Bioavailability and In Vivo Antioxidant Activity of a Standardized Polyphenol Mixture Extracted from Brown Propolis.

Several lines of evidence demonstrate the antioxidant, anti-inflammatory and antimicrobial activities of propolis, mostly ascribed to its polyphenol content. However, little is known regarding the bioavailability of propolis in acute and prolonged settings of oral administration. In this study, we first determined the content of the main polyphenols in a brown propolis extract obtained using a patented extraction method (Multi Dinamic Extraction-M.E.D.) by RP-HPLC-UV-PDA-MSn analysis, followed by the bioavailability of galangin and chrysin, the most abundant polyphenols in the mixture (7.8% and 7.5% respectively), following acute (single bolus of 500 mg/kg containing about 3.65 mg of the polyphenol mixture) and prolonged (100, 250 and 500 mg/kg body for 30 days) oral administration in 30 male 8 weeks old C57BL/6 wild-type mice. In the acute setting, blood was taken at 30 s and 5, 10, 15, 20, 25, 30, 45, 60 and 120 min following the oral bolus. In the prolonged setting, blood samples were obtained after 10, 20 or 30 days of administration. At the end of treatment, expression of antioxidant enzymes (superoxyde dismutase, SOD-1; catalase, CAT; glutathione peroxidase, GSS) was evaluated in liver tissue. Following both acute and prolonged administration, neither galangin nor chrysin were detectable in the plasma of mice, whereas the glucuronide metabolite of galangine was detectable 5 min after acute administration. At the end of the prolonged treatment SOD-1 was found to have increased significantly, unlike CAT and GSS. Overall, these data suggest that oral administration of whole brown propolis extract is followed by rapid absorption and metabolization of galangin followed by adaptations of the antioxidant first line defense system.

Electron Microscope Detection of 5-Methylcytosine on DNA and RNA.

5-Methylcytosine is the major epigenetic modification occurring on DNA. It is known to be involved not only in gene expression regulation but also in the control of chromatin structure. However, this modification is also found on different types of RNA, including mRNA. Generally, biomolecular techniques are applied for studying the epigenetic profile of nucleic acids. Here, we describe the ultrastructural detection of 5-methylcytosine as an unusual approach to localize this modification on chromatin regions and/or RNA single molecules. This tool requires a careful sample preparation to preserve antigen epitopes that will be revealed immunocytochemically by a specific anti-5-methylcytosine antibody. The multiple staining procedures that can be adopted allow the identification of both DNA or RNA. A semiquantitative analysis can also be carried out.

A new platinum-based prodrug candidate: Its anticancer effects in B50 neuroblastoma rat cells.

AIMS: Neuroblastoma is a rare cancer that affects children, mostly under the age of 5. This type of cancer starts in very early forms of immature nerve cells or developing cells found in embryo or fetus. To date cisplatin represents one of the most potent antitumor agent known, however, the onset of systemic side effects and the induction of drug resistance limit its use in the clinic for long-term treatment. In the present study we have analysed the effects of a new compound of platinum(IV) conjugates, named Pt(IV)Ac-POA, which is able to generate a synergistic antineoplastic action when released along with cisplatin upon intracellular Pt(IV) → Pt(II) reduction. MAIN METHODS: To assess the growth inhibition of the compounds under investigation, a cell viability test, i.e. the resazurin reduction assay was used on the B50 neuroblastoma rat cells. Further analysis on the cell cycle and metabolic alterations were carried out through flow cytometry. Morphological changes and activation of different cell death pathways after treatment, were observed at transmission electron microscope and by immunocytochemistry at fluorescence microscopy. Protein expression was examined by western blot analysis. KEY FINDINGS: This compound bearing bioactive axial ligand, such as the active histone deacetylase inhibitor (HDACi) (2-propynyl)octanoic acid (POA), induced cell death through different pathways at a concentration ten times lower than cisplatin. SIGNIFICANCE: The results showed that Pt(IV)Ac-POA could represent a promising improvement of Pt-based chemotherapy against neuroblastoma.

Perichromatin region: a moveable feast.

The perichromatin region is an elusive zone of the cell nucleus located at the periphery of the condensed chromatin areas. This region is visible at the electron microscope level under special staining treatments, otherwise it is merged with the border of condensed chromatin. In this 200 nm-thick area, several fundamental cell processes take place, such as replication, DNA repair and transcription. In addition, RNA processing occurs in the perichromatin region, including 5'-capping and 3'-polyadenylation as well as splicing. Recently, it has become clear that also some epigenetics modifications take place there, such as methylation of DNA and RNA on cytosine and adenosine. In summary, this thin interface between chromatin and the interchromatinic space represents the zone where the majority of the functions of DNA in interphase occur, in a place where there is no steric hindrance of condensed chromatin, the products can easily move away toward their target and the enzymes can freely dock.

Zc3h13/Flacc is required for adenosine methylation by bridging the mRNA-binding factor Rbm15/Spenito to the m6A machinery component Wtap/Fl(2)d.

N6-methyladenosine (m6A) is the most abundant mRNA modification in eukaryotes, playing crucial roles in multiple biological processes. m6A is catalyzed by the activity of methyltransferase-like 3 (Mettl3), which depends on additional proteins whose precise functions remain poorly understood. Here we identified Zc3h13 (zinc finger CCCH domain-containing protein 13)/Flacc [Fl(2)d-associated complex component] as a novel interactor of m6A methyltransferase complex components in Drosophila and mice. Like other components of this complex, Flacc controls m6A levels and is involved in sex determination in Drosophila We demonstrate that Flacc promotes m6A deposition by bridging Fl(2)d to the mRNA-binding factor Nito. Altogether, our work advances the molecular understanding of conservation and regulation of the m6A machinery.

Ultrastructural localization of 5-methylcytosine on DNA and RNA.

DNA methylation is the major epigenetic modification and it is involved in the negative regulation of gene expression. Its alteration can lead to neoplastic transformation. Several biomolecular approaches are nowadays used to study this modification on DNA, but also on RNA molecules, which are known to play a role in different biological processes. RNA methylation is one of the most common RNA modifications and 5-methylcytosine presence has recently been suggested in mRNA. However, an analysis of nucleic acid methylation at electron microscope is still lacking. Therefore, we visualized DNA methylation status and RNA methylation sites in the interphase nucleus of HeLa cells and rat hepatocytes by ultrastructural immunocytochemistry and cytochemical staining. This approach represents an efficient alternative to study nucleic acid methylation. In particular, this ultrastructural method makes the visualization of this epigenetic modification on a single RNA molecule possible, thus overcoming the technical limitations for a (pre-)mRNA methylation analysis.

Osmium Ammine for Staining DNA in Electron Microscopy.

The osmium ammine staining allows the specific detection of DNA in the cell nucleus and represents one of the most used techniques for EM cytochemistry.The procedure is a Feulgen-type reaction, consisting of an acid hydrolysis to obtain free aldehyde groups on DNA followed by their binding to osmium ammine, a Schiff-type reagent. Osmium ammine is polyamminic electron-dense compound commercially available.Here, we describe the staining procedure for ultrathin sections and the different procedures for the preparation of the reagent for acrylic and epoxy sections.

Visualizing RNA at Electron Microscopy by Terbium Citrate.

Although the EDTA regressive technique allows the visualization of RNPs, this widely used method is not intended to be specific for RNA alone. A fine ultrastructural visualization of RNA on ultrathin sections can be obtained with terbium citrate: this method gives a weak contrast but a very fine end product which allows observations at a high resolution level.The procedure is very simple since it consists only of a period of incubation and very short washes, which are the crucial point of this technique to avoid the Tb removal from RNA. This method does not require any special type of fixation and embedding.

Inhibition of the Autophagy Pathway Synergistically Potentiates the Cytotoxic Activity of Givinostat (ITF2357) on Human Glioblastoma Cancer Stem Cells.

Increasing evidence highlighted the role of cancer stem cells (CSCs) in the development of tumor resistance to therapy, particularly in glioblastoma (GBM). Therefore, the development of new therapies, specifically directed against GBM CSCs, constitutes an important research avenue. Considering the extended range of cancer-related pathways modulated by histone acetylation/deacetylation processes, we studied the anti-proliferative and pro-apoptotic efficacy of givinostat (GVS), a pan-histone deacetylase inhibitor, on cell cultures enriched in CSCs, isolated from nine human GBMs. We report that GVS induced a significant reduction of viability and self-renewal ability in all GBM CSC cultures; conversely, GVS exposure did not cause a significant cytotoxic activity toward differentiated GBM cells and normal mesenchymal human stem cells. Analyzing the cellular and molecular mechanisms involved, we demonstrated that GVS affected CSC viability through the activation of programmed cell death pathways. In particular, a marked stimulation of macroautophagy was observed after GVS treatment. To understand the functional link between GVS treatment and autophagy activation, different genetic and pharmacological interfering strategies were used. We show that the up-regulation of the autophagy process, obtained by deprivation of growth factors, induced a reduction of CSC sensitivity to GVS, while the pharmacological inhibition of the autophagy pathway and the silencing of the key autophagy gene ATG7, increased the cell death rate induced by GVS. Altogether these findings suggest that autophagy represents a pro-survival mechanism activated by GBM CSCs to counteract the efficacy of the anti-proliferative activity of GVS. In conclusion, we demonstrate that GVS is a novel pharmacological tool able to target GBM CSC viability and its efficacy can be enhanced by autophagy inhibitory strategies.