100 research outputs found
Regulated interactions of the SUMO ligase RANBP2 with the mitotic spindle and kinetochores during mitotic progression
No full textRANBP2 is a large nucleoporin (NUP) residing at nuclear pore complexes (NPCs) in interphase and plays a role in nucleo-cytoplasmic transport of macromolecules across the NPC. In mitosis, when nuclear envelope (NE) breaks down and NPCs disassemble, RANBP2 localizes on mitotic structures. RANBP2 has SUMO (small ubiquitin-related modifier) E3 ligase and SUMO stabilizing activities and regulates protein SUMO conjugation, a post-translational modification of particular importance in dynamic processes such as the DNA damage response, stress response, various signaling pathways and mitosis. A characterized SUMOylated RANBP2 target is RANGAP1, the GTP-hydrolysis activating factor for theGTPase RAN. RANBP2 and RANGAP1, together with Ubc9 (a SUMO E2 enzyme), form a complex, called RRSU (RANBP2/RANGAP1-SUMO/UBC9), that has enhanced SUMO ligase activity and localizes to kinetochores (KTs) in metaphase with a mechanism that is not completely understood.
The goal of my PhD project was to identify the molecular mechanisms regulating the RRSU complex localization in space and time during mitosis, particularly to KTs, given the importance of these structures as the connecting structures between chromosomes and the mitotic spindle and their crucial role in chromosome segregation.
Both RANBP2 and RANGAP1 are known to interact with nuclear transport receptors, Importin beta and CRM1, during nuclear transport in interphase. In my project I have developed in situ proximity ligation assays (PLA) to visualize their interactions with these transport factors, follow their dynamics during cell division and assess whether nuclear transport receptors have themselves a functional role in the RSSU complex localization in mitotic cells.
PLA results show that the RRSU complex engages in dynamic interactions with Importin beta and CRM1 during mitotic progression: it preferentially interacts with Importin beta in early mitotic stages along the spindle MTs. In metaphase, after MTs attach all KTs, this interaction decreases. Concomitantly, the RRSU complex also interacts with CRM1: this interaction becomes up-regulated in metaphase and becomes visible at MT attached-KTs. Thus, the RRSU complex appears to “switch partners” from prometaphase (prevalent engagement with Importin beta along the spindle) to metaphase (increased PLA signals with CRM1 at KTs), suggesting that protein SUMO conjugation takes place with a spatially and temporally regulated programme in mitosis.
To validate the “switch partner” model I generated inducible cell lines, both for Importin beta and CRM1, to assess whether unbalancing one or the other would influence the RRSU complex localization in mitosis. Results from experiments with the inducible cell lines show that the mitotic localization of the RRSU complex depends on the antagonistic actions of Importin beta and CRM1: indeed, unbalancing each one of them impairs the RSSU complex localization and concomitantly generates segregation defects, suggesting that KT functions are defective. Overall, the results of my project highlight the importance of localized SUMOylation of proteins at the mitotic apparatus and KTs for balanced chromosome segregation, and indicate a role of nuclear transport receptors as upstream regulators in the process. It is of note that several cancer types overexpress these transport factors, which may contribute to the high level of genetic instability observed in these cancers
Common Fragile Sites: updating the causes of their variability in different cell tissues
No full textCommon Fragile Sites (CFSs) are regions in which DNA is prone to gaps, breaks or constrictions visible on metaphase chromosomes when cells are under replicative stressful conditions.
CFSs are characterized by slow/late replication timing mainly due, among other characteristics, to nucleotide sequence which tend to form secondary structure, and to the number of active (or inducible) replication origins. Recent studies indicate that CFSs expression is associated with tissue specificity. In the first part of the work, induction and classification of CFSs in two human lung fibroblast cell line, IMR-90 and MRC-5, has been done. Cytogenetical identification of the most expressed CFSs in both fibroblast cell lines were done: 1p31.1 and 3q13.3, located on chromosome 1 and 3 respectively, are peculiar for this tissue. These regions have typical and confirmed CFSs’ characteristics such as expression higher than 3%, high AT levels and enrichment in large genes.
Using genomic databases, searching for causes of their instability were done comparing percentage of repetitive elements among the CFSs, non-fragile regions (NFRA) and standard genomic sequences. These CFSs are characterized by presence of large genes, NEGR1 found in 1p31.1, LSAMP and ARHGAP31 in the most fragile region of 3q13.3, that could be co-responsible for their genomic instability.
Using probes delimitating fragile regions and combining FISH with IF anti-BrdU, analysis of relationship between replication timing and fragility was done. Furthermore, comparison between replication timing, in normal and stressful condition using APH, was done as well. The results obtained for these fragile regions reflect the replication timing impairments typical of fragile sites, in both normal and stressful conditions.
The same probes when used in lymphocytes result in a normal replication timing, moreover also using CFSs probes specific in lymphocytes on fibroblast, results in normal replication timing.
The results from replication timing analysis are strictly correlated with the structural and functional characteristics that are specific of the tissues in which these CFSs are expressed
Simultaneous production of Q and R bands after staining with chromomycin A3 or olivomycin.
No full textHuman and mouse chromosomes, stained with either chromomycin A3 or olivomycin, which bind preferentially to G - C-rich DNA (where G is guanosine and C is cytosine), exhibit a Q or a reverse banding pattern, depending on the wavelength used for excitation. The two complementary banding patterns can be observed in the same metaphase simply by changing the combination of excitation filters. These data suggest, therefore, that in addition to base composition, other factors are involved in the production of chromosome banding by chromomycin A3 and olivomycin
The base analog 6-N-hydroxylaminopurine (HAP) mutagenesis is dependent on the integrity of the uvsE, uvsF and uvsB genes in Aspergillus nidulans.
No full textDNA methylation in insects
No full textCytosine DNA methylation has been demonstrated in numerous eukaryotic organisms and has been shown to play an important role in human disease. The function of DNA methylation has been studied extensively in vertebrates, but establishing its primary role has proved difficult and controversial. Analysing methylation in insects has indicated an apparent functional diversity that seems to argue against a strict functional conservation. To investigate this hypothesis, we here assess the data reported in four different insect species in which DNA methylation has been analysed more thoroughly: the fruit fly Drosophila melanogaster, the cabbage moth Mamestra brassicae, the peach-potato aphid Myzus persicae and the mealybug Planococcus citri
CSB: An Emerging Actionable Target for Cancer Therapy
No full textThe DNA repair protein Cockayne syndrome group B (CSB) is frequently found overexpressed in cancer cells. High CSB levels favor tumor cell proliferation whilst inhibiting apoptosis. Conversely, the suppression of CSB has significant anticancer effects. In this manuscript we describe CSB downregulation as a potential new therapeutic approach in cancer
Recidiva precoce del morbo di Crohn e lesioni microscopiche dei margini della resezione intestinale.
No full textL'assorbimento della vit. B12 (test di Schilling) in pazienti con M. di Crohn resecati e non, sotto terapia medica (prednisolone e/o salazopirina) di mantenimento (TMM)
No full textOral and topical 5-aminosalicylic acid (mesalazine) in inducing and maintaining remission in mild-moderate relapse of ulcerative colitis: one-year randomised multicentre trial
No full textBackground. The association of oral 5-aminosalicylic acid (mesalazine) and enema is effective in treatment of mild-moderate forms of ulcerative colitis. However no study has been aimed at determining optimal duration of this association in active ulcerative colitis.
Aim. To determine whether longer duration of therapy: 1. increases the rate of patients achieving remission, and 2. reduces relapse rate during the maintenance period in patients in remission.
Patients and methods. A total of 149 patients, (89 male, 60 female), were randomly assigned to a regimen with 5-aminosalicylic acid tablets 2.4 g/day associated with 5-aminosalycilic enema 2 g/day for a 4-week (n=73) or 8-week regimen (n=76). After this acute therapy, patients were submitted to clinical, endoscopic and histological examinations and those in remission were assigned to a follow-up (maintenance) period with. oral mesalazine alone at a dosage of 1. 2 g/day A clinical visit, including laboratory tests, at 6 months and an endoscopic-histological control at 12 months were carried out to exclude symptoms and endoscopic-histological signs of activity. Pelapse of disease, i.e., presence of clinical symptoms or abnormal laboratory tests, was confirmed by endoscopy and histology.
Results. At end of acute phase, clinical, endoscopic and histological remission was comparable in the two groups: 42176 (5596), in the 4-week, and 47173 patients (64%), in the 8-week regimen. No difference was found stratifying patients according to extension of disease. Of these 89 patients in I-emission, 75 (34 from 4-week regimen; 41 from 8-week regimen) completed 12 months' follow-up. At end of follow-up, a similar percentage of patients in the 4-week regimen (50%) and 8-week regimen (51%) were still in remission. No significant difference between cumulative relapse rates of the two groups was found. Stratifying patients according to extension of disease, in the 8-week regimen group, those with left-sided colitis showed a higher remission rate than that of patients with diffuse colitis (66% versus 35%, p<0.05). All regimens were well tolerated by most patients during the entire study period.
Conclusions. An additional 4 weeks of topical treatment does not increase the remission rate in patients with mild-moderate active ulcerative colitis but seems to reduce the probability of relapse in patients with left-sided colitis
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