocytes, Blastocysts, Cattle, lcsh:Q, Reactive Oxygen Species, Developmental Biology
Abstract

Although oocyte cryopreservation has great potentials in the field of reproductive technologies, it still is an open challenge in the majority of domestic animals and little is known on the biochemical transformation induced by this process in the different cellular compartments. Raman micro-spectroscopy allows the non-invasive evaluation of the molecular composition of cells, based on the inelastic scattering of laser photons by vibrating molecules. The aim of this work was to assess the biochemical modifications of both the zona pellucida and cytoplasm of vitrified/warmed in vitro matured bovine oocytes at different post-warming times. By taking advantage of Principal Component Analysis, we were able to shed light on the biochemical transformation induced by the cryogenic treatment, also pointing out the specific role of cryoprotective agents (CPs). Our results suggest that vitrification induces a transformation of the protein secondary structure from the α-helices to the β-sheet form, while lipids tend to assume a more packed configuration in the zona pellucida. Both modifications result in a mechanical hardening of this cellular compartment, which could account for the reduced fertility rates of vitrified oocytes. Furthermore, biochemical modifications were observed at the cytoplasmic level in the protein secondary structure, with α-helices loss, suggesting cold protein denaturation. In addition, a decrease of lipid unsaturation was found in vitrified oocytes, suggesting oxidative damages. Interestingly, most modifications were not observed in oocytes exposed to CPs, suggesting that they do not severely affect the biochemical architecture of the oocyte. Nevertheless, in oocytes exposed to CPs decreased developmental competence and increased reactive oxygen species production were observed compared to the control. A more severe reduction of cleavage and blastocyst rates after in vitro fertilization was obtained from vitrified oocytes. Our experimental outcomes also suggest a certain degree of reversibility of the induced transformations, which renders vitrified oocytes more similar to untreated cells after 2 h warming.

Published
2017

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