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Ivanov Y.,University Professor Dr. Assen Zlatarov Burgas | Marinov I.,University Professor Dr. Assen Zlatarov Burgas | Portaccio M.,The Second University of Naples | Portaccio M.,Italian National Institute of Biosystems and Biostructures | And 5 more authors.
Biotechnology and Biotechnological Equipment | Year: 2012

A flow-injection system with integrated amperometric biosensor featuring an easily replaceable immobilzed acetylcholinesterase (AChE) membrane was studied. The amperometric biosensor was constructed on the basis of site-specific immobilization of AChE on a hybrid polymer membrane with integrated multi-walled carbon nanotubes. Multistage modification of the membrane and immobilization of the enzyme was proved by Fourier transform infrared spectroscopy. The optimum flow-rate of the flowinjection analysis (FIA) system was 0.5 mL/min. It gave a linear response to acetylthiocholine chloride from 2 μM to 100 μM, with an average RSD of 3.0% (n = 6). The sensitivity of the constructed biosensor was 0.093 μA/μM·cm2. The Km app value of the immobilized AChE was 1.15 mM and the linear correlation coefficient R2, 0.9949. The method had a low detection limit for three organophosphorus pesticides (OPs) in model pesticide solutions -paraoxon ethyl (0.9×10-12 M), monocroptophos (1.8×10-12 M) and dichlorvos (2.0×10-12 M). This indicated that the action of multi-walled nanotubes and controlled site-specific enzyme immobilization ensured high electrocatalytic activity and selectivity of the biosensor towards pesticides. It was found that the biosensor can be reused 15 operation cycles. After storage for 30 days the enzyme membrane retained over 80% of its initial response. The FIA system was used for detection of anti-cholinesterase activity of two binary OP mixtures. The results for paraoxon + monocroptophos and paraoxon + dichlorvos showed that the total inhibition activity was not simply additive, but was lower than the sum of the individual inhibition values. Moreover, the difference between the sum of the individual inhibition values and the real results for the mixture was bigger for the binary system paraoxon and dichlorvos (7-10%) compared with that for paraoxon and monocroptophos (5-7%). The developed biosensor system is an ideal tool for monitoring of organophosphate pesticides. © 2012 American Scientific Publishers.


Ponti D.,University of Rome La Sapienza | Bellenchi G.C.,Institute of Genetics and Biophysics ABT | Puca R.,University of Rome La Sapienza | Bastianelli D.,University of Rome La Sapienza | And 6 more authors.
PLoS ONE | Year: 2014

EGR1 is an immediate early gene with a wide range of activities as transcription factor, spanning from regulation of cell growth to differentiation. Numerous studies show that EGR1 either promotes the proliferation of stimulated cells or suppresses the tumorigenic growth of transformed cells. Upon interaction with ARF, EGR1 is sumoylated and acquires the ability to bind to specific targets such as PTEN and in turn to regulate cell growth. ARF is mainly localized to the periphery of nucleolus where is able to negatively regulate ribosome biogenesis. Since EGR1 colocalizes with ARF under IGF-1 stimulation we asked the question of whether EGR1 also relocate to the nucleolus to interact with ARF. Here we show that EGR1 colocalizes with nucleolar markers such as fibrillarin and B23 in the presence of ARF. Western analysis of nucleolar extracts from HeLa cells was used to confirm the presence of EGR1 in the nucleolus mainly as the 100 kDa sumoylated form. We also show that the level of the ribosomal RNA precursor 47S is inversely correlated to the level of EGR1 transcripts. The EGR1 is effective to regulate the synthesis of the 47S rRNA precursor. Then we demonstrated that EGR1 binds to the Upstream Binding Factor (UBF) leading us to hypothesize that the regulating activity of EGR1 is mediated by its interaction within the transcriptional complex of RNA polymerase I. These results confirm the presence of EGR1 in the nucleolus and point to a role for EGR1 in the control of nucleolar metabolism. © 2014 Ponti et al.


Siervo M.,University of Naples Federico II | Ruggiero D.,Institute of Genetics and Biophysics ABT | Sorice R.,Institute of Genetics and Biophysics ABT | Nutile T.,Institute of Genetics and Biophysics ABT | And 6 more authors.
Nutrition | Year: 2012

Objectives: Childhood obesity is associated with an increased risk of atherosclerosis, which can be mediated by an increase in angiogenesis and inflammation. The objective was to investigate the association between body mass index (BMI) and circulating biomarkers of angiogenesis, inflammation, and cardiac dysfunction in children and adolescents. Methods: The Genetic Park Study is a highly inclusive survey conducted in three isolated villages of southern Italy. One hundred fifty-one children and adolescents (age range 5-17 y, 45% male) were included and categorized as obese (BMI z-score ≥ 1.64, n = 38) or non-obese (n = 113). Metabolic and cardiovascular biomarkers included glucose, triacylglycerol, total cholesterol, high-density lipoprotein, vascular endothelial growth factor (VEGF), placental growth factor, soluble feline sarcoma virus (fms)-like tyrosine kinase-1, highly sensitive C reactive protein (hs-CRP), highly sensitive troponin T (hs-TnT), and N-terminal prohormone brain natriuretic peptide (NT-proBNP). Results: Obese subjects had higher levels of triacylglycerol (P = 0.03) and hs-CRP (P = 0.02) after adjustment for age and gender. Circulating levels of VEGF were directly associated with BMI z-score (r = 0.22, P = 0.007) and hs-CRP (r = 0.33, P < 0.001). BMI z-score was not associated with biomarkers of cardiac dysfunction (hs-TnT and NT-proBNP). Conclusion: Increasing BMI was associated with plasma levels hs-CRP and VEGF, which are involved in the initiation and progression of atherosclerosis. The lack of association between BMI and markers of cardiac damage (hs-TnT) or ventricular volume overload (NT-proBNP) suggest that atherosclerotic risk may still at a preclinical stage in this population of obese but otherwise healthy young individuals. Collectively, this suite of biomarkers could provide mechanistic insights into the physiopathologic progression of cardiovascular risk associated with childhood obesity. © 2012 Elsevier Inc.


Mita L.,Italian National Institute of Biosystems and Biostructures | Mita L.,The Second University of Naples | Sica V.,The Second University of Naples | Guida M.,University of Naples Federico II | And 14 more authors.
Journal of Molecular Catalysis B: Enzymatic | Year: 2010

A planar bioreactor, equipped with a polypropylene membrane on which a lipase was immobilised, has been employed in a bioremediation process involving water polluted by dimethylphthalate (DMP), a model for a class of endocrine disruptors. The dependence of enzyme activity on pH, temperature and DMP concentration has been characterised under isothermal conditions, whereas the kinetics parameters have been studied under non-isothermal conditions. The following sequence was found for the values of lipase affinity, Km, towards the DMP: Km free < Km, non-isoth imm < Km, isoth imm. A comparison of the results obtained under isothermal and non-isothermal conditions indicated that there was an advantage in using non-isothermal bioreactors in the environmental field. These advantages in particular resulted in: (i) an increase in the enzyme activity proportional to the applied transmembrane temperature difference and (ii) a reduction in the bioremediation times and, consequently, the process costs. The advantages in using bioremediation processes in the place of classical membrane processes, such as ultrafiltration or reverse osmosis, are also discussed. © 2009 Elsevier B.V. All rights reserved.


Catapane M.,Institute of Genetics and Biophysics ABT | Catapane M.,Italian National Institute of Biosystems and Biostructures | Nicolucci C.,Italian National Institute of Biosystems and Biostructures | Nicolucci C.,The Second University of Naples | And 11 more authors.
Journal of Hazardous Materials | Year: 2013

A fluidized bed reactor, filled with laccase-based beads, has been employed to bioremediate aqueous solutions polluted by endocrine disruptors belonging to the alkylphenols (APs) class. In particular Octylphenol and Nonylphenol have been studied. The catalytic activity of free and immobilized laccase from Trametes versicolor has been characterized as a function of pH, temperature and substrate concentration in the reaction medium. In view of practical applications for each substrate concentration the removal efficiency (RE), the time to halve the initial concentration (τ50), and the tc=0, i.e. the time to reach complete pollutant removal, have been calculated. The immobilized laccase exhibited a lower affinity for octylphenol (Km=1.11mM) than for Nonylphenol (Km=0.72mM), but all the other parameters of applicative interest resulted more significant for octylphenol. For example, the times to reach the complete removal of octylphenol compared to those for nonylphenol at the same concentration is shorter of about 15% (at low concentrations) up to 40% (at high concentrations). The study of cell proliferation with MPP89 cells, a human mesothelioma cell line, and the assay with the YES test indicated the loss of estrogenic activity of the APs solutions after laccase treatment. © 2013.

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