A.Finazzi Agrò

miR-143 regulates hexokinase 2 expression in cancer cells

Tumor cells activate pathways that facilitate and stimulate glycolysis even in the presence of adequate levels of oxygen in order to satisfy their continuous need of molecules, such as nucleotides, ATP and fatty acids, necessary to support their rapid proliferation. Accordingly, a variety of human tumors are characterized by elevated expression levels of the hexokinase 2 isoform (HK2). Although different molecular mechanisms, including genetic and epigenetic mechanisms, have been suggested to account for the altered expression of HK2 in tumors, the potential role of microRNAs (miRNAs) in the regulation of HK2 expression has not been evaluated. Here, we report that miR-143 inhibits HK2 expression via a conserved miR-143 recognition motif located in the 3′-untranslated region (3′UTR) of HK2 mRNA. We demonstrate that miR143 inhibits HK2 expression both in primary keratinocytes and in head and neck squamous cell carcinoma (HNSCC)-derived cell lines. Importantly, we found that miR-143 inversely correlates with HK2 expression in HNSCC-derived cell lines and in primary tumors. We also report that the miRNA-dependent regulation of hexokinase expression is not limited to HK2 as miR-138 targets HK1 via a specific recognition motif located in its 3′UTR. All these data unveil a new miRNA-dependent mechanism of regulation of hexokinase expression potentially important in the regulation of glucose metabolism of cancer cells.

Hydrogen peroxide is an intermediate in the platelet activation cascade triggered by collagen, but not by thrombin.

Human blood platelets produce oxidant species when stimulated by collagen and thrombin. The oxidative burst of platelets has been studied by cytofluorimetry taking advantage of the fluorogenic dye DCFH2-DA, which is taken up and deacetylated by platelets and then oxidized to the fluorescent derivative DCF. The oxidation of DCFH2 is induced by stimulation with collagen but not with thrombin and inhibited by external catalase. Catalase also inhibited the aggregation induced by collagen, but not that induced by thrombin. Aspirin and indomethacin inhibited the formation of the fluorochrome only when platelets were stimulated by thrombin. Externally added H2O2 increased the cytoplasmic calcium content as probed by the fluorescence of Indo-1. The present data suggest that collagen induces the production of H2O2, which in turn may stimulate the aggregation of platelets through a calcium mobilization. Instead the stimulation by thrombin does not require the intermediacy of H2O2.

Pyridoxal phosphate as a prosthetic group of pig kidney diamine oxidase

Abstract Pig kidney diamine oxidase was obtained in homogeneous form by the criteria of gel electrophoresis and ultracentrifugation. A new modification of the purification procedure is described. The presence of pyridoxal phosphate in the enzyme was demonstrated by the following studies: ( 1 ) spectrophotometric observations on the enzyme treated with carbonyl reagents; ( 2 ) chromatography of pyridoxal phosphate semicarbazone obtained from the enzyme; and ( 3 ) reactivation of apo-aspartate aminotransferase by diamine oxidase derivatives.

The fine structure of luminescence spectra of azurin.

The spectra of azurin absorption, fluorescence, phosphorescence and fluorescence excitation have been measured in aqueous solutions at ordinary and liquid nitrogen temperatures. The fluorescence spectra of azurin even at ordinary temperatures have a well resolved fine vibrational structure. The frequency analysis reveals practically the same wave number distances between the main structure peaks in fluorescence spectra at room and low temperatures and in phosphorescence spectra. The comparison of the protein absorption and excitation spectra shows that all the energy absorbed by tyrosine residues is transferred onto indole chromophore. These data suggest an unusual tryptophan environment in this protein, which is characterized by the absence of any hydrogen bonding or other polar interaction of tryptophan with its environment. The problem of the possibility of contributions of two electronic transitions (1La in equilibrium A and 1Lb in equilibrium A) in absorption and emission spectra of azurin tryptophan arising from their mirror symmetry is discussed.

Lentil seedlings amine oxidase: Preparation and properties of the copper-free enzyme

The reaction of copper-free lentil seedlings amine oxidase with substrates has been studied. While devoid of catalytic activity, this enzyme preparation is still able to oxidize two moles of substrate and to release two moles of aldehyde and two moles of ammonia per mole of dimeric protein. The same stoichiometry has been determined on the native enzyme in the absence of oxygen. Although copper is essential for the reoxidation of the reduced enzyme, a binding of oxygen to the copper-free protein has been demonstrated.

Intracellular localization of superoxide dismutase and its relation to the distribution and mechanism of hydrogen peroxide-producing enzymes

Abstract Superoxide dismutase has been localized exclusively in the soluble fraction of rat liver homogenates. In view of this result, the generation of O2 - by different H2O2-producing enzymes has been examined. It appeared that O2 - is generated only by xanthine oxidase, which is localized in the same compartment as the dismutase. Other enzymes, which are localized in other subcellular fractions, do not produce O2-, but H2O2 directly.

BINDING OF HYDROPHOBIC COMPOUNDS TO APOFERRITIN SUBUNITS Effects on the polymerization state

Apoferritin is a very stable polymer made up by 24 identical subunits forming a nearly spherical, hollow shell in which a ferric hydroxide core is enclosed. Dissociation of apoferritin into subunits occurs only at extremes of pH; thus, in the acid range, dissociation begins below pH 2.8 and the subunits reassociation begins above pH 3.5 [ 11. The importance of hydrophobic interactions at the intersubunit contact regions has been clearly established [ 1,2] and binding of hydrophobic molecules by the subunits could be anticipated. This work reports experiments on the binding of two hydrophobic compounds to apoferritin subunits. ANS was chosen because of its widespread use as a hydrophobic fluorescent probe; BZ, which is an antiinflammatory drug, since it has been shown to bind to hydrophobic sites in serum albumin [3]. Both compounds bind to apoferritin subunits, but not to the native protein, and induce their polymerization.

Adriamycin-catalyzed aerobic photooxidation of NAD dimers to NAD+

The photooxidation of the dimers of nicotinamide adenine dinucleotide, (NAD)2, is catalyzed by adriamycin under aerobic conditions. (NAD)2 and O2 react in 1:1 molar ratio to yield 2 mol of NAD+. Experiments carried out by irradiating at 340 and 485 nm, corresponding to the absorption maxima of (NAD)2 and adriamycin, respectively, clearly indicate that the process is primed by photoexcitation of adriamycin. The key step of the process is the redox reaction between (NAD)2 and adriamycin with formation of the semiquinone radical anion, identified by the EPR spectrum. The semiquinone is then oxidized back to adriamycin by oxygen with formation of the superoxide radical.

Interaction of terbium with human platelets

The effect of terbium on platelets has been studied by aggregation experiments and by fluorescence measurements. TbCl3 does not substitute for CaCl2 in the aggregation of platelets induced by ADP, but it may even inhibit, probably by a competition mechanism. It was impossible to observe a sensitized emission of Tb3+ in the presence of platelets. Instead the lanthanide, like Ca2+, significantly increases the aggregation of platelets induced by A23187. The fluorescence yield of this compound is greater in the presence of platelets than in buffer alone. Energy transfer appears to take place from the aromatic amino acids of the platelet membrane to the bound ionophore.