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260


Fenton's pre-treatment of mature landfill leachate

Lopez, Antonio; Pagano, Michele; Volpe, Angela; Di Pinto, Appio Claudio
The aim of this study was to check the effectiveness of the Fenton's reagent (Fe2+ + H2O2 + H+) for the pre-treatment of a municipal landfill leachate with the objective of improving its overall biodegradability, evaluated in terms of BOD5/COD ratio, up to a value compatible with biological treatment. The leachate came from a municipal sanitary landfill located in southern Italy and the average values of its main parameters were: pH=8.2; COD=10,540 mgl(-1); BOD5=2,300 mgl(-1); TOC=3,900 mgl(-1); NH4-N=5210 mgl(-1); conductivity=45,350 microScm(-1); alkalinity=21,470 mgl(-1) CaCO3. The effect of initial pH value on the pre-treatment effectiveness was evaluated by titrating the amount of acidic by-products formed. The extent of leachate oxidation was monitored and controlled by both pH and redox potential measurements. The best operational conditions for achieving the desired goal (i.e., BOD5/COD> or =0.5) resulted: Fe2+=275 mgl(-1); H2O2=3,300 mgl(-1); initial pH=3; reaction time=2 h. At the end of the Fenton's pre-treatment, in order to permit a subsequent biological treatment, residual ferric ions were removed increasing the pH up to 8.5 by adding 3 gl(-1) of Ca(OH)2 and 3 mgl(-1) of a cationic polyelectrolyte, the latter as an aid to coagulation. This final step also resulted in a further modest removal of residual COD due to co-precipitation phenomena
PMID: 14637358
ISSN: 0045-6535
CID: 64226

Degradation of Cdc25A by beta-TrCP during S phase and in response to DNA damage

Busino, Luca; Donzelli, Maddalena; Chiesa, Massimo; Guardavaccaro, Daniele; Ganoth, Dvora; Dorrello, N Valerio; Hershko, Avram; Pagano, Michele; Draetta, Giulio F
The Cdc25A phosphatase is essential for cell-cycle progression because of its function in dephosphorylating cyclin-dependent kinases. In response to DNA damage or stalled replication, the ATM and ATR protein kinases activate the checkpoint kinases Chk1 and Chk2, which leads to hyperphosphorylation of Cdc25A. These events stimulate the ubiquitin-mediated proteolysis of Cdc25A and contribute to delaying cell-cycle progression, thereby preventing genomic instability. Here we report that beta-TrCP is the F-box protein that targets phosphorylated Cdc25A for degradation by the Skp1/Cul1/F-box protein complex. Downregulation of beta-TrCP1 and beta-TrCP2 expression by short interfering RNAs causes an accumulation of Cdc25A in cells progressing through S phase and prevents the degradation of Cdc25A induced by ionizing radiation, indicating that beta-TrCP may function in the intra-S-phase checkpoint. Consistent with this hypothesis, suppression of beta-TrCP expression results in radioresistant DNA synthesis in response to DNA damage--a phenotype indicative of a defect in the intra-S-phase checkpoint that is associated with an inability to regulate Cdc25A properly. Our results show that beta-TrCP has a crucial role in mediating the response to DNA damage through Cdc25A degradation
PMID: 14603323
ISSN: 1476-4687
CID: 42121

Proteasome-Mediated Degradation of p21 via N-Terminal Ubiquitinylation

Bloom, Joanna; Amador, Virginia; Bartolini, Francesca; DeMartino, George; Pagano, Michele
We examined the mechanism responsible for the degradation of p21, a negative regulator of the cell division cycle. We found that p21 proteolysis requires functional ubiquitin and Nedd8 systems. Ubiquitinylated forms of p21 and p21(K0), a p21 mutant missing all lysines, are detected in vivo and in vitro, showing that the presence of lysines is dispensable for p21 ubiquitinylation. Instead, the free amino group of the N-terminal methionine of p21 is a site for ubiquitinylation in vivo. Although wild-type p21 is more abundantly ubiquitinylated than p21(K0) mutant due to the presence of internal lysine residues, their rates of proteolysis are indistinguishable. These results demonstrate that proteasomal degradation of p21 is regulated by the ubiquitin pathway and suggest that the site of the ubiquitin chain is critical in making p21 a competent substrate for the proteasome
PMID: 14532004
ISSN: 0092-8674
CID: 38122

When protein destruction runs amok, malignancy is on the loose

Pagano, Michele; Benmaamar, Ramla
Ubiquitin-dependent proteolysis ensures that specific protein functions are turned off at the right time, in the right place, and in a unidirectional fashion. The high substrate specificity of the system is determined by a large family of ubiquitin ligases, which competes with the protein kinases to be the largest family of enzymes in mammals. Given the crucial function of the proteolytic machinery, altered degradation of cellular regulators contributes to the unchecked proliferation typical of cancer cells. Here we review the aberrant activity of a variety of ubiquitin ligases in human cancer, hence the prospect of targeting them in cancer therapy
PMID: 14585352
ISSN: 1535-6108
CID: 38583

Olive husk: an alternative sorbent for removing heavy metals from aqueous streams

Volpe, Angela; Lopez, Antonio; Pagano, Michele
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
PMID: 14512634
ISSN: 0273-2289
CID: 64227

Role of the SCFSkp2 ubiquitin ligase in the degradation of p21Cip1 in S phase

Bornstein, Gil; Bloom, Joanna; Sitry-Shevah, Danielle; Nakayama, Keiko; Pagano, Michele; Hershko, Avram
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
PMID: 12730199
ISSN: 0021-9258
CID: 64228

Altered expression of p27 and Skp2 proteins in prostate cancer of African-American patients

Drobnjak, Marija; Melamed, Jonathan; Taneja, Samir; Melzer, Kate; Wieczorek, Rosemary; Levinson, Benjamin; Zeleniuch-Jacquotte, Anne; Polsky, David; Ferrara, Jay; Perez-Soler, Roman; Cordon-Cardo, Carlos; Pagano, Michele; Osman, Iman
PURPOSE: The purpose is to investigate the clinical relevance of altered patterns of p27 and Skp2 expression in African-American patients with localized prostate cancer. The abundance of p27, an inhibitor of cell proliferation, is controlled by Skp2-dependent proteolysis. EXPERIMENTAL DESIGN: A well-characterized cohort of 162 African-Americans who underwent radical prostatectomy at the Veterans Affairs Medical Center of New York between 1990 and 2000 was studied. We analyzed p27 and Skp2 expression by immunohistochemistry. Altered expression of p27 (defined as <40% tumor cells expressing the protein) and Skp2 (defined as > or ==' BORDER='0'>20% tumor cells expressing the protein) were correlated with clinicopathological parameters and time to prostate-specific antigen (PSA) recurrence. RESULTS: Altered expression of p27 and Skp2 was observed in 112 of 162 (69.1%) and 93 of 162 (57.4%) cases, respectively. Inverse patterns of Skp2 and p27 protein expression were seen in 87 of 162 (53.7%) cases. A marginally significant association was found between Skp2 overexpression and extracapsular extension (P = 0.065). Moreover, patients with Skp2 overexpression had a 2.77 years decreased median time to PSA recurrence compared with patients with low Skp2 expression; however, the difference was not statistically significant. In multivariate analysis, only tumor grade and stage independently predicted PSA recurrence in this cohort. CONCLUSIONS: Our data suggest a role for Skp2 overexpression in prostate cancer pathogenesis that might not be exclusively related to p27 degradation. More studies are needed to determine the mechanistic role of Skp2 in prostate cancer
PMID: 12855638
ISSN: 1078-0432
CID: 38153

Control of meiotic and mitotic progression by the F box protein beta-Trcp1 in vivo

Guardavaccaro, Daniele; Kudo, Yasusei; Boulaire, Jerome; Barchi, Marco; Busino, Luca; Donzelli, Maddalena; Margottin-Goguet, Florence; Jackson, Peter K; Yamasaki, Lili; Pagano, Michele
SCF ubiquitin ligases, composed of three major subunits, Skp1, Cul1, and one of many F box proteins (Fbps), control the proteolysis of important cellular regulators. We have inactivated the gene encoding the Fbp beta-Trcp1 in mice. beta-Trcp1(-/-) males show reduced fertility correlating with an accumulation of methaphase I spermatocytes. beta-Trcp1(-/-) MEFs display a lengthened mitosis, centrosome overduplication, multipolar metaphase spindles, and misaligned chromosomes. Furthermore, cyclin A, cyclin B, and Emi1, an inhibitor of the anaphase promoting complex, are stabilized in mitotic beta-Trcp1(-/-) MEFs. Indeed, we demonstrate that Emi1 is a bona fide substrate of beta-Trcp1. In contrast, stabilization of beta-catenin and IkappaBalpha, two previously reported beta-Trcp1 substrates, does not occur in the absence of beta-Trcp1 and instead requires the additional silencing of beta-Trcp2 by siRNA. Thus, beta-Trcp1 regulates the timely order of meiotic and mitotic events
PMID: 12791266
ISSN: 1534-5807
CID: 39205

Deregulated degradation of the cdk inhibitor p27 and malignant transformation

Bloom, Joanna; Pagano, Michele
p27 acts as a critical negative regulator of the cell cycle by inhibiting the activity of cyclin/cdk complexes during G0 and G1. Degradation of p27 is a critical event for the G1/S transition and occurs through ubiquitination by SCF(Skp2) and subsequent degradation by the 26S-proteasome. A tumor suppressing function of p27 has been demonstrated in mouse models and studies of human tumors. More recent evidence suggests that Skp2, the specific recognition factor for p27 ubiquitination, has oncogenic properties. This review will focus on the regulation of p27 proteolysis and its consequences for tumorigenesis
PMID: 12507555
ISSN: 1044-579x
CID: 39340

Novel p27(kip1) C-terminal scatter domain mediates Rac-dependent cell migration independent of cell cycle arrest functions

McAllister, Sandra S; Becker-Hapak, Michelle; Pintucci, Giuseppe; Pagano, Michele; Dowdy, Steven F
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
PMCID:140659
PMID: 12482975
ISSN: 0270-7306
CID: 64229