Purification and enzymatic properties of a peroxidase from leaves of Phytolacca dioica L. (Ombú tree)

A peroxidase (PD-cP; 0.47 mg/100 g leaves) was purified from autumn leaves of Phytolacca dioica L. and characterized. PD-cP was obtained by acid precipitation followed by gel-filtration and cation exchange chromatography. Amino acid composition and N-terminal sequence of PD-cP up to residue 15 were similar to that of Spinacia oleracea (N-terminal pairwise comparison showing four amino acid differences). PD-cP showed a molecular mass of approx. 36 kDa by SDS-PAGE, pH and temperature optima at 3.0 and 50.0°C, respectively and seasonal variation. The Michaelis-Menten constant (K(M)) for H(2)O(2) was 5.27 mM, and the velocity maximum (V(max)) 1.31 nmol min(-1), while the enzyme turnover was 0.148 s(-1). Finally, the presence of Ca(2+) and Mg(2+) enhanced the PD-cP activity, with Mg(2+) 1.4-fold more effective than Ca(2+)     

A new potentially toxic Azadinium species (Dinophyceae) from the Mediterranean Sea,A. dexteroporum sp. nov.

A new photosynthetic planktonic marine dinoflagellate, Azadinium dexteroporum sp. nov., is described from the Gulf of Naples (South Tyrrhenian Sea, Mediterranean Sea). The plate formula of the species, Po, cp, X, 4′, 3a, 6″, 6C, 5?S, 6? and 2″″, is typical for this recently described genus. Azadinium dexteroporum is the smallest rep‐resentative of the genus (8.5 μm average length, 6.2 μm average width) and shares the presence of a small antapical spine with the type species A. spinosum and with A. polongum. However, it differs from all other Azadinium species for the markedly asymmetrical Po plate and the position of the ventral pore, which is located at the right posterior end of the Po plate. Another peculiarity of A. dexteroporum is the pronounced concavity of the second intercalary plate (2a), which appears collapsed with respect to the other plates. Phylogenetic analyses based on the large subunit 28S rDNA (D1/D2) and the internal transcribed spacer (ITS rDNA) support the attribution of A. dexteroporum to the genus Azadinium and its separation from the other known species. LC/MS‐TOF analysis shows that Azadinium dex‐teroporum produces azaspiracids in low amounts. Some of them have the same molecular weight as known compounds such as azaspiracid‐3 and ‐7 and Compound 3 from Amphidoma languida, as well as similar fragmentation patterns in some cases. This is the first finding of a species producing azapiracids in the Mediterranean Sea.     

A Novel Role for Tm7sf2 Gene in Regulating TNFα Expression

We have explored the role of Tm7sf2 gene, which codifies for 3β-hydroxysterol Δ14-reductase, an endoplasmic reticulum resident protein, in the sensitivity to endoplasmic reticulum stress and in the resulting inflammatory response. We used mouse embryonic fibroblasts, derived from Tm7sf2^+/+ and Tm7sf2^−/− mice, to determine the in vitro effects of thapsigargin on NF-κB activation. Our results show that the Tm7sf2 gene controls the launch of the unfolded protein response and presides an anti-inflammatory loop thus its absence correlates with NF-κB activation and TNFα up-regulation. Our data also show that Tm7sf2 gene regulates liver X receptor activation and its absence inhibits LXR signalling. By expressing the hTm7sf2 gene in KO MEFs and observing a reduced NF-κB activation, we have confirmed that Tm7sf2 gene is linked to NF-κB activation. Finally we used genetically modified mice in an in vivo model of ER stress and of inflammation. Our results show a significant increase in renal TNFα expression after tunicamycin exposure and in the oedematogenic response in Tm7sf2^−/− mice. In conclusion, we have shown that the Tm7sf2 gene, to date involved only in cholesterol biosynthesis, also controls an anti-inflammatory loop thereby confirming the existence of cross talk between metabolic pathways and inflammatory response.     

Sustained release of PI3K inhibitor from PHA nanoparticles and in vitro growth inhibition of cancer cell lines

The phosphoinositide-3-kinases (PI3Ks) are a conserved family of lipid kinases that phosphorylate the 3-hydroxyl group of phosphatidylinositols in response to extracellular stimuli. PI3K pathway is enrolled in different kinds of human cancer and plays a prominent role in cancer cell growth and survival. Several PI3K inhibitors have been recently identified but some PI3K inhibitors with high potency in vitro do not show satisfactory effects in animal cancer models because of the poor pharmaceutical properties in vivo such as poor solubility, instability, and fast plasma clearance rate. In this study, we developed a sustained release system of PI3K inhibitor (TGX221) based on polyhydroxyalkanoate nanoparticles (NP) and used it to block proliferation of cancer cell lines. TGX221 was gradually released from PHA-based NP and growth of cancer cell lines was significantly slower in NP-TGX221-treated cells than in either negative controls or in cells receiving free TGX221. Since poor bioavailability and limited in vivo half-life are common features of hydrophobic PI3K inhibitors, our results open the way to similar formulation of other PI3K blockers and to new strategies in cancer treatment.     

Cancerous tissues in forensic genetic analysis

Microsatellites or short tandem repeats (STRs) markers are important tools for mapping disease-causing genes by linkage, for performing investigations in forensic medicine, for population genetic studies and for studying genetic modifications in tumors. In forensic applications neoplastic tissues can be used as a source of genetic information for personal identification or paternity testing when no other specimen is available. Cancer tissues can show microsatellite instability (MSI) and loss of heterozygosity (LOH) also for the STRs used in the forensic field. In this study, we screened 56 sporadic gastrointestinal carcinomas in order to provide further data for the evaluation of the incidence of allelic alterations for 15 STR loci and the suitability of using cancerous tissues in forensic applications. Sixty-six percent of the cancerous tissues were found to possess allelic alterations of the microsatellites analyzed with a high incidence of MSI-L (microsatellite instability low) when compared to the corresponding normal tissue. The most frequently altered loci were D18S51, VWA, and FGA. From a forensic perspective, great care must be taken in evaluating the DNA typing results obtained from cancerous tissue samples.