Polyamine oxidase is involved in spermidine reduction of transglutaminase type 2-catalyzed βh-crystallins polymerization in calcium-induced experimental cataract

In an in vitro Ca2+-induced cataract model, the progression of opacification is paralleled by a rapid decrease of the endogenous levels of spermidine (SPD) and an increase of transglutaminase type 2 (TG2, EC 2.3.2.13)-catalyzed lens crystallins cross-linking by protein-boundN(1)-N-8-bis(gamma-glutamyl) SPD. This pattern was reversed adding exogenous SPD to the incubation resulting in a delayed loss of transparency of the rabbit lens. The present report shows evidence on the main incorporation of SPD by the catalytic activity of TG2, toward beta H-crystallins and in particular to the beta B2- and mostly in beta B3-crystallins. The increase of endogenous SPD in the cultured rabbit lens showed the activation of a flavin adenine dinucleotide (FAD)-dependent polyamine oxidases (PAO EC 1.5.3.11). As it is known that FAD-PAO degrades theN(8)-terminal reactive portion ofN(1)-mono(gamma-glutamyl) SPD, the protein-boundN(8)-mono(gamma-glutamyl) SPD was found the mainly available derivative for the potential formation of beta B3-crystallins cross-links by protein-boundN(1)-N-8-bis(gamma-glutamyl)SPD. In conclusion, FAD-PAO degradation of theN(8)-terminal reactive residue of the crystallins boundN(1)-mono(gamma-glutamyl)SPD together with the increased concentration of exogenous SPD, leading to saturation of glutamine residues on the substrate proteins, drastically reducesN(1)-N-8-bis(gamma-glutamyl)SPD crosslinks formation, preventing crystallins polymerization and avoiding rabbit lens opacification. The ability of SPD and MDL 72527 to modulate the activities of TG2 and FAD-PAO involved in the mechanism of lens opacification suggests a potential strategy for the prevention of senile cataract

Flavonoids: a myth or a reality for cancer therapy?

Nutraceuticals are biologically active molecules present in foods; they can have beneficial effects on health, but they are not available in large enough quantities to perform this function. Plant metabolites, such as polyphenols, are widely diffused in the plant kingdom, where they play fundamental roles in plant development and interactions with the environment. Among these, flavonoids are of particular interest as they have significant effects on human health. In vitro and/or in vivo studies described flavonoids as essential nutrients for preventing several diseases. They display broad and promising bioactivities to fight cancer, inflammation, bacterial infections, as well as to reduce the severity of neurodegenerative and cardiovascular diseases or diabetes. Therefore, it is not surprising that interest in flavonoids has sharply increased in recent years. More than 23,000 scientific publications on flavonoids have described the potential anticancer activity of these natural molecules in the last decade. Studies, in vitro and in vivo, show that flavonoids exhibit anticancer properties, and many epidemiological studies confirm that dietary intake of flavonoids leads to a reduced risk of cancer. This review provides a glimpse of the mechanisms of action of flavonoids on cancer cells

Plasmonic DNA: Towards Genetic Diagnosis Chips

"Plasmonics and DNA" special issueInternational audienc

Involvement of cell surface TG2 in the aggregation of K562 cells triggered by gluten

Gluten-induced aggregation of K562 cells represents an in vitro model reproducing the early steps occurring in the small bowel of celiac patients exposed to gliadin. Despite the clear involvement of TG2 in the activation of the antigen-presenting cells, it is not yet clear in which compartment it occurs. Herein we study the calcium-dependent aggregation of these cells, using either cell-permeable or cell-impermeable TG2 inhibitors. Gluten induces efficient aggregation when calcium is absent in the extracellular environment, while TG2 inhibitors do not restore the full aggregating potential of gluten in the presence of calcium. These findings suggest that TG2 activity is not essential in the cellular aggregation mechanism. We demonstrate that gluten contacts the cells and provokes their aggregation through a mechanism involving the A-gliadin peptide 31-43. This peptide also activates the cell surface associated extracellular TG2 in the absence of calcium. Using a bioinformatics approach, we identify the possible docking sites of this peptide on the open and closed TG2 structures. Peptide docks with the closed TG2 structure near to the GTP/GDP site, by establishing molecular interactions with the same amino acids involved in stabilization of GTP binding. We suggest that it may occur through the displacement of GTP, switching the TG2 structure from the closed to the active open conformation. Furthermore, docking analysis shows peptide binding with the β-sandwich domain of the closed TG2 structure, suggesting that this region could be responsible for the different aggregating effects of gluten shown in the presence or absence of calcium. We deduce from these data a possible mechanism of action by which gluten makes contact with the cell surface, which could have possible implications in the celiac disease onset

Polymerase-chain reaction: analysis of DNA/DNA hybridization by capillary electrophoresis

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Surface plasmon resonance for detection of genetically modified organisms in the food supply

A review is presented demonstrating that biospecific interaction analysis, using surface plasmon resonance (SPR) and biosensor technologies is a simple, rapid, and automatable approach to detect genetically modified organisms (GMOs). Using SPR, we were able to monitor in real-time the hybridization between oligonucleotide or polymerase chain reaction (PCR)-generated probes and target single-stranded PCR products obtained by using as substrates DNA isolated from normal or transgenic soybean and maize. This procedure allows a one-step, nonradioactive detection of GMOs. PCR-generated probes are far more efficient in detecting GMOs than are oligodeoxyribonucleotide probes. This is expected to be a very important parameter, because information on low percentage of GMOs is of great value. Determination of the ability of SPR-based analysis to quantify GMOs should be considered a major research field for future studies, especially for the analyses of food supplies

Surface plasmon resonance based biosensor technology for real-time detection of PCR products

In this chapter, we reviewed different experimental approaches for PCR-based biospecific interaction analysis (BIA) employing surface plasmon resonance (SPR) and biosensor technologies: choice of primers and probes, characterization of PCR-products immobilized on flow cells, use of PNA as molecular probes, injection of asymmetric PCR products to flow cells carrying specific probes. We discussed of the advantages of SPR-based BIA for characterization of PCR products, problems and possible solutions and future perspectives

Surface Plasmon Resonance-Based Biosensor Technology for Real-Time Detection of PCR Products.

The major advantages of using SPR-based analysis employing optical-based biosensors are (1) analysis is performed in a few minutes, (2) no label is required, and (3) in protocols employing immobilization of molecular probes on the flow cells, the sensor chip can be reused several times. The main limitation is the high cost for most of the commercially available instruments. With respect to the use of (RUfin – RUi) or (RUres – RUi) values, the choice depends on the ability of the employed probes in discriminating target DNA during the association phase of the BIA analysis. In this case, the higher (RUfin – RUi) values are preferably used for analytical determinations, with highly reproducible results (for instance in the detection of GMO or HIV-1 sequences). We cannot generalize on the use of (RUfin – RUi) values for the detection of point mutations; this strongly depends on the length of the probes and the secondary structures of PCR-generated single-stranded target DNAs. In most instances, when (RUfin – RUi) values are not informative for detecting point mutations, (RUres – RUi) values should be taken into consideration