Size-dependent toxicity and cell interaction mechanisms of gold nanoparticles on mouse fibroblasts.

Gold nanoparticles (AuNPs) are currently used in several fields including biomedical applications, although no conclusive information on their cytotoxicity is available. For this reason this work has investigated the effects of AuNPs in vitro on Balb/3T3 mouse fibroblasts. Results obtained exposing cells for 72 h to AuNPs 5 and 15 nm citrate stabilized, revealed cytotoxic effects only for AuNPs 5 nm at concentration ≥ 50 µM if measured by colony forming efficiency (CFE). To understand the differences in cytotoxicity observed for the two AuNPs sizes, we investigated the uptake and the intracellular distribution of the nanoparticles. By TEM it was observed that 5 and 15 nm AuNPs are internalized by Balb/3T3 cells and located within intracellular endosomal compartments. Quantification of the uptake by ICP-MS showed that AuNPs internalization enhanced even up to 72 h. Disruption of the actin cytoskeleton was evident, with cell footprints narrow and contracted; effects more remarkable in cells exposed to 5 nm AuNP. The mechanism of NPs cell internalization was investigated using immunocytochemistry and western blot. No significant effect was observed in the expression level of caveolin, while reduction of the expression and degradation of the clathrin heavy chain was observed in cells exposed for 72 h to AuNPs.

Neurogenic-committed human pre-adipocytes express CYP1A isoforms.

Stem cell models offer an opportunity both for therapeutic use and for the assessment of alternative in vitro models. Human lipoaspirate is a source of adult stem cells (pre-adipocytes), which are able to differentiate into various phenotypes, such as neurogenic lineage. Here, we analyse the suitability of these in vitro models in screening exogenous compounds, such as environmental pollutants, that may affect adipose cells and neurogenic development. To evaluate neurogenic differentiation, we analysed expression of cholinergic system and acetylcholinesterase immunoreactivity. Heterocyclic derivatives of polycyclic aromatic hydrocarbons (PAHs) are often significant components of environmental contaminants. As they contain inducers of cytochrome P450 1A1 (CYP1A1), we explored the activity of CYP1A1-related enzymes, i.e. 7-ethoxycoumarin- and 7-ethoxyresorufin-O-deethylase (ECOD and EROD) in both cell systems in basal conditions and after exposure to non-cytotoxic doses of beta-naphthoflavone (BNF), a well-known PAH-type inducer. Both cell models showed basal and inducible levels of ECOD. Analysis of CYP1A1 protein expression and EROD-related enzyme activity confirmed the inducibility of the CYP1A1 isoform by BNF. These results demonstrate that mesenchymal adult stem cells can constitute innovative models. We therefore propose the use of pre-adipocytes and their neurogenic derivates to evaluate the cytotoxic/biological effects of unintended exposure to contaminants.

Pre-adipocytes commitment to neurogenesis 1: preliminary localisation of cholinergic molecules.

A great effort has recently been made to obtain human stem cells able to differentiate into cholinergic neurons, as a number of diseases are associated to the cholinergic neuron loss, degeneration or incorrect function (Alzheimer's disease and motor neuron disease). A stem cell population (i.e. pre-adipocytes) is present in the adipose stromal compartment. Pre-adipocytes, like the mesodermic derivative cells, retain high plasticity and potentiality to convert in vitro from one phenotype into many others, and they can be isolated from adult adipose tissue. Pre-adipocytes committed in vitro to neural differentiation were followed up to the acquisition of neural morphology. Acetylcholinesterase and choline acetyltransferase are expressed from the native cell stage, with different localisations and roles during neural commitment. Western blots show the beginning of a new synthesis of these enzymes at 4 weeks of culture of neurogenic pre-adipocytes, in parallel with neural morphology. The passage of the choline-acetyltransferase immunoreactivity from cytoplasmic to membrane localisation shows the possible onset of catalytic activity and the histochemical reaction confirms the activity of acetylcholinesterase. This explains the possibility of obtaining cholinergic-like phenotype from pre-adipocytes.

Differential expression of nuclear lamins in normal and cancerous prostate tissues.

The process of carcinogenesis is characterized by definite changes in the protein composition of the nuclear matrix. We have recently found that lamins form, in addition to the nuclear lamina, an intranuclear web of thin fibrils. This finding prompted us to address the question of whether changes in the expression of lamins occur in the course of tumor development. In prostate cancer, lamin B undergoes a significant increase; interestingly, its nuclear content strongly correlates with tumor differentiation. Moreover, all the lamins show reproducible alterations in the distribution of the isoelectric variants, suggesting that dephosphorylation events could trigger changes in the pattern of gene expression by inducing structural rearrangements of the nuclear scaffold.

SCN- binding to the charged lysines of histones end domains mimics acetylation and shows the major histone-DNA interactions involved in eu and heterochromatin stabilization.

SCN- binds to the charged amino group of lysines, inducing local changes in the electrostatic free energy of histones. We exploited this property to selectively perturb the histone-DNA interactions involved in the stabilization of eu and heterochromatin. Differential scanning calorimetry (DSC) was used as leading technique in combination with trypsin digestion that selectively cleaves the histone end domains. Euchromatin undergoes progressive destabilization with increasing KSCN concentration from 0 to 0.3 M. Trypsin digestion in the presence of 0.2 M KSCN show that the stability of the linker decreases as a consequence of the competitive binding of SCN- to the amino groups located in the C and N-terminal domain of H1 and H3, respectively; likewise, the release of the N-terminal domain of H4 induces an appreciable depression in both the temperature and enthalpy of melting of core particle DNA. Unfolding of heterochromatin requires, in addition to further cleavage of H4, extensive digestion of H2A and H2B, strongly suggesting that these histones stabilize the higher order structure by forming a protein network which extends throughout the heterochromatin domain.

Personal daily exposures to benzo(a)pyrene of taxi drivers in Genoa, Italy.

Twenty-four hour personal exposures (both occupational and environmental) to benzo(a)pyrene (BaP) of 15 taxi drivers working in Genoa, Italy, were monitored in May-July 1998 (15 samplings), February 1999 (7 samplings) and June 1999 (7 samplings). The mean BaP exposures measured at these different times were 1.4 ng/m3, 1.23 ng/m3 and 1.22 ng/m3, respectively, values were significantly greater than the levels found in controls (0.16+/-0.2 ng/m3). Mean daily personal BaP exposures of taxi drivers were not statistically different from the mean daily airborne BaP concentrations measured by fixed samplers during the same sampling periods.

DNA supercoiling in apoptotic chromatin.

In a previous paper, we have reported that in rat thymocyte apoptosis chromatin undergoes a specific structural change as well as an appreciable increase in the unacetylated forms of histones H3 and H4. Here, we show that H3 and H4 deacetylation bears no relation to chromatin condensation, and present new ultrastructural and topological observations that largely clarify the organization of the condensed state. The texture of the latter corresponds to a closely woven network of negatively supercoiled 11 nm fibers, as shown by both ultrastructural observations and relaxation experiments using ethidium bromide. Circularly closed chromatin loops undergoing apoptotic condensation, clearly showing nucleosome compact dimers or higher oligomers, as well as long stretches of supercoiled DNA, have also been detected. All of these modifications are strongly reminiscent of the alterations induced in nucleosome bearing plasmids by the chromatin remodeling factors SWI/SNF and RSC.

An intranuclear frame for chromatin compartmentalization and higher-order folding.

Recent ultrastructural, immunoelectron, and confocal microscopy observations done in our laboratory [Barboro et al. [2002] Exp. Cell. Res. 279:202-218] have confirmed that lamins and the nuclear mitotic apparatus protein (NuMA) are localized inside the interphase nucleus in a polymerized form. This provided evidence of the existence of a RNA stabilized lamin/NuMA frame, consisting of a web of thin ( approximately 3 and approximately 5 nm) lamin filaments to which NuMA is anchored mainly in the form of discrete islands, which might correspond to the minilattices described by Harborth et al. [1999] (EMBO. J. 18:1689-1700). In this article we propose that this scaffold is involved in the compartmentalization of both chromatin and functional domains and further determines the higher-order nuclear organization. This hypothesis is strongly supported by the scrutiny of different structural transitions which occur inside the nucleus, such as chromatin displacement and rearrangements, the collapse of the internal nuclear matrix after RNA digestion and the disruption of chromosome territories induced by RNase A and high salt treatment. All of these destructive events directly depend on the loss of the stabilizing effect exerted on the different levels of structural organization by the interaction of RNA with lamins and/or NuMA. Therefore, the integrity of nuclear RNA must be safeguarded as far as possible to isolate the matrix in the native form. This material will allow for the first time the unambiguous ultrastructural localization inside the INM of the components of the functional domains, so opening new avenues of investigation on the mechanisms of gene expression in eukaryotes.