Eugenio Lendaro

Modulation of mitochondrial respiration by nitric oxide: investigation by single cell fluorescence microscopy

With the electro‐driven import of rhodamine 123, we used single cell fluorescence microscopy to single out the contribution of nitric oxide (NO) in controlling mitochondrial membrane potential expressed by (stationary growing) rhabdomyosarcoma and neuroblastoma cells in culture. The experimental design and the computer‐aided image analysis detected and quantitated variations of fluorescence signals specific to mitochondria. We observed that 1) the two cell lines display changes of fluorescence dependent on mitochondrial energization states; 2) mitochondrial fluorescence decreases after exposure of the cells to a NO releaser; 4) the different fluorescence intensity measured under stationary growing conditions, or after activation and inhibition of constitutive NO synthase, is consistent with a steady‐state production of NO. Direct comparison of single cell fluorescence with bulk cytofluorimetry proved that the results obtained by the latter method may be misleading because of the intrinsic‐to‐measure lack of information about distribution of fluorescence within different cell compartments. The kinetic parameters describing the reactions between cytochrome oxidase, NO, and O2 may account for the puzzling (20‐fold) increase of the KM for O2 reported for cells and tissues as compared to purified cytochrome c oxidase, allowing an estimate of in vivo NO flux.—Sarti, P., Lendaro, E., Ippoliti, R., Bellelli, A., Benedetti, P. A., Brunori, M. Modulation of mitochondrial respiration by nitric oxide: investigation by single cell fluorescence microscopy. FASEB J. 13, 191–197 (1999)

The crystal structure of saporin SO6 from Saponaria officinalis and its interaction with the ribosome.

The 2.0 Å resolution crystal structure of the ribosome inactivating protein saporin (isoform 6) from seeds of Saponaria officinalis is presented. The fold typical of other plant toxins is conserved, despite some differences in the loop regions. The loop between strands β7 and β8 in the C‐terminal region which spans over the active site cleft appears shorter in saporin, suggesting an easier access to the substrate. Furthermore we investigated the molecular interaction between saporin and the yeast ribosome by differential chemical modifications. A contact surface inside the C‐terminal region of saporin has been identified. Structural comparison between saporin and other ribosome inactivating proteins reveals that this region is conserved and represents a peculiar motif involved in ribosome recognition.

Evaluation of different extraction methods from pomegranate whole fruit or peels and the antioxidant and antiproliferative activity of the polyphenolic fraction

Pomegranate is a functional food of great interest, due to its multiple beneficial effects on human health. This fruit is rich in anthocyanins and ellagitannins, which exert a protective role towards degenerative diseases. The aim of the present work was to optimize the extraction procedure, from different parts of the fruit, to obtain extracts enriched in selected polyphenols while retaining biological activity. Whole fruits or peels of pomegranate cultivars, with different geographic origin, were subjected to several extraction methods. The obtained extracts were analyzed for polyphenolic content, evaluated for antioxidant capacity and tested for antiproliferative activity on human bladder cancer T24 cells. Two different extraction procedures, employing ethyl acetate as a solvent, were useful in obtaining extracts enriched in ellagic acid and/or punicalagins. Antioxidative and antiproliferative assays demonstrated that the antioxidant capability is directly related to the phenolic content, whereas the antiproliferative activity is to be mainly attributed to ellagic acid.

A ribosomal protein is specifically recognized by saporin, a plant toxin which inhibits protein synthesis

Many plants express enzymes which specifically remove an adenine residue from the skeleton of the 28 S RNA in the major subunit of the eukaryotic ribosome (ribosome inactivating proteins, RIPs). The site of action of RIPs (A4324 in the rRNA from rat liver) is in a loop structure whose nucleotide sequence all around the target adenine is also conserved in those species which are completely or partially insensitive to RIPs. In this paper we identify a covalent complex between saporin (the RIP extracted from Saponaria officinalis) and ribosomal proteins from yeast (Saccharomyces cerevisiae), by means of chemical crosslinking and immunological or avidin‐biotin detection. The main complex (mol. wt. ≈ 60 kDa) is formed only with a protein from the 60 S subunit of yeast ribosomes, and is not detected with ribosomes from E. coli, a resistant species. This observation supports the hypotesis for a molecular recognition mechanism involving one or more ribosomal proteins, which could provide a ‘receptor’ site for the toxin and favour optimal binding of the target adenine A4324 to the active site of the RIP.

Endocytosis of a chimera between human pro-urokinase and the plant toxin saporin: an unusual internalization mechanism

A fluorescent derivative of a chimeric toxin between human pro‐urokinase and the plant ribo‐some‐inactivating protein saporin (p‐uPA‐SapTRITC), has been prepared in order to study the endocytosis of this potentially antimetastatic conjugate in the murine model cell line LB6 clone19 (Cl19) transfected with the human urokinase receptor gene. The physiological internalization of urokinase‐inhibitor complexes is triggered by the interaction of plasminogen inhibitors (PAIs) with receptors belonging to the low density lipoprotein‐related receptor protein (LRP) family, and involves a macro‐quaternary structure including uPAR, LRP, and PAIs. However, in contrast to this mechanism, we observed a two‐step process: first, the urokinase receptor (uPAR) acts as the anchoring factor on the plasma membrane; subsequently, LRP acts as the endocytic trigger. Once the chimera is bound to the plasma membrane by interaction with uPAR, we suggest that a possible exchange may occur to transfer the toxin to LRP via the saporin moiety and begin the internalization. So an unusual endocytic process is described, where the toxin enters the cell via a receptor different from that used to bind the plasma membrane.—Ippoliti, R., Lendaro, E., Benedetti, P. A., Torrisi, M. R., Belleudi, F., Carpani, D., Soria, M. R., Fabbrini, M. S. Endocytosis of a chimera between human pro‐urokinase and the plant toxin saporin: an unusual internalization mechanism. FASEB J. 14, 1335–1344 (2000)

The effect of monensin and chloroquine on the endocytosis and toxicity of chimeric toxins

Abstract. The toxicity of two conjugates containing ribosome-inactivating proteins (RIPs, i.e. saporin and ricin-A chain x-linked to transferrin) has been measured on a prostatic cancer line (PC3) naturally overexpressing the transferrin receptor, in the presence of monensin and chloroquine. This paper investigates whether the increased toxicity of Tf-RIPs induced by monensin and chloroquine may be due to alterations of the normal endocytotic pathway of the complexes mediated by the transferrin receptor. Monensin, besides inducing alkalinization of normally acid intracellular compartments, causes an accumulation of the receptor-bound Tf-RIP in a perinuclear region contiguous to the cisternae of the trans-Golgi network. Chloroquine, though increasing the intracellular pH, seems not to modify the endocytotic pathway of these chimeric molecules. We believe that the enhanced toxicity of the Tf-RIPs may be related to intracellular alkalinization (i.e. endosomal or lysosomal pH) rather than to the effects on the recycling of transferrin receptor-bound toxins. We conclude that the efficacy of chimeric toxins may be modulated not only by the carrier used for their engineering but also by addition of drugs able to influence the stability and activation of the toxins inside the cell.

5-S-Cysteinyl-dopamine effect on the human dopaminergic neuroblastoma cell line SH-SY5Y

In recent years a catechol-thioether metabolite of dopamine, 5-S-cysteinyl-dopamine, has been identified in certain dopaminergic regions of the brain, notably the Substantia Nigra. 5-S-Cysteinyl-dopamine has received great attention in view of its possible significance as an index of oxidative stress in aging and in neurodegenerative processes, particularly in Parkinsons disease. In the present study the effect of 5-S-cysteinyl-dopamine on human dopaminergic neuroblastoma SH-SY5Y cells is investigated. The substance is highly cytotoxic, even at a concentration as low as 30 microM. Treatment of the cells with 5-S-cysteinyl-dopamine induce the following intracellular responses: a decrease of the mitochondrial transmembrane potential, an increase in reactive oxygen species such as superoxide anion and peroxides, a marked decrease of reduced glutathione and an inhibition of the complex I activity. Caspase-3-like protease activation and oligonucleosomal DNA fragmentation have also been observed. These data are indicative of the onset of apoptotic processes due to 5-S-cysteinyl-dopamine.

P-glycoprotein inserted in planar lipid bilayers formed by liposomes opened on amorphous carbon and Langmuir–Blodgett monolayer

The insertion of proteins into planar lipid layers is of outstanding interest as the resulting films are suitable for the investigation of protein structure and aggregation in a lipid environment and/or the development of biotechnological applications as biosensors. In this study, purified P-glycoprotein (P-gp), a membrane drug pump, was incorporated in model membranes deposited on solid supports according to the method by Puu and Gustafson, Biochim. Biophys. Acta 1327 (1997) 149-161. The models were formed by a double lipid layer obtained by opening P-gp-containing liposomes onto two hydrophobic supports: amorphous carbon films and Langmuir-Blodgett (L-B) lipid monolayers, which were then observed by transmission electron microscopy and atomic force microscopy, respectively. Before the opening of liposomes, the P-gp structure and functionality were verified by circular dichroism spectroscopy and enzymatic assay. Our micrographs showed that liposomes containing P-gp fuse to the substrates more easily than plain liposomes, which keep their rounded shape. This suggests that the protein plays an essential role in the fusion of liposomes. To localize P-gp, the immunogold labeling of two externally exposed protein epitopes was carried out. Both imaging techniques confirmed that P-gp was successfully incorporated in the model membranes and that the two epitopes preserved the reactivity with specific mAbs, after sample preparation. Model membranes obtained on L-B monolayer incorporated few molecules with respect to those incorporated in the model membrane deposited onto amorphous carbon, probably because of the different mechanism of proteoliposome opening. Finally, all particles appeared as isolated units, suggesting that P-gp molecules were present as monomers.

Extra Virgin Olive Oil Phenols Suppress Migration and Invasion of T24 Human Bladder Cancer Cells Through Modulation of Matrix Metalloproteinase-2

The consumption of extra virgin olive oil (EVOO), a common dietary habit of the Mediterranean people, seems to be related to a lower incidence of certain types of cancer including bladder neoplasm. Metastases are the major cause of bladder cancer-related deaths and targeting cell motility has been proposed as a therapeutic strategy to prevent cancer spread. This study aimed to investigate the potential antimetastatic effect of total phenols extracted from EVOO against the human transitional bladder carcinoma cell line T24. We also aimed at verifying that EVOO extract exerts cytotoxic effect on tumor cells without affecting normal urothelial fibroblasts. Our results show that EVOO extract can significantly inhibit the proliferation and motility of T24 bladder cells in a dose-dependent manner. In the same experimental conditions fibroblast proliferation and motility were not significantly modified. Furthermore the enzymatic activity of MMP-2 was inhibited at nontoxic EVOO extract doses only in T24 cells. The qRT-PCR revealed a decrease of the MMP-2 expression and a simultaneous increase of the tissue inhibitors of metalloproteinases expression. Our results may support the epidemiological evidences that link olive oil consumption to health benefits and may represent a starting point for the development of new anticancer strategies.

Crystallization and preliminary X-ray study of saporin, a ribosome-inactivating protein from Saponaria officinalis.

Single crystals of the protein saporin isolated from the seeds of S. officinalis have been grown by the vapor-diffusion method using ammonium sulfate as precipitant. The crystals are tetragonal, space group P4122 (P4322), with cell dimensions a = b = 67.53 and c = 119. 67 A, and diffract to 2.0 A resolution on a rotating-anode X-ray source. The asymmetric unit contains one molecule, corresponding to a volume of the asymmetric unit per unit mass (Vm) of 2.38 A3 Da-1.