Faculty Bibliography

Sorption properties of olive husk were investigated under equilibrium (batch tests) and dynamic (column tests) conditions in order to assess the possibility of using such a waste material for removing heavy metals from aqueous streams. Husk samples were contacted, at 25 degrees C, with aqueous solutions of nitric salts of Pb, Cd, Cu, and Zn. Sorption isotherms obtained from equilibrium data were fitted and interpreted by the Freundlich model. Metals-saturated husk samples resulting from column tests were air-dried and incinerated to simulate combustion in order to assess the fate of sorbed metals. The results demonstrated that, under both equilibrium and dynamic conditions, metal sorption capacity of the husk was in the sequence Pb>Cd>Cu>Zn. For all the metals, calculated Freundlich constants decreased by increasing initial metal concentration or decreasing solution pH. In dynamic tests, a significant reduction of sorption capacity was recorded (except for copper) when a metal was fed simultaneously to the others: Pb (77%); Cd (93%); Zn (68%). Combustion tests carried out on metals-saturated husk samples showed that the average losses of lead and cadmium, as volatile species, were always three to four times greater than the losses of copper and zinc, in both single-metal- and multimetal-saturated samples

The cyclin-dependent kinase inhibitor p21Cip1 has important roles in the control of cell proliferation, differentiation, senescence, and apoptosis. It has been observed that p21 is a highly unstable protein, but the mechanisms of its degradation remained unknown. We show here that p21 is a good substrate for an SCF (Skp1-Cullin1-F-box protein) ubiquitin ligase complex, which contains the F-box protein Skp2 (S phase kinase-associated protein 2) and the accessory protein Cks1 (cyclin kinase subunit 1). A similar ubiquitin ligase complex has been previously shown to be involved in the degradation of a related cyclin-dependent kinase inhibitor, p27Kip1. The levels of Skp2 oscillate in the cell cycle, reaching a maximum in S phase. The ubiquitylation of p21 in vitro required the supplementation of all components of the SCF complex as well as of Cks1 and Cdk2-cyclin E. The protein kinase Cdk2-cyclin E acts both by the phosphorylation of p21 on Ser-130 and by the formation of a complex with p21, which is required for its presentation to the ubiquitin ligase. As opposed to the case of p27, the phosphorylation of p21 stimulates its ubiquitylation but is not absolutely required for this process. Levels of p21 are higher in Skp2-/- mouse embryo fibroblasts than in wild-type fibroblasts in the S phase, and the rates of the degradation of p21 are slower in cells that lack Skp2. It is suggested that SCFSkp2 participates in the degradation of p21 in the S phase

Hepatocyte growth factor (HGF) signaling via its receptor, the proto-oncogene Met, alters cell proliferation and motility and has been associated with tumor metastasis. HGF treatment of HepG2 human hepatocellular carcinoma cells induces cell migration concomitant with increased levels of the p27(kip1) cyclin-cdk inhibitor. HGF signaling resulted in nuclear export of endogenous p27 to the cytoplasm, via Ser-10 phosphorylation, where it colocalized with F-actin. Introduction of transducible p27 protein (TATp27) was sufficient for actin cytoskeletal rearrangement and migration of HepG2 cells. TATp27 mutational analysis identified a novel p27 C-terminal domain required for cell migration, distinct from the N-terminal cyclin-cyclin-dependent kinase (cdk) binding domain. Loss or disruption of the p27 C-terminal domain abolished both actin rearrangement and cell migration. The cell-scattering activity of p27 occurred independently of its cell cycle arrest functions and required cytoplasmic localization of p27 via Ser-10 phosphorylation. Furthermore, Rac GTPase was necessary for p27-dependent migration but alone was insufficient for HepG2 cell migration. These results predicted a migration defect in p27-deficient cells. Indeed, p27-deficient primary fibroblasts failed to migrate, and reconstitution with TATp27 rescued the motility defect. These observations define a novel role for p27 in cell motility that is independent of its function in cell cycle inhibition

Skp1 is involved in a variety of crucial cellular functions, among which the best understood is the formation together with Cul1 of Skp1-cullin-F-box protein ubiquitin ligases. To investigate the role of Skp1, we generated transgenic (Tg) mice expressing a Cul1 deletion mutant (Cul1-N252) able to sequestrate and inactivate Skp1. In vivo interference with Skp1 function through expression of the Cul1-N252 mutant into the T-cell lineage results in lymphoid organ hypoplasia and reduced proliferation. Nonetheless, after a period of latency, Cul1-N252 Tg mice succumb to T-cell lymphomas with high penetrance (>80%). Both T-cell depletion and the neoplastic phenotype of Cul1-N252 Tg mice are largely rescued in Cul1-N252, Skp1 double-Tg mice, indicating that the effects of Cul1-N252 are due to a sequestration of the endogenous Skp1. Analysis of Cul1-N252 lymphomas demonstrates striking karyotype heterogeneity associated with c-myc amplification and c-Myc overexpression. We show that the in vitro expression of the Cul1-N252 mutant causes a pleiotrophic phenotype, which includes the formation of multinucleated cells, centrosome and mitotic spindle abnormalities, and impaired chromosome segregation. Our findings support a crucial role for Skp1 in proper chromosomal segregation, which is required for the maintenance of euploidy and suppression of transformation

The protein expression of the cyclin-dependent kinase inhibitor p27 is often deregulated in human tumors. In lymphomas the inactivation of p27 is achieved through either increased degradation(1) or sequestration via D cyclins,(2) and p27 protein levels have been shown to have a prognostic significance.(1,3) Recently, S-phase kinase-associated protein 2 (Skp2) has been proved to mediate p27 degradation in normal cells(4-7) and to have oncogenetic properties.(8,9) In this study, B-, T-, and myeloid hematopoietic cell lines and a well-characterized panel of human lymphomas (n = 244) were studied for the expression of Skp2. In human lymphomas, the expression of Skp2 strongly related to the grade of malignancy, being low in indolent tumors and very high in aggressive lymphomas. Moreover, the percentages of Skp2- and S-phase-positive cells, as measured by DNA content or BrdU labeling, strictly matched and closely parallel that of Ki-67 and cyclin A. An inverse correlation between Skp2 and p27 was found in the majority of lymphoma subtypes. Nonetheless, most mantle cell lymphomas and a subset of diffuse large cell lymphomas failed to show this correlation, suggesting that alternative pathway(s) for the regulation of p27 might exist. The detection of Skp2 protein either by flow cytometry or by immunohistochemistry represents a simple method to precisely assess the S phase of lymphomas. The potential diagnostic and prognostic value of Skp2 is discussed

OBJECTIVE: Knowledge of alimentary habits among populations permits a better definition of appropriate public health interventions. We designed the epidemiological project 'Ventimiglia di Sicilia' to characterize the risk profile in a rural village with low total cholesterol levels and low early cardiovascular mortality but with a large prevalence of overweight and obesity, which previously have been significantly associated with total mortality. METHODS: 488 individuals of age 20 to 69 years were included in the dietary survey conducted by a seven-day food record. RESULTS: Alimentary habits were characterized by high consumption of total and complex carbohydrates (respectively 52.5 +/- 7.6% and 46.6 +/- 8.2% of daily energy) and by a low cholesterol intake (92.5 +/- 35.0 mg/1000 kcal/day). Fat intake was 34.7 +/- 7.7% of daily energy due to a higher consumption of monounsaturated fats in respect to saturated fats (respectively 20.5 +/- 5.1% and 10.2 +/- 2.9% of daily energy). In particular, in this population there was a large consumption of bread, pasta, fresh vegetables, olive oil and fruits. We also observed an excess of total calories (about 2900 kcal/day in men and 2100 kcal/day in women) not balanced by a high degree of physical activity levels. Furthermore we found a significant higher total and saturated fat consumption in the youngest individuals and in people with higher educational levels. CONCLUSIONS: Dietary habits of Ventimiglia di Sicilia still follow the nutritional characteristics typical of the Mediterranean diet. The high total calorie intake indicates a quantitative more than qualitative problem, which may account the large prevalence of overweight and obesity and may represent a public health issue that needs to be corrected in such a rural population

The Cdc25 dual-specificity phosphatases control progression through the eukaryotic cell division cycle by activating cyclin-dependent kinases. Cdc25 A regulates entry into S-phase by dephosphorylating Cdk2, it cooperates with activated oncogenes in inducing transformation and is overexpressed in several human tumors. DNA damage or DNA replication blocks induce phosphorylation of Cdc25 A and its subsequent degradation via the ubiquitin-proteasome pathway. Here we have investigated the regulation of Cdc25 A in the cell cycle. We found that Cdc25 A degradation during mitotic exit and in early G(1) is mediated by the anaphase-promoting complex or cyclosome (APC/C)(Cdh1) ligase, and that a KEN-box motif in the N-terminus of the protein is required for its targeted degradation. Interestingly, the KEN-box mutated protein remains unstable in interphase and upon ionizing radiation exposure. Moreover, SCF (Skp1/Cullin/F-box) inactivation using an interfering Cul1 mutant accumulates and stabilizes Cdc25 A. The presence of Cul1 and Skp1 in Cdc25 A immunocomplexes suggests a direct involvement of SCF in Cdc25 A degradation during interphase. We propose that a dual mechanism of regulated degradation allows for fine tuning of Cdc25 A abundance in response to cell environment

Estrogen receptor (ER) expression and Her-2 amplification define specific subsets of breast tumors for which specific therapies exist. The S-phase kinase-associated protein Skp2 is required for the ubiquitin-mediated degradation of the cdk-inhibitor p27 and is a bona fide proto-oncoprotein. Using microarray analysis and immunohistochemistry, we determined that higher levels of Skp2 are present more frequently in ER-negative tumors than in ER-positive cases. Interestingly, the subset of ER-negative breast carcinomas overexpressing Skp2 are also characterized by high tumor grade, negativity for Her-2, basal-like phenotype, high expression of certain cell cycle regulatory genes, and low levels of p27 protein. We also found that Skp2 expression is cell adhesion-dependent in normal human mammary epithelial cells but not in breast cancer cells and that an inhibition of Skp2 induces a decrease of adhesion-independent growth in both ER-positive and ER-negative cancer cells. Finally, forced expression of Skp2 abolished effects of antiestrogens, suggesting that deregulated Skp2 expression might play a role in the development of resistance to antiestrogens. We conclude that Skp2 has oncogenic potential in breast epithelial cells and is overexpressed in a subset of breast carcinomas (ER- and Her-2 negative) for which Skp2 inhibitors may represent a valid therapeutic option