The selection of serum-independent PC12 cells for a more-reliable manganese cytotoxicity test

A major issue concerning the protocols of heavy metal cytotoxicity tests with PC12 cells was the hypothesis that serum in the culture medium might sequester the metal, thus altering the results obtained. However, serum withdrawal impairs the viability of PC12 cells themselves, thus impeding cytotoxicity testing in the absence of serum. In this study, we repeatedly selected undifferentiated, totally non-adherent PC12 cells in Petri dishes. Surprisingly, we discovered that these cells could survive and proliferate in serum-free medium. Moreover, features such as NGF-responsiveness, resazurin reduction potential, doubling rate, protein content, and basal caspase-3 enzyme activity, were equivalent to those exhibited by standard PC12 cultures. Further experiments aimed at fully characterising these serum-independent PC12 cells are in progress. These cells enabled cytotoxicity experiments to be conducted with manganese, both in serum-supplemented and in serum-deprived medium. The results demonstrated that serum removal decreased the LC50 of manganese from 250microM to 32microM, without affecting the internalisation of the metal. The data exclude an early competitive effect of serum on metal internalisation; rather, they suggest a late protective mechanism mediated by serum against the cytotoxic effect of the already-internalised metal.     

PTX3 interacts with inter-alpha-trypsin inhibitor: implications for hyaluronan organization and cumulus oophorus expansion

Pentraxin 3 (PTX3) and heavy chains (HCs) of inter-alpha-trypsin inhibitor (IalphaI) are essential for hyaluronan (HA) organization within the extracellular matrix of the cumulus oophorus, which is critical for in vivo oocyte fertilization and female fertility. In this study, we examined the possibility that these molecules interact and cooperate in this function. We show that HCs and PTX3 colocalize in the cumulus matrix and coimmunoprecipitate from cumulus matrix extracts. Coimmunoprecipitation experiments and solid-phase binding assays performed with purified human IalphaI and recombinant PTX3 demonstrate that their interaction is direct and not mediated by other matrix components. PTX3 does not bind to IalphaI subcomponent bikunin and, accordingly, bikunin does not compete for the binding of PTX3 to IalphaI, indicating that PTX3 interacts with IalphaI subcomponent HC only. Recombinant PTX3-specific N-terminal region, but not the PTX3-pentraxin C-terminal domain, showed the same ability as full-length protein to bind to HCs and to enable HA organization and matrix formation by Ptx3(-/-) cumulus cell oocyte complexes cultured in vitro. Furthermore, a monoclonal antibody raised against PTX3 N terminus, which inhibits PTX3/IalphaI interaction, also prevents recombinant full-length PTX3 from restoring a normal phenotype to in vitro-cultured Ptx3(-/-) cumuli. These results indicate that PTX3 directly interacts with HCs of IalphaI and that such interaction is essential for organizing HA in the viscoelastic matrix of cumulus oophorus, highlighting a direct functional link between the two molecules.     

Expression of cyclo-oxygenase-2 in macrophages associated with cutaneous melanoma at different stages of progression

The biological significance of the almost constant presence of macrophages in the tumoral microenvironment is an issue debated by several authors. The major difficulty in understanding the role played by tumor-associated macrophages (TAMs) in tumor progression is due to the contrasting effects of TAMs found in different studies. In addition, there is a limited information on which of the many biological activities expressed by TAMs are critical in inducing stimulatory or inhibitory effect on tumor growth. The aim of our study was: (a) to explore to what extent cyclo-oxygenase-2 (COX-2) in TAMs associated with human melanoma is expressed at different stages of tumor progression; and (b) to explore whether COX-2 expression in TAMs is stimulated by melanoma cells. In order to answer this question, we determined COX-2 positive TAMs associated with cutaneous melanocytic nevi, in situ, invasive and metastatic melanoma. In addition, we investigated whether COX-2 is expressed in peritoneal thioglycollate-elicited macrophages after co-cultivation with murine B16 melanoma cells. We found that COX-2-positive TAMs, as revealed by immunohistochemical analysis, were rare in common nevi and "dysplastic nevi", but present in a high percentage in in situ and thin melanoma. COX-2-positive TAMs were also found in more advanced tumors and metastatic melanoma, although at a significantly lower percentage in these latter. The in vitro protocol revealed that COX-2 was expressed in peritoneal macrophages upon contact with B16 murine melanoma cells, but not with normal murine fibroblasts. On the whole, the results of in vivo and in vitro studies suggest that COX-2 expressed in TAMs appears to act as an effective biomarker of melanoma progression, and melanoma cells themselves might stimulate COX-2 in macrophages.     

Cross-presentation of caspase-cleaved apoptotic self antigens in HIV infection

We found that the proteome of apoptotic T cells includes prominent fragments of cellular proteins generated by caspases and that a high proportion of distinct T cell epitopes in these fragments is recognized by CD8+ T cells during HIV infection. The frequencies of effector CD8+ T cells that are specific for apoptosis-dependent epitopes correlate with the frequency of circulating apoptotic CD4+ T cells in HIV-1-infected individuals. We propose that these self-reactive effector CD8+ T cells may contribute to the systemic immune activation during chronic HIV infection. The caspase-dependent cleavage of proteins associated with apoptotic cells has a key role in the induction of self-reactive CD8+ T cell responses, as the caspase-cleaved fragments are efficiently targeted to the processing machinery and are cross-presented by dendritic cells. These findings demonstrate a previously undescribed role for caspases in immunopathology.     

Oligothiophene molecular beacons

Oligomers of thiophene are widely studied compounds for their electronic and optoelectronic properties. Despite their strong fluorescence, their use as markers for biomolecules, especially for oligonucleotides (ONs), is still largely unexplored. Here, we describe the synthesis of a series of ON molecular beacons employing different oligothiophenes as fluorescent probes and discuss their fluorescence emissions in comparative experiments with and without dabcyl as a quencher, in their hairpin and linear conformations, and as duplexes after hybridization with a complementary target.     

Feijoa sellowiana derived natural Flavone exerts anti-cancer action displaying HDAC inhibitory activities

Curative properties of some medicinal plants such as the Feijoa sellowiana Bert. (Myrtaceae), have been often claimed, although the corresponding molecular mechanism(s) remain elusive. We report here that the Feijoa acetonic extract exerts anti-cancer activities on solid and hematological cancer cells. Feijoa extract did not show toxic effects on normal myeloid progenitors thus displaying a tumor-selective activity. In the Feijoa acetonic extract, fractionation and subsequent purification and analyses identified Flavone as the active component. Flavone induces apoptosis which is accompanied by caspase activation and p16, p21 and TRAIL over-expression in human myeloid leukemia cells. Use of ex vivo myeloid leukemia patients blasts confirms that both the full acetonic Feijoa extract and its derived Flavone are able to induce apoptosis. In both cell lines and myeloid leukemia patients blasts the apoptotic activity of Feijoa extract and Flavone is accompanied by increase of histone and non-histone acetylation levels and by HDAC inhibition. Our findings show for the first time that the Feijoa apoptotic active principle is the Flavone and that this activity correlates with the induction of HDAC inhibition, supporting the hypothesis of its epigenetic pro-apoptotic regulation in cancer systems.     

Nitric oxide has dual opposite roles during early and late phases of apoptosis in cerebellar granule neurons

The involvement and the role of nitric oxide (NO) as a signaling molecule in the course of neuronal apoptosis, whether unique or modulated during the progression of the apoptotic program, has been investigated in a cellular system consisting of cerebellar granule cells (CGCs) where apoptosis can be induced by lowering extracellular potassium. Several parameters involved in NO signaling pathway, such as NO production, neuronal nitric oxide synthase (nNOS) expression, and cyclic GMP (cGMP) production were examined in the presence or absence of different inhibitors. We provide evidence that nitric oxide has dual and opposite effects depending on time after induction of apoptosis. In an early phase, up to 3 h of apoptosis, nitric oxide supports survival of CGCs through a cGMP-dependent mechanism. After 3 h, nNOS expression and activity decreased resulting in shut down of NO and cGMP production. Residual NO then contributes to the apoptotic process by reacting with rising superoxide anions leading to peroxynitrite production and protein inactivation. We conclude that whilst NO over-production protects neurons from death in the early phase of neuronal damage, its subsequent reduction may contribute to neuronal degeneration and ultimate cell death.     

Presymbiotic growth and sporal morphology are affected in the arbuscular mycorrhizal fungus Gigaspora margarita cured of its endobacteria

Some arbuscular mycorrhizal fungi contain endocellular bacteria. In Gigaspora margarita BEG 34, a homogenous population of beta-Proteobacteria is hosted inside the fungal spore. The bacteria, named Candidatus Glomeribacter gigasporarum, are vertically transmitted through fungal spore generations. Here we report how a protocol based on repeated passages through single-spore inocula caused dilution of the initial bacterial population eventually leading to cured spores. Spores of this line had a distinct phenotype regarding cytoplasm organization, vacuole morphology, cell wall organization, lipid bodies and pigment granules. The absence of bacteria severely affected presymbiotic fungal growth such as hyphal elongation and branching after root exudate treatment, suggesting that Ca. Glomeribacter gigasporarum is important for optimal development of its fungal host. Under laboratory conditions, the cured fungus could be propagated, i.e. could form mycorrhizae and sporulate, and can therefore be considered as a stable variant of the wild type. The results demonstrated that - at least for the G. margarita BEG 34 isolate - the absence of endobacteria affects the spore phenotype of the fungal host, and causes delays in the growth of germinating mycelium, possibly affecting its ecological fitness. This cured line is the first manipulated and stable isolate of an arbuscular mycorrhizal fungus.