Diet and primary prevention of stroke: Systematic review and dietary recommendations by the ad hoc Working Group of the Italian Society of Human Nutrition.

BACKGROUND AND AIMS: To systematically review the latest evidence on established and emerging nutrition-related risk factors for incidence of and mortality from total, ischemic and haemorrhagic strokes. The present review was conducted in the framework of the work carried out through 2015 and 2016 for the preparation of the Italian Guidelines for the Prevention and Treatment of Stroke, 8th Edition, by ISO-SPREAD (Italian Stroke Organization and the Stroke Prevention and Educational Awareness Diffusion). METHODS AND RESULTS: Systematic review of articles focused on primary prevention of stroke published between January 2013 to May 2016 through an extensive search of the literature using MEDLINE/PUBMED, EMBASE and the Cochrane Library. Articles were ranked according to the SIGN methodology while the GRADE system was used to establish the strength of recommendations. As a result of our literature search, we examined 87 meta-analyses overall (mainly of prospective studies), a few isolated more recent prospective studies not included in the meta-analyses, and a smaller number of available randomized controlled trials and case-control studies. Based on the analysis of the above articles, 36 Syntheses of the available evidence and 36 Recommendations were eventually prepared. The present document was developed by organizing the available evidence into three individual areas (nutrients, food groups and dietary patterns) to provide a systematic and user-friendly overview of the available evidence on the relationship between nutrition and primary prevention of stroke. Yet analysis of foods and food patterns allowed translating the information about nutrients in a tool more amenable to use in daily life also in the light of the argument that people eat foods rather than nutrients. CONCLUSIONS: The present literature review and dietary recommendations provide healthcare professionals and all interested readers with a useful overview for the reduction of the risk of total, ischemic and haemorrhagic stroke through dietary modifications.

Skeletal muscle differentiation: role of dehydroepiandrosterone sulfate.

Dehydroepiandrosterone (DHEA) and its sulfonated form dehydroepiandrosterone sulfate (DHEAS) are the main circulating steroid hormones and many epidemiological studies show an inverse relationship between DHEA/DHEAS levels and muscle loss for which the primary cause is the accelerated protein breakdown. The aim of this work was to determine whether DHEA/DHEAS supplementation in differentiating C2C12 skeletal muscle cells might influence the expression of the atrophy-related ubiquitin ligase, MuRF-1, and thereby impact key molecules of the differentiation program. DHEA is the prohormone crucial for sex steroid synthesis, and DHEAS is thought to be its reservoir. However, our preliminary experiments showed that DHEAS, but not DHEA, is able to influence MuRF-1 expression. Therefore, we treated differentiating C2C12 cells with various concentrations of DHEAS and analyzed the expression of MuRF-1, Hsp70, myosin heavy chain (MHC), myogenin, and the activity of creatine kinase. We observed that DHEAS at physiological concentrations downregulates MuRF-1 expression and affects muscle differentiation, as shown by the increased levels of MHC, which is a sarcomeric protein that undergoes MuRF-1-dependent degradation, and also by an increase in creatine kinase activity and myogenin expression, which are two other well-known markers of differentiation. Moreover, we found that DHEAS might have a protective effect on differentiating cells as suggested by the augmented levels of Hsp70, a member of heat shock proteins family that, besides its cytoprotective action, seems to have a regulatory role on key atrophy genes such as MuRF-1. In conclusion, our data shed light on the role of DHEAS at physiologic concentrations in maintaining muscle mass.

Trans-plasma membrane electron transport in human blood platelets.

The plasma membrane redox (PMR) system is important for cell metabolism and survival; it is also crucial for blood coagulation and thrombosis. This review will give an update on the PMR system, with a particular regard to platelets, and on the role of antioxidant vitamins belonging to this system.

SVCT1 and SVCT2: key proteins for vitamin C uptake.

Vitamin C is accumulated in mammalian cells by two types of proteins: sodium-ascorbate co-transporters (SVCTs) and hexose transporters (GLUTs); in particular, SVCTs actively import ascorbate, the reduced form of this vitamin. SVCTs are surface glycoproteins encoded by two different genes, very similar in structure. They show distinct tissue distribution and functional characteristics, which indicate different physiological roles. SVCT1 is involved in whole-body homeostasis of vitamin C, while SVCT2 protects metabolically active cells against oxidative stress. Regulation at mRNA or protein level may serve for preferential accumulation of ascorbic acid at sites where it is needed. This review will summarize the present knowledge on structure, function and regulation of the SVCT transporters. Understanding the physiological role of SVCT1 and SVCT2 may lead to develop new therapeutic strategies to control intracellular vitamin C content or to promote tissue-specific delivery of vitamin C-drug conjugates.

Luteolin-7-glucoside inhibits IL-22/STAT3 pathway, reducing proliferation, acanthosis, and inflammation in keratinocytes and in mouse psoriatic model.

The epidermis is a dynamic tissue in which keratinocytes proliferate in the basal layer and undergo a tightly controlled differentiation while moving into the suprabasal layers. The balance between keratinocyte proliferation, differentiation, and death is essential, and its perturbation can result in pathological changes. Some common skin diseases, such as psoriasis, are characterized by hyperproliferation accompanied by inflammatory reactions, suggesting that molecules with topical anti-inflammatory and ROS scavenging abilities may be useful for their treatment. Here we investigate the potential of the flavone Luteolin-7-glucoside (LUT-7G) as a treatment for psoriasis. We show that LUT-7G leads to a modification of the cell cycle and the induction of keratinocyte differentiation, with modification of energy, fatty acid, and redox metabolism. LUT-7G treatment also neutralizes the proliferative stimulus induced by the proinflammatory cytokines IL-22 and IL-6 in HEKn. Moreover, in the Imiquimod (IMQ) mouse model of psoriasis, topical administration of LUT-7G leads to a marked reduction of acanthosis and re-expression of epidermal differentiation markers. Dissection of the IL-22 signalling pathway, activated by IMQ treatment, demonstrates that LUT-7G impairs the nuclear translocation of phosphorylated (activated) STAT3, blocking the IL-22 signalling cascade. Thus LUT-7G appears to be a promising compound for the treatment of hyperproliferative and inflammatory skin diseases, such as psoriasis.

Ascorbic acid maintenance in HaCaT cells prevents radical formation and apoptosis by UV-B.

We have investigated the enzymatic reduction and accumulation of vitamin C in HaCaT epithelial cells. The subcellular localization and the activities of ascorbyl free radical reductase and dehydroascorbate reductase showed that mitochondrial, microsomal and plasma membranes fractions express high levels of ascorbyl free radical reductase activity, whereas dehydroascorbate reductase activity was found at low levels only in the post microsomal supernatant. We have also investigated cell proliferation and vitamin C accumulation induced by ascorbic acid 2-phosphate. This derivative caused no inhibition of cell growth, was uptaken from the extracellular medium and accumulated as ascorbic acid in mM concentrations. These results show that HaCaT cells possess very efficient systems to maintain high levels of both intracellular and extracellular ascorbic acid. The regeneration and uptake of ascorbic acid from extracellular medium contributes to the intracellular antioxidant capacity, as evaluated by 2',7'-dihydrodichlorofluorescein staining. Consequently, cells became more resistant to free radical generation and cell death induced by UV-B irradiation.

Cell death by oxidative stress and ascorbic acid regeneration in human neuroectodermal cell lines.

In this paper, we show that human neuroectodermal cells exposed to 1-5 mM hydrogen peroxide or 10 nM-1 mM ascorbate die by programmed cell death induced by oxidative stress. The cell death by peroxide occurs within 4 h and involves approximately 80% of B-mel melanoma cells, while ascorbate causes cell death of approximately 86% of B-mel cells within 24 h. SK-N-BE(2) neuroblastoma cells are more resistant, 32% and 43% cell death for peroxide and ascorbate, respectively. In all cases, cell death causes hypodiploic DNA staining, evaluated by flow cytometry. Both cell lines can efficiently metabolise ascorbate due to significant levels of NADH-dependent semidehydroascorbate reductase and glutathione-dependent dehydroascorbate reductase. The cell death observed suggests a pro-oxidant, rather than anti-oxidant, role for ascorbic acid at physiological concentrations under these experimental conditions.

Hydrogen peroxide is an intermediate in the platelet activation cascade triggered by collagen, but not by thrombin.

Human blood platelets produce oxidant species when stimulated by collagen and thrombin. The oxidative burst of platelets has been studied by cytofluorimetry taking advantage of the fluorogenic dye DCFH2-DA, which is taken up and deacetylated by platelets and then oxidized to the fluorescent derivative DCF. The oxidation of DCFH2 is induced by stimulation with collagen but not with thrombin and inhibited by external catalase. Catalase also inhibited the aggregation induced by collagen, but not that induced by thrombin. Aspirin and indomethacin inhibited the formation of the fluorochrome only when platelets were stimulated by thrombin. Externally added H2O2 increased the cytoplasmic calcium content as probed by the fluorescence of Indo-1. The present data suggest that collagen induces the production of H2O2, which in turn may stimulate the aggregation of platelets through a calcium mobilization. Instead the stimulation by thrombin does not require the intermediacy of H2O2.

Ascorbic acid recycling in N-myc amplified human neuroblastoma cells.

The present study investigated the ability of two neuroblastoma cell lines (SK-N-SH, with one copy of N-myc, and SK-N-BE(2), with over 150 copies of N-myc) to recycle ascorbate by quantifying semidehydroascorbate reductase and dehydroascorbate reductase activities. Both cell lines expressed dehydroascorbate activity (SK-N-SH 28.4 +/- 9.8, SK-N-BE(2) 21.7 +/- 5.2 nmol/min/mg protein). Intracellular semidehydroascorbate activity was present only in SK-N-BE(2) cells (4.7 +/- 1.2 nmol/min/mg protein). Extracellular ascorbate was regenerated by semidehydroascorbate membrane activity, the activity of SK-N-BE(2) being twice that of SK-N-SH cells. The present data may explain the ability of the tumor to progress or regress through mechanisms involving both myc oncogene and apoptosis.

Catalytic and spectroscopic properties of cytochrome-c, horseradish peroxidase, and ascorbate oxidase embedded in a sol-gel silica matrix as a function of gelation time.

In this study, we investigated the optical features of the redox metal-dependent proteins cytochrome-c, horseradish peroxidase (HRP), and ascorbate oxidase embedded in a sol-gel-processed silica matrix as a function of gelation time. Circular dichroism, absorbance, and fluorescence spectroscopies revealed that the sol-gel process affects the complex structure of the dimeric ascorbate oxidase (although the prosthetic coppers still remain bound to the enzyme) but not that of monomeric cytochrome-c and HRP. Any modifications in ascorbate oxidase occurred in the initial gelation phase; the drying process induced no further alterations and the enzyme remained stable for months. Unfolding-refolding experiments on cytochrome-c revealed severely restricted motility in the protein moiety in the xerogel, the concentrated matrix that forms after drying. The diffusion time of the solvent within the matrix, which regulated the enzyme-substrate reaction rate, depended on the thickness of the monolith, not on the dryness of the specimen.

The interaction of the polyphenylacetylene surface with biological environments studied by XPS, RAIRS and biological tests.

A pi-conjugated polymer, polyphenylacetylene or PPA, has been tested for its possible applications as biosensor or biomaterial. Protein adsorption was investigated by incubating PPA films in solutions of bovine serum albumin (BSA) dissolved in phosphate buffer (PBS) having increasing protein concentration. Investigations on the PPA films were carried out by means of two surface analysis techniques, X-ray photoelectron spectroscopy (XPS) and reflection-absorption infrared spectroscopy (RAIRS). Desorption of BSA from the PPA surface was also investigated. Finally, the cytototoxicity of the PPA surface was checked by measuring viability and proliferation of lymphoma macrophages and SAOS osteoblasts grown in the presence of the polymer.

Full, reversible copper removal from ascorbate oxidase.

Anaerobic treatment with cyanide of reduced ascorbate oxidase causes total depletion of copper. No significant amount of the metal is reincorporated when the apo-enzyme is incubated with cupric ions, but it is upon incubation with a stoichiometric amount (eight mol per mol of native enzyme) of a Cu(I) complex stable in air [Cu(I)(thiourea)3]Cl. The yield in reconstituted protein is higher under anaerobic conditions (85-90%) than in air (70-75%). By treatment with less than stoichiometric amounts of [Cu(I)(thiourea)3]Cl the apo-protein binds copper preferentially at the blue copper site. As a consequence the recovery of enzymatic activity is percentually lower than copper reincorporation.

Dehydroascorbic acid uptake in a human keratinocyte cell line (HaCaT) is glutathione-independent.

Vitamin C plays an important role in neutralizing toxic free radicals formed during oxidative metabolism or UV exposure of human skin. This study was performed to investigate the mechanisms that regulate the homoeostasis of vitamin C in HaCaT cells by identifying the events involved in the transport and in the reduction of dehydroascorbic acid. Dehydroascorbic acid accumulated to a greater extent and faster compared with ascorbic acid; its transport appeared to be mediated by hexose transporters and was entirely distinct from ascorbic acid transport. Dehydroascorbate reductase activity was unaffected by glutathione depletion, although it was sensitive to thiol protein reagents. These observations, as well as the subcellular distribution of this enzymic activity and the cofactor specificity, indicate that thioredoxin reductase and lipoamide dehydrogenase play an important role in this reduction process. HaCaT cells were able to enhance their dehydroascorbic acid reductase activity in response to oxidative stress.

Redox buffering ability of lymphoid cells evaluated by the oxidation of 2',7'-dichlorofluorescin.

The redox buffering activity of several lymphoid cells against endogenous and exogenous H2O2 has been evaluated using 2',7'-dichlorofluorescin diacetate (DCFH2-DA). The mechanism of 2',7'-dichlorofluorescin (DCFH2) oxidation has also been investigated. It was found that while the oxidation by external H2O2 is completely inhibited by azide or cyanide, the oxidation by endogenous species is still present, even under anaerobic conditions. The data herein reported indicate that autoxidation and peroxidation of DCFH2 are distinct reactions. Hence only by addition of increasing concentrations of exogenous hydrogen peroxide, the fluorescence of DCF can be used to evaluate the cellular ability of scavenging H2O2. By this method we have found that the erythroleukaemia cell line K562 and promyelocytic line HL-60 show a faster rate of DCFH2 oxidation than peripheral blood leukocytes (PBL), mature T-cells (MOLT-3 and MOLT-4) and B-cells (DAUDI). Using this method the balance between antioxidant enzymes activity and the redox state of the cell can be easily assessed by fluorescence both in single cells and in cell populations.

The role of copper in the stability of ascorbate oxidase towards denaturing agents.

The susceptibility of native, type-2 Cu-depleted and fully Cu-depleted ascorbate oxidase to thermal and chemical denaturation has been probed by differential scanning calorimetry, fluorimetry and circular dichroism. The data indicate that copper affects the stability, but not the protein conformation. The unfolding of ascorbate oxidase is characterized by a single endotherm. Calorimetric domains revealed by deconvolution are consistent with the domains identified by X-ray crystallography.

Unmediated heterogeneous electron transfer reaction of ascorbate oxidase and laccase at a gold electrode.

The unmediated electrochemistry of two large Cu-containing proteins, ascorbate oxidase and laccase, was investigated by direct-current cyclic voltammetry. Rapid heterogeneous electron transfer was achieved in the absence of promoters or mediators by trapping a small amount of protein within a solid, electrochemically inert, tributylmethyl phosphonium chloride membrane coating a gold electrode. The problems typical of proteins in solution, such as adsorption on the electrode surface, were avoided by this procedure. In anaerobic conditions, the cyclic voltammograms, run at a scan rate of up to 200 mV/s, showed the electron transfer process to be quasi-reversible and diffusion-controlled. The pH-dependent redox potentials (+360 mV and +400 mV against a normal hydrogen electrode at pH7.0 for ascorbate oxidase and laccase respectively and +390 mV and +410 mV at pH5.5) were similar to those of the free proteins. The same electrochemical behaviour was recorded for the type 2 Cu-depleted derivatives, which contain reduced type 3 Cu, whereas the apoproteins were electrochemically inactive. Under aerobic conditions the catalytic current intensity of holoprotein voltammograms increased up to approx. 2-fold at a low scanning rate, with unchanged redox potentials. The voltammograms of type 2 Cu-depleted proteins and of apoproteins were unaffected by the presence of oxygen. This suggests that electron uptake at the electrode surface involves type 1 Cu and that only in the presence of oxygen is the intramolecular electron transfer to other protein sites rapid enough to be observed. The analogy with available kinetic results is discussed.

Reassessment of copper stoichiometry in ascorbate oxidase.

A very pure ascorbate oxidase solution was obtained by dissolving a crystalline sample of the enzyme. The ratio between 280 and 610 nm absorbancies was 22.5. It contained 8.0 +/- 0.2 Cu ions, 50% EPR detectable, per dimeric molecule (140,000 M.W.) with a molar extinction coefficient of 10,000 cm-1 at 610 nm. Two Cu ions were removed by treatment with N,N-diethyldithiocarbamate. The optical blue absorption band was unaffected, while two EPR detectable Cu ions were lost, with disappearance of the type 2 Cu signal. It is concluded that native ascorbate oxidase contains two type 1, two type 2, and four type 3 Cu ions.

Dynamic fluorescence in copper proteins. Selected examples.

The fluorescence properties of three copper proteins, namely human superoxide dismutase, Pseudomonas aeruginosa azurin and Thiobacillus versutus amicyanin have been studied. All these proteins show a non-exponential decay of fluorescence, though the tryptophanyl residues responsible for the emission are very differently located in the three proteins. All the three decays can be fitted by at least two lifetimes or better with one or two lorentzian-shaped, continuous distributions of lifetime. In each case the removal of copper affects the quantum yield of fluorescence without affecting the shape of the emission.

NADH oxidase from the extreme thermophile Thermus aquaticus YT-1. Purification and characterisation.

A protein with NADH oxidase activity from the extreme thermophile Thermus aquaticus YT-1 was purified and characterised. The enzyme was found to have a relative molecular mass of 110,000 and be composed of two subunits of identical size. FAD was found to be present at a concentration of 0.7 mol/mol dimer and was required for activity. During the oxidation of NADH, oxygen uptake takes place with the production of hydrogen peroxide. The enzyme had, with the exception of a higher glutamic acid and tryptophan content, a similar amino acid composition as the NADH oxidase isolated from the mesophile Bacillus megaterium. Purified NADH oxidase was found to have a Km of 39 microM for beta-NADH and a Vmax of 4.68 mumol NADH mg-1 min-1 and was still active at 95 degrees C. Enzymatic activity was found to be independent of pH between 5.0 and 10.5.