Carnosine modulates the Sp1-Slc31a1/Ctr1 copper-sensing system and influences copper homeostasis in murine CNS-derived cells.

Carnosine (CAR) is an endogenous dipeptide physiologically present in excitable tissues, such as central nervous system (CNS) and muscle. CAR is acknowledged as a substrate involved in many homeostatic pathways and mechanisms and, due to its biochemical properties, as a molecule intertwined with the homeostasis of heavy metals such as copper (Cu). In CNS, Cu excess and dysregulation imply oxidative stress, free-radical production, and functional impairment leading to neurodegeneration. Here, we report that CAR intercepts the regulatory routes of Cu homeostasis in nervous cells and tissues. Specifically, in a murine neuron-derived cell model, i.e., the B104 neuroblastoma cells, extracellular CAR exposure up to 24 h influenced intracellular Cu entry and affected (downregulated) the key Cu-sensing system, consisting of the gene coding for the Slc31a1 transmembrane Cu importer (alias Ctr1), and the gene coding for the Cu-responsive transcription factor Sp1 ( Sp1). Also, CAR exposure upregulated CAR biosynthesis ( Carns1), extracellular degradation ( Cndp1), and transport ( Slc15a4, alias Pht1) genes and elicited CAR intracellular accumulation, contributing to the outline of functional association between CAR and Cu within the cell. Interestingly, the same gene modulation scheme acting in vitro operates in vivo in brains of mice undergoing dietary administration of CAR in drinking water for 2 wk. Overall, our findings describe for the first time a regulatory interaction between CAR and Cu pathways in CNS and indicate CAR as a novel active molecule within the network of ligands and chaperones that physiologically regulate Cu homeostasis.

Effect of the flame retardant tris (1,3-dichloro-2-propyl) phosphate (TDCPP) on Na+-K+-ATPase and Cl- transport in HeLa cells.

Tris (1, 3-dichloro-2-propyl) phosphate (TDCPP) is one of the most diffused phosphorus flame retardants in the environment and is highly persistent and abundant in residential dust samples. To date the cellular targets and mechanisms underlying its toxic effects are not completely understood. The aim of this work was to study the effects of TDCPP on ion transport mechanisms fundamental for the cellular ionic homeostasis, such as Na+-K+-ATPase and Cl- transport. HeLa cells were used as experimental model. TDCPP showed a dose-dependent effect on cell viability in cells exposed for 24 h as assessed by MTT test (IC50 = 52.5 µM). The flame retardant was able to exert a dose and time-dependent inhibition on the Na+-K+-ATPase activity. A short-term exposure (1 h) was able to exert a significant inhibition at 75 and 100 µM TDCPP, suggesting that TDCPP is able to directly interfere with the Na+-K+-ATPase phosphate catalytic activity. The sensitivity of the pump to lower TDCPP concentrations increased with the increase of the time of exposure. Following 24 h exposure a significant inhibition of about 40% was evident already at 10 µM and the IC50 value observed was 12.8 ± 6.0 µM. Moreover, TDCPP was also able to impair the NKCC mediated Cl- transport in HeLa cells, as assessed in YFP-H148Q/I152L-expressing HeLa cells. Following 1 h exposure TDCPP significantly inhibited the transport by about 30%. The kinetic analysis demonstrated a noncompetitive mechanism of inhibition. In conclusion, results demonstrated the impairment of ion transport mechanisms fundamental for ion homeostasis by TDCPP on HeLa cells.

Annurca (Malus pumila Miller cv. Annurca) apple as a functional food for the contribution to a healthy balance of plasma cholesterol levels: results of a randomized clinical trial.

BACKGROUND: Recent human studies have evaluated the effect of daily apple consumption on plasma cholesterol level, which is recognized as an important risk factor for cardiovascular disease (CVD). Nevertheless, slightly significant effects have been generally registered although consuming more than two apples a day for several weeks. RESULTS: This study describes the influence of daily consumption of Annurca apples on the cholesterol levels of mildly hypercholesterolaemic healthy subjects. A monocentric, randomized, parallel-group, placebo-controlled, 4-month study was conducted. The subjects (n = 250) were randomly assigned to five treatment groups (each one of 50 subjects: 28 men and 22 women). Four groups were administered one apple per day among the following: Red Delicious, Granny Smith, Fuji, Golden Delicious. The fifth group was asked to consume two Annurca apples per day, since the weight of this cultivar is on average half that of the commercial ones considered in this study. Comparing results, Annurca led to the most significant outcome, allowing a reduction in total and low-density lipoprotein cholesterol levels by 8.3% and 14.5%, respectively, and an increase in high-density lipoprotein cholesterol levels by 15.2% (all P < 0.001). CONCLUSION: Our data would reasonably indicate Annurca apple as a useful tool to contribute to the prevention of CVD risk through normal diet. © 2016 Society of Chemical Industry.

Behind the Link between Copper and Angiogenesis: Established Mechanisms and an Overview on the Role of Vascular Copper Transport Systems.

Angiogenesis critically sustains the progression of both physiological and pathological processes. Copper behaves as an obligatory co-factor throughout the angiogenic signalling cascades, so much so that a deficiency causes neovascularization to abate. Moreover, the progress of several angiogenic pathologies (e.g. diabetes, cardiac hypertrophy and ischaemia) can be tracked by measuring serum copper levels, which are being increasingly investigated as a useful prognostic marker. Accordingly, the therapeutic modulation of body copper has been proven effective in rescuing the pathological angiogenic dysfunctions underlying several disease states. Vascular copper transport systems profoundly influence the activation and execution of angiogenesis, acting as multi-functional regulators of apparently discrete pro-angiogenic pathways. This review concerns the complex relationship among copper-dependent angiogenic factors, copper transporters and common pathological conditions, with an unusual accent on the multi-faceted involvement of the proteins handling vascular copper. Functions regulated by the major copper transport proteins (CTR1 importer, ATP7A efflux pump and metallo-chaperones) include the modulation of endothelial migration and vascular superoxide, known to activate angiogenesis within a narrow concentration range. The potential contribution of prion protein, a controversial regulator of copper homeostasis, is discussed, even though its angiogenic involvement seems to be mainly associated with the modulation of endothelial motility and permeability.

Role of the cellular prion protein in the neuron adaptation strategy to copper deficiency.

Copper transporter 1 (CTR1), cellular prion protein (PrP(C)), natural resistance-associated macrophage protein 2 (NRAMP2) and ATP7A proteins control the cell absorption and efflux of copper (Cu) ions in nervous tissues upon physiological conditions. Little is known about their regulation under reduced Cu availability, a condition underlying the onset of diffused neurodegenerative disorders. In this study, rat neuron-like cells were exposed to Cu starvation for 48 h. The activation of Caspase-3 enzymes and the impairment of Cu,Zn superoxide dismutase (Cu,Zn SOD) activity depicted the initiation of a pro-apoptotic program, preliminary to the appearance of the morphological signs of apoptosis. The transcriptional response related to Cu transport proteins has been investigated. Notably, PrP(C) transcript and protein levels were consistently elevated upon Cu deficiency. The CTR1 protein amount was stable, despite a two-fold increase in the transcript amount, meaning the activation of post-translational regulatory mechanisms. NRAMP2 and ATP7A expressions were unvaried. The up-regulated PrP(C) has been demonstrated to enhance the cell Cu uptake ability by about 50% with respect to the basal transport, and so sustain the Cu delivery to the Cu,Zn SOD cuproenzymes. Conclusively, the study suggests a pivotal role for PrP(C) in the cell adaptation to Cu limitation through a direct activity of ion uptake. In this view, the PrP(C) accumulation observed in several cancer cell lines could be interpreted as a molecular marker of cell Cu deficiency and a potential target of therapeutic interventions against disorders caused by metal imbalances.

Fluorimetric analysis of copper transport mechanisms in the b104 neuroblastoma cell model: a contribution from cellular prion protein to copper supplying.

Dysregulated body copper homeostasis can negatively impact neuronal functions, but full knowledge of the mechanisms underlying the cell metal distribution has not been achieved yet. The high-affinity copper transporter 1 (Ctr1) is considered the main route for cell copper entry, while the cellular prion protein (PrP(C)) is presumed to be involved in the same process. Anchored to the outer side of the plasma membrane, this protein has the ability to bind copper ions and undergo internalization. To provide indications about the contribution of Ctr1 and PrP(C) proteins in cell copper transport, we used a fluorimetric method to characterize the kinetic properties of ion internalization in a neuroblastoma cell model, overexpressing prion protein (B104). Biochemical characteristics of intake delineated in the presence of other metal ions and an excess of extracellular potassium were compatible with PrP(C)-mediated endocytotic transport. Accordingly, inhibition of clathrin-dependent endocytosis by hypertonic shock and enzymatic removal of surface prion protein reduced copper influx by the same extent. On the whole, experimental evidence collected in a neuron-like cell model sustains a role for PrP(C) in mediating copper uptake by clathrin-dependent endocytosis.

Bioenergetics profile of CD4(+) T cells in relapsing remitting multiple sclerosis subjects.

Multiple sclerosis (MS) is a chronic inflammatory autoimmune demyelinating disease of the central nervous system. There are four clinical forms of MS, the most common of which is characterized by a relapsing remitting course (RRMS). The etiology of MS is unknown, but many studies suggested that genetic, environmental and infectious agents may contribute to the development of this disease. In experimental autoimmune encephalomyelitis (EAE), the animal model for MS, it has been shown that CD4(+) T cells play a key role in MS pathogenesis. In fact, these cells are able to cross the blood-brain barrier and cause axonal damage with neuronal death. T cell activation critically depends on mitochondrial ATP synthesis and reactive oxygen species (ROS) production. Interestingly, lots of studies linked the oxidative damage arising from mitochondrial changes to neurodegenerative disorders, such as MS. Based on these evidences, this work focused on the metabolic reprogramming of CD4(+) T cells in MS subjects, being this cell population directly implicated in pathogenesis of disease, paying attention to mitochondrial function and response to oxidative stress. Such aspects, once clarified, may open new opportunities for a therapeutic metabolic modulation of MS disorder.

Coffee and Depression: A Short Review of Literature.

Coffee is among the most widespread and healthiest beverages in the world. It is known to be a highly rich source of biologically active natural metabolites which possess therapeutic effects (i.e. caffeine) and functional properties (i.e. chlorogenic acids). Therefore, coffee can be considered a drink which has different positive effects on human health such as cardioprotective, neuroprotective, hepatoprotective, nephroprotective, etc. However, heavy coffee consumption may be related to some unpleasant symptoms, mainly anxiety, headache, increased blood pressure, nausea, and restlessness. During the past two decades, several studies have indicated that there is a close correlation between consumption of coffee and incidence of depression. In addition, phytochemical studies showed that caffeine is the main responsible constituent for antidepressant effects of coffee through multiple molecular mechanisms. The aim of the present paper was to collect the latest literature data (from 1984 to 2014) on the positive and negative impacts of coffee consumption on the major depressive disorders and to clarify the role of bioactive constituents of coffee in the related different clinical trials. To the best of our knowledge, this the first review on this topic.

Evaluation of thermal and non-thermal effects of UHF RFID exposure on biological drugs.

The Radio Frequency Identification (RFID) technology promises to improve several processes in the healthcare scenario, especially those related to traceability of people and things. Unfortunately, there are still some barriers limiting the large-scale deployment of these innovative technologies in the healthcare field. Among these, the evaluation of potential thermal and non-thermal effects due to the exposure of biopharmaceutical products to electromagnetic fields is very challenging, but still slightly investigated. This paper aims to setup a controlled RF exposure environment, in order to reproduce a worst-case exposure of pharmaceutical products to the electromagnetic fields generated by the UHF RFID devices placed along the supply chain. Radiated powers several times higher than recommended by current normative limits were applied (10 W and 20 W). The electric field strength at the exposed sample location, used in tests, was as high as 100 V/m. Non-thermal effects were evaluated by chromatography techniques and in vitro assays. The results obtained for a particular case study, the ActrapidTM human insulin preparation, showed temperature increases lower than 0.5 °C and no significant changes in the structure and performance of the considered drug.

Cuprizone neurotoxicity, copper deficiency and neurodegeneration.

Cuprizone is used to obtain demyelination in mice. Cuprizone-treated mice show symptoms similar to several neurodegenerative disorders such as severe status spongiosus. Although it has a simple chemical formula, its neurotoxic mechanism is still unknown. In this work, we examined both physico-chemical properties and biological effects of cuprizone. Our results indicate that cuprizone has very complicated and misunderstood solution chemistry. Moreover, we show here the inability of cuprizone to cross neither the intestinal epithelial barrier nor the neuronal cell membrane, as well its high tolerability by cultured neurons. If added to mice diet, cuprizone does not accumulate in liver or in brain. Therefore, its neurotoxic effect is explainable only in terms of its capability to chelate copper, leading to chronic copper deficiency.

Real-time monitoring of copper ions-induced cytotoxicity by EIS cell chips.

An important goal of biomedical research is the development of tools for high-throughput evaluation of drug effects and cytotoxicity tests. Here we demonstrate EIS cell chips able to monitor cell growth, morphology, adhesion and their changes as a consequence of treatment with drugs or toxic compounds. As a case study, we investigate the uptake of copper ions and its effect on two cell lines: B104 and HeLa cells. For further understanding, we also carried out in parallel with EIS studies, a complete characterization of cell morphology and changes induced by copper ions through complementary methodologies (including state-of-the-art AFM, viability test and Western blot). Our results reveal a strong correlation between EIS data and both MTT test and AFM characterization so our chip can be used as powerful tools in all biology lab in combination with other standard methods giving additional information that can be useful in a complete and deep investigation of a biological process. This chip can be used even alone replacing in vitro drug tests based on conventional biochemical methods, being very cheap and reusable and allowing to perform cytotoxicity tests without using any expensive reagent or equipment.

Confocal microscopy evidence of prion protein fragment hPrP[173-195] internalization in rat B104 neuroblastoma cell line.

The cytotoxicity of hPrP[173-195] prion peptide against a neuroblastoma cell model was found independent of its tendency to aggregate over time. Cytosolic and nuclear inclusions of peptide were highlighted by confocal microscopy, suggesting a role as a transcription factor in activating signal transduction pathways involved in cell toxicity.

The prion protein: Structural features and related toxic peptides.

Prion diseases are characterized by the conversion of the physiological cellular form of the prion protein (PrP(C)) into an insoluble, partially protease-resistant abnormal scrapie form (PrP(Sc)). PrP(C) is normally expressed in mammalian cell and is highly conserved among species, although its role in cellular function remains elusive. The conversion of PrP(C) to PrP(Sc) parallels a conformational change of the polypeptide from a predominantly alpha-helical to a highly beta-sheet secondary structure. The pathogenesis and molecular basis of the consequent nerve cell loss are not understood. Limited structural information is available on aggregate formation by this protein as the possible cause of these diseases and on its toxicity. This brief overview focuses on the large amount of structure-activity studies based on the prion fragment approach, hinging on peptides derived from the unstructured N-terminal and globular C-terminal domains. It is well documented that most of the fragments with regular secondary structure, with the exception of helices 1 and 3, possess a high beta-sheet propensity and tendency to form beta-sheet-like aggregates. In this context, helix 2 plays a crucial role because it is able to adopt both misfolded and partially helical conformation. However, only a few mutants are able to display its intrinsic neurotoxicity.