Abstract: HIV-Encephalopathy (HIVE) is a common neurological disorder associated with HIV-1 infection and AIDS. The activity of the HIV trans-activating protein Tat is thought to contribute to neuronal pathogenesis. While Tat proteins from primary virus isolates consist of 101 or more amino acids, 72 and 86 amino acids forms of Tat are commonly used for in vitro studies. Although Tat72 contains the minimal domain required for viral replication, other activities of Tat appear to vary according to its length, sub-cellular localization, cell type and the stage of cellular differentiation. In this study, we investigated the stability of intracellular Tat101 during proliferation and differentiation of neuronal cells in culture. We have utilized rat neuronal progenitors as a model of neuronal cell proliferation and differentiation, as well as rat primary cortical neurons as a model of fully differentiated cells. Our results indicate that, upon internalization, Tat101 was degraded more rapidly in proliferating cells than in cells which either underwent neuronal differentiation or were fully differentiated. Intracellular degradation of Tat was prevented by the calpain 1 inhibitor, ALLN, in both proliferating and differentiated cells. Inhibition of calpain 1 by calpastatin peptide also prevented Tat cleavage. In vitro calpain digestion and mass spectrometry analysis further demonstrated that the sequence of Tat sensitive to calpain cleavage was located in the C-terminus of this viral protein, between amino acids 68 and 69. Moreover, cleavage of Tat101 by calpain 1 increased neurotoxic effect of this viral protein and presence of the calpain inhibitor protected neuronal cells from Tat-mediated toxicity.

Abstract: Docosahexaenoic acid (DHA) is a well known chemopreventive nutrient within diet formulations, but it may also exert toxic effects on cultured cells, while this is limited when also another relevant nutrient as vitamin E is present. This effect, beside the involvement of the two nutrients in oxidative processes, likely affects the expression of specific genes. To obtain information on combined activities of DHA and vitamin E on some gene products previously resulted to be in vivo regulated from dietary unsaturated fats, the effect of the two nutrients was evaluated in human cell line HepG2. Independently, DHA and vitamin E resulted to affect only slightly UDP-glucuronosyltransferase 1A1 (UGT1A1) mRNA expression. Nevertheless, their combination produced a considerable reduction of this mRNA. DHA also downregulated stearoyl-CoA desaturase (SCD) and sterol regulatory element binding protein (SREBP-1) expression, while vitamin E did not affect these products. However, their combination abolished the downregulation of SCD but did not affect that of SREBP-1. Therefore the effect of the two nutrients is related to specific gene regulation processes resulting in a cooperation which might be related to their physiological effects as dietary components.

Abstract: MicroRNAs (miRs) are short endogenous RNAs that regulate gene expression by incomplete pairing with messenger RNAs. An increasing number of studies show that mammalian microRNAs play fundamental roles in various aspects of cellular function including differentiation, proliferation, and cell death. Recent findings demonstrating the presence of microRNAs in mature neuronal dendrites suggest their possible involvement in controlling local protein translation and synaptic function. HIV-1 Encephalopathy (HIVE) is a manifestation of HIV-1 infection that often results in neuronal damage and dysfunction. While neurons are rarely, if ever, infected by HIV-1, they are exposed to cytotoxic viral and cellular factors including the HIV-1 transactivating factor Tat. In this study, we show that Tat deregulates expression levels of selected microRNAs, including the neuronal mir-128, in primary cortical neurons. We further show that mir-128a inhibits expression of the pre-synaptic protein SNAP25, whereas the anti-mir-128a partially restores Tat/mir-128a-induced downregulation of SNAP25 expression. Altogether, our data provide a novel mechanism by which HIV-Tat perturbs neuronal activity.

Abstract: In this study we analysed the regulation of gene expression by arvanil and anandamide in human peripheral blood mononuclear cells (PBMCs) to clarify their immunosuppressive properties. PBMCs were activated, leading to CD36 down regulation, that was normalized by arvanil and anandamide. We used microarray technology to identify a regulatory pattern associated with cell proliferation in the presence of both substances. CD3-CD28 stimulated PBMCs showed a pattern of up-regulated and down-regulated genes after treatment with these substances. We selected and analysed several genes chosen by their function in the regulation of cell proliferation. We showed a transcriptional control of the CD36 gene by arvanil and anandamide associated with an increased protein expression, thus suggesting a possible role of CD36 in anandamide and arvanil anti-inflammatory pattern.

Abstract: BACKGROUND AND AIM: The lipid content of Mediterranean diet is mostly accounted for its disease preventive action. We investigated whether the short term nutritional effect of a fat quota mainly derived from olive and fish oil affects liver mRNA expression profile in rats. METHODS AND RESULTS: The study was carried out using DNA microarray techniques. The effect was evaluated at liver mRNA expression level to identify genes whose expression was regulated by dietary modifications. Two groups of six rats were alternatively supplied for two weeks with either a control or with an experimental diet. Both diets were semisynthetic and isocaloric, with identical major nutrients composition (protein 20%, carbohydrates 56% and lipids 22% of total energy) being different only in the quality of fats. The lipid quota of the control diet contained exclusively saturated animal fats, derived from butter, while in the experimental diet some unsaturated fats were present, being derived also from olive and fish oil (10% and 6% of total energy, respectively). Out of 26,334 genes analyzed, 11,292 were found expressed in the liver, 72 were induced and 180 were inhibited from the experimental diet. Out of these, 33 of the induced and 59 of the inhibited species have a well known function. CONCLUSIONS: The diet with olive and fish oil modulates several genes related to lipolysis or lipogenesis and newly identified responders from other metabolisms. Some of these genes are also reported to be similarly modulated by the action of fibrates, but without the complete gene activation typical of these PPARalpha ligands.

Abstract: OBJECTIVE: Farnesyl diphosphate synthase (FPPs) produces FPP which is considered a branch-point intermediate in the synthesis of sterols and isoprenylated cellular metabolites. In this study we investigated whether detectable FPPs activity was present in human colorectal cancer (CRC), also evaluating in vitro the role of this enzyme in the growth and apoptosis of CRC cells by using Pamidronate (PAM), a FPPs activity inhibitor. METHODS: The activity level of FPPs was determined in CRC and the normal surrounding mucosa of 50 patients by radiochemical assay. The FPPs mRNA expression was investigated in 15 of 50 patients by quantitative reverse transcriptase polymerase chain reaction (RT-PCR). K-ras mutation was evaluated using PCR and restriction enzyme analysis. Cell growth and apoptosis, after PAM treatment, in human CRC cell line DLD-1 were measured by MTT test and DNA fragmentation, respectively. RESULTS: FPPs activity was detectable in human CRC. FPPs activity and its mRNA were significantly more abundant in cancer samples than in normal mucosa. In vitro PAM resulted in a significant reduction of cell growth and also gave rise to a marked proapoptotic effect. CONCLUSIONS: This study provides the first evidence of the presence of FPPs activity in human CRC. Moreover, FPPs enzyme was found to play a significant role in colon cancer proliferation.