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Papers by vittorio tomasi

Biochemical and Biophysical Research Communications, 1969

Adenyl cyclase has been shown to be specifically localized in the plasma membrane of rat liver. T... more Adenyl cyclase has been shown to be specifically localized in the plasma membrane of rat liver. The activity of this enzyme is stimulated by glucagon and epinephrine in isolated plasma membrane systems. The stimulation by epinephrine has a lag period of about 10 minutes. Epinephrine shows selective binding to isolated plasma membranes. The results indicate that epinephrine binds to a receptor protein rather than interacting directly with the enzyme adenyl cyclase. Calcium ions also stimulate adenyl cyclase in isolated plasma membranes but the effect shows appreciable variability. The biological significance of cyclic AMP (adenosine-3',5'-monophosphate)

Biochimica et Biophysica Acta (BBA) - Biomembranes, 1970

Membrane bound adenyl cyclase in isolated rat liver plasma membranes exhibits a requirement for M... more Membrane bound adenyl cyclase in isolated rat liver plasma membranes exhibits a requirement for Mg 2+ and has optimal activity near pH 8.0. The optimal concentration of ATP is approx. 0.3 mM at a Mg ~+ concentration of i.o raM. Ca 2+ at a concentration range of 0.03-0.3 mM stimulates the basal activity of this enzyme. The enzyme activity is abolished by I rain heating at IOO °. Detergents such as Triton X-ioo and sodium dodecyl sulfate alter the membrane structure and slightly enhance the enzyme actively, whereas sodium deoxycholate slightly inhibits the enzyme. Glucagon and epinephrine stimulate whereas insulin inhibits adenyl cyclase activity. When studied under identical conditions on the same membrane preparation the glucagon stimulation is seen earlier and at lower hormone concentration (I-lO-6 M) than is the epinephrine stimulation which requires lO .5 M hormone. The inhibition by insulin is seen at I. lO-5 M or greater. The stimulation by glucagon is inhibited by i-lO-4-1 • lO-5 M Ca 2+ but the stimulation by epinephrine is enhanced by I. IO-4-I-IO-~ M Ca ~+. Moreover, epinephrine enhances, whereas glucagon inhibits the binding of Ca 2+ to the membrane. Studies on the combined effects of the hormones show that the stimulation by glucagon plus epinephrine is not additive and that insulin antagonizes the glucagon stimulation of adenyl cyclase. Na + inhibits the basal activity of adenyl cyclase but K + stimulates the enzyme. F-inhibits the enzyme. These results point to a complex interplay of hormones and metal ions with the membrane bound adenyl cyclase. * The validity of the two-dimensional chromatographic assay for cyclic AMP is based on the following: (a) co-chromatography with authentic 3',5'-cyclic AMP, (b) the hydrolysis of cyclic AMP to 5'-AMP by brain phosphodiesterase and (c) the complete separation of cyclic AMP from ATP, ADP, 5'-AMP, adenosine, adenine, hypoxanthine, inosine, inosinic acid, ribose and ribose phosphate.

British journal of cancer, Jan 28, 2010

Cyclooxygenase-2 (COX-2) overexpression is strongly associated with colorectal tumourigenesis. It... more Cyclooxygenase-2 (COX-2) overexpression is strongly associated with colorectal tumourigenesis. It has been demonstrated that the chronic use of non-steroidal anti-inflammatory drugs (COX inhibitors) partially protects patients from colorectal cancer (CRC) development and progression but induces severe cardiovascular side effects. New strategies for selective COX-2 blockade are required. We developed an improved technique, based on RNA interference (RNAi), to gain a selective COX-2 silencing in CRC cells by a tumour-dependent expression of anti-COX-2 short-hairpin RNA (shCOX-2). Anti-COX-2 shRNA-expressing vectors were delivered in CRC cells (in vitro) and in colon tissues (ex vivo) using engineered Escherichia coli strains, capable of invading tumour cells (InvColi). A highly tumour-dependent shCOX-2 expression and a significant COX-2 silencing were observed in CRC cells following InvColi strain infection. Cyclooxygenase-2 silencing was associated with a strong reduction in both pro...

British Journal of Cancer, 2006

Silencing those genes that are overexpressed in cancer and contribute to the survival and progres... more Silencing those genes that are overexpressed in cancer and contribute to the survival and progression of tumour cells is the aim of several researches. Cyclooxygenase-2 (COX-2) is one of the most intensively studied genes since it is overexpressed in most tumours, mainly in colon cancer. The use of specific COX-2 inhibitors to treat colon cancer has generated great enthusiasm. Yet, the side effects of some inhibitors emerging during long-term treatment have caused much concern. Genes silencing by RNA interference (RNAi) has led to new directions in the field of experimental oncology. In this study, we detected sequences directed against COX-2 mRNA, that potently downregulate COX-2 gene expression and inhibit phorbol 12-myristate 13-acetate-induced angiogenesis in vitro in a specific, nontoxic manner. Moreover, we found that the insertion of a specific cassette carrying anti-COX-2 short hairpin RNA sequence into a viral vector (pSUPER.retro) greatly increased silencing potency in a colon cancer cell line (HT29) without activating any interferon response. Phenotypically, COX-2 deficient HT29 cells showed a significant impairment of their in vitro malignant behaviour. Thus, the retroviral approach enhancing COX-2 knockdown, mediated by RNAi, proved to be an useful tool to better understand the role of COX-2 in colon cancer. Furthermore, the higher infection efficiency we observed in tumour cells, if compared to normal endothelial cells, may disclose the possibility to specifically treat tumour cells without impairing endothelial COX-2 activity.

Immunity & Ageing, 2010

Predictive diagnostics and prevention of chronic degenerative disease Bologna, Italy.

Journal of Biomedicine and Biotechnology, 2006

It has been reported that cellular prion protein (PrPc) is enriched in caveolae or caveolae-like ... more It has been reported that cellular prion protein (PrPc) is enriched in caveolae or caveolae-like domains with caveolin-1 (Cav-1) participating to signal transduction events by Fyn kinase recruitment. By using the Glutathione-S-transferase (GST)-fusion proteins assay, we observed that PrPc strongly interacts in vitro with Cav-1. Thus, we ascertained the PrPc caveolar localization in a hypothalamic neuronal cell line (GN11), by confocal microscopy analysis, flotation on density gradient, and coimmunoprecipitation experiments. Following the anti-PrPc antibody-mediated stimulation of live GN11 cells, we observed that PrPc clustered on plasma membrane domains rich in Cav-1 in which Fyn kinase converged to be activated. After these events, a signaling cascade through p42/44 MAP kinase (Erk 1/2) was triggered, suggesting that following translocations from rafts to caveolae or caveolae-like domains PrPc could interact with Cav-1 and induce signal transduction events.

Cell Biochemistry and Biophysics, 2003

Cellular prion protein (PrPc), the normal cellular counterpart of the infectious prion called PrP... more Cellular prion protein (PrPc), the normal cellular counterpart of the infectious prion called PrP scrapie (PrPsc, see ref. 1), is not a structural component typical of evolutionary advanced organisms, but the product of a long evolutionary history (2). Its adaptation to promote the emergence of new traits, at least in yeasts (3), suggests that many functions are still to be discovered. So far, the physiologic roles attributed to PrPc are rather disparate and include: (a) function as a membrane receptor; (b) regulator of apoptosis; (c) carrier or binding protein for copper ions; (d) effectors in signal transduction mechanisms; (e) regulator of synaptic transmission; and (f) transcription factor. This redundancy reflects a complete lack of understanding regarding two crucial aspects, the topological organization and the subcellular localization of PrPc. Studies carried out in some laboratories, ours included, are throwing

Angiogenesis Assays, 2006

ABSTRACT The concept that cell signalling is topologically restricted and vectorially orientated ... more ABSTRACT The concept that cell signalling is topologically restricted and vectorially orientated has stimulated much work. Restriction has been explained by the identification of signalling protein complexes in membrane microdomains (rafts and caveolae). The participation of endothelial cell caveolae to angiogenesis has been well reported and a distinction between normal and tumour-induced angiogenesis has been clearly documented. These advancements in the comprehension of signal transduction events preceding or concomitant to the angiogenic process required dramatic advancements in the methodology employed. We will describe the main techniques employed to evaluate the connection of endothelial cell signal transduction to angiogenesis, focusing on their strength and limitations.

Nowadays, no data are available concerning the potential use of dual COX/5-LOX inhibitors as anti... more Nowadays, no data are available concerning the potential use of dual COX/5-LOX inhibitors as anticancer agents in colon cancer treatment. Here we report, for the first time, that the dual COX/5-LOX inhibitor licofelone triggers apoptosis in a dose-and time-dependent manner in HCA-7 colon cancer cells. Induction of apoptosis was related to the recruitment of the intrinsic mitochondrial apoptotic pathway, as shown by loss in mitochondrial membrane potential, cytochrome c release, caspase-9 and 3 activation and poly-(ADPribose)polymerase-1 cleavage. Moreover, licofelone induced the cleavage of the full length p21 Bax into p18 Bax , a more potent inducer of the apoptotic process than the uncleaved form. Pre-treatment of HCA-7 cells with the pan-caspase inhibitor z-VAD-fmk significantly blocked licofelone-induced apoptosis, confirming that this process occurred primarily in a caspase-dependent pathway. We also present evidences that licofelone was able to affect the arachidonic acid cascade, as it blocked the activity of 5-LOX and COXs enzymes, and it induced, through the phosphorylation of cytoplasmic phospholipase A 2 , the release of unesterified arachidonic acid from HCA-7 membrane phospholipids. However, apoptosis induction was not related to the ability of licofelone to affect the arachidonic acid cascade, since neither exogenous PGE 2 and LTB 4 addition, nor pharmacological inhibition of cytoplasmic phospholipase A 2 , were able to rescue HCA-7 cells from apoptosis. Even if further studies are needed to clarify the mechanism of licofelone-induced apoptosis, this study suggest that this drug, as well as similar dual COX/5-LOX inhibitors, may represent a novel and promising approach in colon cancer treatment.