No abstract

The 2.0 A î 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 L L7 and L L8 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.z 2000 Federation of European Biochemical Societies.

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.

Epidemiological data indicate that the daily consumption of extra-virgin olive oil (EVOO), a common dietary habit of the Mediterranean area, lowers the incidence of certain types of cancer, in particular bladder neoplasm. The aim of the present study was to evaluate the antiproliferative activity of polyphenols extracted from EVOO on bladder cancer (BCa), and to clarify the biological mechanisms that trigger cell death. Furthermore, we also evaluated the ability of low doses of extra-virgin olive oil extract (EVOOE) to modulate the in vitro activity of paclitaxel or mitomycin, two antineoplastic drugs used in the management of different types of cancer. Our results showed that EVOOE significantly inhibited the proliferation and clonogenic ability of T24 and 5637 BCa cells in a dose-dependent manner. Furthermore, cell cycle analysis after EVOOE treatment showed a marked growth arrest prior to mitosis in the G2/M phase for both cell lines, with the subsequent induction of apoptosis only in the T24 cells. Notably, simultaneous treatment of mitomycin C and EVOOE reduced the drug cytotoxicity due to inhibition of ROS production. Conversely, the co-treatment of T24 cells with paclitaxel and the polyphenol extract strongly increased the apoptotic cell death at each tested concentration compared to paclitaxel alone. Our results support the epidemiological evidence indicating that olive oil consumption exerts health benefits and may represent a starting point for the development of new anticancer strategies.

Human transferrin (Tf) and saporin-6 (Sap), a ribosome inactivating protein from Saponaria officinalis, were chemically conjugated: the reaction generated two chimeras (called Tf-Sap) that proved to be cytotoxic to HepG2 cells. Electrophoretic and chromatographic analysis revealed that the two conjugates contained saporin and Tf in a 2:1 or 1:1 molar ratio (140 and 110 KDa, respectively). Free saporin is essentially nontoxic, whereas Tf-Sap efficiently kills HepG2 cells, although its ID50 (= 6 nM) is 1000-fold greater than that of ricin. Intracellular transport of these toxins was followed by in vivo fluorescence video microscopy, preparing the conjugates starting from rhodamine isothiocyanate-labeled saporin. Image analysis of living HepG2 cells exposed to fluorescent Tf-Sap revealed that the endocytotic pathway involving passage through secondary endosomes is dictated by Tf and is different from that of ricin (the dimeric toxin from Ricinus communis), which is delivered to the Golgi apparatus, the probable site of activation. We discuss whether differences in toxicity between ricin and Tf-Sap can be attributed to the different mechanisms of transport and activation.

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.

No abstract

Many plants express enzymes which specifically remove an adenine residue from the skeleton of the 28 S RNA in the major subunit of thccukaryotic ribosomc (ribosome inactivating proleins, RIPS). The site of action of RIPS (A4324 in the rRNA from rdt liver) is in a loop structure whose nucleotidc 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 3 covalent complex between saporin (the RIP extracted from Saporlrrirr oflcinak) and ribosomal proteins from yeast (Saccharorr~yces cereeisiae), 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 detcctcd with ribosomes from E. cdl, 3 resistant spies. This observation supports the hypotesis for 3 molecular recognition mechanism involving one or more ribosomal proteins, which could provide a 'receptor' site for the toxin nnd favour optimal binding of the target adcnine A4324 to the active site of the RIP.