Mechanistic studies on two dinuclear organogold(III) compounds showing appreciable antiproliferative properties and a high redox stability

Two dinuclear oxo-bridged organogold(III) compounds, namely [(N,N,C)(2)Au(2)(μ-O)][PF(6)](2) (with N,N,CH = 6-(1-methylbenzyl)-2,2'-bipyridine, Au(2)O1; or 6-(1,1-dimethylbenzyl)-2,2'-bipyridine, Au(2)O2), were previously prepared and characterised. Their solution chemistry under physiological-like conditions has been investigated here as well as their in vitro antiproliferative properties. Notably, these compounds reveal a marked redox stability even in the presence of effective biological reductants such as ascorbic acid and glutathione. The two dinuclear gold(iii) compounds were evaluated for cytotoxic actions against a representative panel of 12 human tumor cell lines, in comparison to respective mononuclear parent compounds [(N,N,C)AuOH][PF(6)], and appreciable biological activity could be highlighted. The reactions of Au(2)O1 and Au(2)O2 with a few model proteins were studied and the ability to form metallodrug-protein adducts monitored through ESI MS methods. Typical adducts were identified where the protein is associated to monometallic gold fragments; in these adducts gold remains in the oxidation state +3 and conserves its organic ligand. A direct comparison of the biological profiles of these binuclear organogold(III) compounds with those previously reported for a series of dinuclear oxo-bridged complexes [(N,N)(2)Au(2)(μ-O)(2)][PF(6)](2) (N,N = 6(6')-substituted 2,2'-bipyridines) named Auoxo's was carried out. It emerges that the greater cytotoxicity of the latter is mainly due to the greater oxidising power of their gold(III) centres and to propensity to generate gold(i) species; in contrast, the here described bimetallic organogold(III) complexes manifest a far higher redox stability in the biological milieu coupled to lower, but still significant, antiproliferative properties. Different molecular mechanisms are thus hypothesised for these two classes of dinuclear gold(III) agents.     

Clinical features predicting identification of CDKN2A mutations in Italian patients with familial cutaneous melanoma

CDKN2A is the most common, most penetrant gene whom germline mutations predisposing to cutaneous familial melanoma (FAM). Multiple primary melanoma (MPM), early age at onset, >2 affected members and pancreatic cancer are consistent features predicting positive test. However, the impact that cumulative clinical features have on the likelihood of molecular testing is unknown. In this work, genotype-phenotype correlations focused on selected clinical features were performed in 100 Italian FAM unrelated patients. Molecular studies of CDKN2A mutations were performed by direct sequencing. Statistical study included multiple correspondence analysis, uni- and multivariate analyses, and individual patient's probability calculation. MPM, >2 affected family members, Breslow thickness >0.4mm, and age at onset ≤41 years were the unique independent features predicting positive CDKN2A screening. The rate of positive testing ranged from 93.2% in the presence of all of them, to 0.4% in their absence. The contribution of each of them was quantified accordingly, with MPM being the most significant. These findings confirm previous data and add novel insights for the role of accurate patients' selection in CDKN2A screening.     

Premature decline of serum total testosterone in HIV-infected men in the HAART-era

Background

Testosterone (T) deficiency remains a poorly understood issue in men with Human Immunodeficiency Virus (HIV). We investigated the gonadal status in HIV-infected men in order to characterize T deficiency and to identify predictive factors for low serum T.

Methodology/Principal Findings

We performed a cross-sectional, observational study on 1325 consecutive HIV male outpatients, most of them having lipodystrophy. Serum total T<300 ng/dL was used as the threshold for biochemical T deficiency. Morning serum total T, luteinizing hormone (LH), estradiol, HIV parameters, and body composition parameters by CT-scan and Dual-Energy-X-ray-Absorptiometry were measured in each case. Sexual behavior was evaluated in a subset of 247 patients. T deficiency was found in 212 subjects, especially in the age range 40–59, but was frequent even in younger patients. T deficiency occurred mainly in association with low/normal serum LH. Adiposity was higher in subjects with T deficiency (p<0.0001) and both visceral adipose tissue and body mass index were the main negative predictors of serum total T. Osteoporosis and erectile dysfunction were present in a similar percentage in men with or without T deficiency.

Conclusions/Significance

Premature decline of serum T is common (16%) among young/middle-aged HIV-infected men and is associated with inappropriately low/normal LH and increased visceral fat. T deficiency occurs at a young age and may be considered an element of the process of premature or accelerated aging known to be associated with HIV infection. The role of HIV and/or HIV infection treatments, as well as the role of the general health state on the gonadal axis, remains, in fact, to be elucidated. Due to the low specificity of signs and symptoms of hypogonadism in the context of HIV, caution is needed in the diagnosis of hypogonadism in HIV-infected men with biochemical low serum T levels.     

PKCδ sensitizes neuroblastoma cells to L-buthionine-sulfoximine and etoposide inducing reactive oxygen species overproduction and DNA damage

Neuroblastoma is a type of pediatric cancer. The sensitivity of neuroblastoma (NB) cancer cells to chemotherapy and radiation is inhibited by the presence of antioxidants, such as glutathione (GSH), which is crucial in counteracting the endogenous production of reactive oxygen species (ROS). We have previously demonstrated that cells depleted of GSH undergo apoptosis via oxidative stress and Protein kinase C (PKC) δ activation. In the present study, we transfected PKCδ in NB cells resistant to oxidative death induced by L-buthionine-S,R-sulfoximine (BSO), a GSH-depleting agent. Cell responses, in terms of ROS production, apoptosis and DNA damage were evaluated. Moreover, PKCδ activation was monitored by analyzing the phosphorylation status of threonine 505 residue, carrying out PKC activity assay and investigating the subcellular localization of the kinase. The cell responses obtained in BSO-resistant cells were also compared with those obtained in BSO-sensitive cells subjected to the same experimental protocol. Our results demonstrate, for the first time, that PKCδ induces DNA oxidation and ROS overproduction leading to apoptosis of BSO-resistant NB cells and potentiates the cytotoxic effects induced by BSO in sensitive cells. Moreover, PKCδ overexpression enhances the sensitivity of NB cells to etoposide, a well-characterised drug, commonly used in neuroblastoma therapy. Altogether our data provide evidence of a pro-oxidant role of PKCδ that might be exploited to design new therapeutic strategies aimed at selective killing of cancer cells and overcoming drug resistance. However, it becomes evident that a more detailed understanding of ROS-mediated signaling in cancer cells is necessary for the development of redox-modulated therapeutic approaches.     

Surface localization of glucosylceramide during Cryptococcus neoformans infection allows targeting as a potential antifungal

Cryptococcus neoformans (Cn) is a significant human pathogen that, despite current treatments, continues to have a high morbidity rate especially in sub-Saharan Africa. The need for more tolerable and specific therapies has been clearly shown. In the search for novel drug targets, the gene for glucosylceramide synthase (GCS1) was deleted in Cn, resulting in a strain (Δgcs1) that does not produce glucosylceramide (GlcCer) and is avirulent in mouse models of infection. To understand the biology behind the connection between virulence and GlcCer, the production and localization of GlcCer must be characterized in conditions that are prohibitive to the growth of Δgcs1 (neutral pH and high CO(2)). These prohibitive conditions are physiologically similar to those found in the extracellular spaces of the lung during infection. Here, using immunofluorescence, we have shown that GlcCer localization to the cell surface is significantly increased during growth in these conditions and during infection. We further seek to exploit this localization by treatment with Cerezyme (Cz), a recombinant enzyme that metabolizes GlcCer, as a potential treatment for Cn. Cz treatment was found to reduce the amount of GlcCer in vitro, in cultures, and in Cn cells inhabiting the mouse lung. Treatment with Cz induced a membrane integrity defect in wild type Cn cells similar to Δgcs1. Cz treatment also reduced the in vitro growth of Cn in a dose and condition dependent manner. Finally, Cz treatment was shown to have a protective effect on survival in mice infected with Cn. Taken together, these studies have established the legitimacy of targeting the GlcCer and other related sphingolipid systems in the development of novel therapeutics.     

IPHEN—a real-time network for phenological monitoring and modelling in Italy

This paper aims to describe the Italian PHEnology Network (IPHEN), a cooperative project started in 2006 with the aim of producing nationwide maps of analysis and forecast of plants phenological stages mainly used to satisfy the needs of agriculture, health and environmental care. Iphen is a data processing system composed of the following main segments (a) collection of atmospheric and phenological data, (b) processing of data with suitable phenological and geo-statistical models and (c) phenological maps of analysis and forecast. In more detail, IPHEN maps of analysis (featuring phenological stages reached at the date of processing) are produced with models based on a Normal Heat Hours approach which weighs hourly air temperature effectiveness for plant phenological progression applied to national grids of hourly temperature derived from the operational agro-meteorological network of CRA-CMA. A correction scheme based on phenological surveys provided by volunteer observers is applied to the first guess maps of analysis to obtain final maps. Forecast maps (prediction of the days of occurrence of relevant phenological stages) are produced on the basis of GFS model medium range forecasts and climatic data. Freeware IPHEN maps for grapevine, common and Arizona cypress, black elder, olive and locust trees are broadcasted weekly on the CRA-CMA website. The positive operational results of IPHEN are testified by 150 maps broadcasted during the 2011 season for the above-mentioned species. The system performances and reliability have been analysed focusing on the analysis of phenological simulation errors and on the sensitivity of phenological maps to anomalous atmospheric circulation patterns. The error analysis shows that phenological models are characterized by advances/delays that justify the adoption of an observation based correction scheme. The sensitivity analysis highlights that the system is responsive to the effects of circulation blocking systems leading to phenological advances and delays.     

The P2X7 receptor as a route for non‐exocytotic glutamate release: dependence on the carboxyl tail

P2X7 receptors trigger Ca²⁺‐dependent exocytotic glutamate release, but also function as a route for non‐exocytotic glutamate release from neurons or astrocytes. To gain an insight into the mechanisms involving the P2X7 receptor as a direct pathway for glutamate release, we compared the behavior of a full‐length rat P2X7 receptor, a truncated rat P2X7 receptor in which the carboxyl tail had been deleted, a rat P2X7 receptor with the 18‐amino acid cysteine‐rich motif of the carboxyl tail deleted, and a rat P2X2 receptor, all of which are expressed in HEK293 cells. We found that the P2X7 receptor function as a route for glutamate release was antagonized in a non‐competitive way by extracellular Mg²⁺, did not require the recruitment of pore‐forming molecules, and was dependent on the carboxyl tail. Indeed, the truncated P2X7 receptor and the P2X7 receptor with the deleted cysteine‐rich motif both lost their function as a pathway for glutamate release, while still evoking intracellular Ca²⁺ elevation. No glutamate efflux was observed through the P2X2 receptor. Notably, HEK293 cells (lacking the machinery for Ca²⁺‐dependent exocytosis), when transfected with P2X7 receptors, appear to be a suitable model for investigating the P2X7 receptor as a route for non‐exocytotic glutamate efflux.     

De novo trisomy 20p characterized by array comparative genomic hybridization: Report of a novel case and review of the literature

We report on a boy with speech delay, mental retardation, motor clumsiness, hyperactivity, dysmorphic facial features, brachytelephalangy and short stature. Electrocardiogram, echocardiography, renal ultrasound, electroencephalogram, fundoscopic exam and auditory brainstem responses were all normal. Brain magnetic resonance imaging showed a left temporal arachnoid cyst and a small pineal gland cyst.