The Effect of Sulforaphane on Glyoxalase I Expression and Activity in Peripheral Blood Mononuclear Cells.

Studies demonstrate that the potential health-beneficial effect of sulforaphane (SR), a compound formed in broccoli, is the result of a number of mechanisms including upregulation of phase two detoxification enzymes. Recent studies suggest that SR increases expression/activity of glyoxalase 1 (Glo1), an enzyme involved in the degradation of methylglyoxal, is major precursor of advanced glycation end products. Those compounds are associated with diabetes complications and other age-related diseases. In this study, the effect of SR on the expression/activity of Glo1 in peripheral blood mononuclear cells (PBMCs) from 8 healthy volunteers was investigated. PBMCs were isolated and incubated with SR (2.5 µM-concentration achievable by consuming a broccoli portion) for 24 h and 48 h. Glo1 activity/expression, reduced glutathione (GSH), and glutathione-S-transferase gene expression were measured. Glo1 activity was not affected while after 48 h a slight but significant increase of its gene expression (1.03-fold) was observed. GSTP1 expression slightly increased after 24 h incubation (1.08-fold) while the expressions of isoform GSTT2 and GSTM2 were below the limit of detection. GSH sharply decreased, suggesting the formation of GSH-SR adducts that may have an impact SR availability. Those results suggest that a regular exposure to SR by broccoli consumption or SR supplements may enhance Glo1.

Measuring Activity of Native Plant Sirtuins - The Wheat Mitochondrial Model.

Sirtuins are NAD+-dependent deacetylase enzymes that have gained considerable interest in mammals for their recognized importance in gene silencing and expression and in cell metabolism. Conversely, knowledge about plant sirtuins remains limited, although a sirtuin-mediated regulation of mitochondrial energy metabolism has been recently reported in Arabidopsis. However, so far, no information is available about direct measurement of intracellular plant sirtuin activity, i.e., in cell extracts and/or subcellular organelles. In this study, a novel approach was proposed for reliable evaluation of native sirtuin activity in plant samples, based on (i) an adequate combinatory application of enzymatic assays very different for chemical basis and rationale and (ii) a comparative measurement of activity of a recombinant sirtuin isoform. In particular, two sirtuin assays were applied, based on bioluminescence emission and Homogeneous Time-Resolved Fluorescence (HTRF®) technology, and the human SIRT1 isoform (hSIRT1) was used for comparison. For the first time in plants, this new approach allowed measuring directly a high and nicotinamide-sensitive sirtuin activity in highly purified mitochondrial fraction obtained from durum wheat (WM). WM-sirtuin activity was 268 ± 10 mU⋅mg-1 protein, as measured by HTRF® assay, and 166 ± 12 ng hSIRT1 eq.⋅mg-1 protein, as evaluated by the bioluminescent assay and calculated on the basis of the hSIRT1 calibration curve. Moreover, effects of resveratrol and quercetin, reported as potent hSIRT1 activators, but whose activation mechanism is still debated, were also studied. No effect of resveratrol was found on both WM-sirtuin and hSIRT1 activities, while only a slight increase, up to about 20%, of hSIRT1 activity by quercetin was observed. In the whole, results of this study indicate that WM may represent a good system for studying native plant sirtuins. In fact, the high yield of purified WM and their high sirtuin activity, together with use of microplate readers, allow performing a large number of measurements from the same preparation, so qualifying the approach for application to large-scale high-throughput screening. Moreover, WM may also represent an excellent tool to investigate physiological role and modulation of plant sirtuins under experimental conditions more physiologically relevant with respect to recombinant purified enzymes.

Different effectiveness of two pastas supplemented with either lipophilic or hydrophilic/phenolic antioxidants in affecting serum as evaluated by the novel Antioxidant/Oxidant Balance approach.

Effectiveness in improving serum antioxidant status of two functional pastas was evaluated by the novel Antioxidant/Oxidant Balance (AOB) parameter, calculated as Antioxidant Capacity (AC)/Peroxide Level ratio, assessed here for the first time. In particular, Bran Oleoresin (BO) and Bran Water (BW) pastas, enriched respectively with either lipophilic (tocochromanols, carotenoids) or hydrophilic/phenolic antioxidants extracted from durum wheat bran, were studied. Notably, BO pasta was able to improve significantly (+65%) serum AOB during four hours after intake similarly to Lisosan G, a wheat antioxidant-rich dietary supplement. Contrarily, BW pasta had oxidative effect on serum so as conventional pasta and glucose, thus suggesting greater effectiveness of lipophilic than hydrophilic/phenolic antioxidants under our experimental conditions. Interestingly, no clear differences between the two pastas were observed, when AC measurements of either serum after pasta intake or pasta extracts by in vitro assays were considered, thus strengthening effectiveness and reliability of AOB approach.

Serum antioxidant capacity and peroxide level of seven healthy subjects after consumption of different foods.

This article reports experimental data related to the research article entitled "Different effectiveness of two pastas supplemented with either lipophilic or hydrophilic/phenolic antioxidants in affecting serum as evaluated by the novel Antioxidant/Oxidant Balance approach" (M.N. Laus, M. Soccio, M. Alfarano, A. Pasqualone, M.S. Lenucci, G. Di Miceli, D. Pastore, 2016) [1]. Antioxidant status of blood serum of seven healthy subjects was evaluated during four hours after consumption of two functional pastas, supplemented with either bran oleoresin or bran water extract obtained from durum wheat. For comparison, the effect of a non-supplemented reference pasta was also evaluated, as well as the effects of glucose, of the wheat grain dietary supplement Lisosan G, and of the reference pasta consumed together with Lisosan G. Serum antioxidant status was evaluated by measuring both the serum antioxidant capacity, using LOX-FL, ORAC and TEAC methods, and the serum oxidant status, assessed as peroxide level.

Modulation of Potassium Channel Activity in the Balance of ROS and ATP Production by Durum Wheat Mitochondria-An Amazing Defense Tool Against Hyperosmotic Stress.

In plants, the existence of a mitochondrial potassium channel was firstly demonstrated about 15 years ago in durum wheat as an ATP-dependent potassium channel (PmitoKATP). Since then, both properties of the original PmitoKATP and occurrence of different mitochondrial potassium channels in a number of plant species (monocotyledonous and dicotyledonous) and tissues/organs (etiolated and green) have been shown. Here, an overview of the current knowledge is reported; in particular, the issue of PmitoKATP physiological modulation is addressed. Similarities and differences with other potassium channels, as well as possible cross-regulation with other mitochondrial proteins (Plant Uncoupling Protein, Alternative Oxidase, Plant Inner Membrane Anion Channel) are also described. PmitoKATP is inhibited by ATP and activated by superoxide anion, as well as by free fatty acids (FFAs) and acyl-CoAs. Interestingly, channel activation increases electrophoretic potassium uptake across the inner membrane toward the matrix, so collapsing membrane potential (ΔΨ), the main component of the protonmotive force (Δp) in plant mitochondria; moreover, cooperation between PmitoKATP and the K(+)/H(+) antiporter allows a potassium cycle able to dissipate also ΔpH. Interestingly, ΔΨ collapse matches with an active control of mitochondrial reactive oxygen species (ROS) production. Fully open channel is able to lower superoxide anion up to 35-fold compared to a condition of ATP-inhibited channel. On the other hand, ΔΨ collapse by PmitoKATP was unexpectedly found to not affect ATP synthesis via oxidative phosphorylation. This may probably occur by means of a controlled collapse due to ATP inhibition of PmitoKATP; this brake to the channel activity may allow a loss of the bulk phase Δp, but may preserve a non-classically detectable localized driving force for ATP synthesis. This ability may become crucial under environmental/oxidative stress. In particular, under moderate hyperosmotic stress (mannitol or NaCl), PmitoKATP was found to be activated by ROS, so inhibiting further large-scale ROS production according to a feedback mechanism; moreover, a stress-activated phospholipase A2 may generate FFAs, further activating the channel. In conclusion, a main property of PmitoKATP is the ability to keep in balance the control of harmful ROS with the mitochondrial/cellular bioenergetics, thus preserving ATP for energetic needs of cell defense under stress.

Two dimensional gel phosphoproteome of peripheral blood mononuclear cells: comparison between two enrichment methods.

BACKGROUND: Protein phosphorylation is considered a key event in signal transduction. Peripheral blood mononuclear cells (PBMCs) are a critical component of the immune system. The analysis of PBMCs phosphoproteome might help elucidate the signaling pathways essential to their biological role in health, immunological diseases and cancer. Enrichment of phosphoproteins becomes a prerequisite for phosphoproteome analysis and conventionally requires a multi-step procedure and sophisticated equipments. In this study, we standardized 2D-PAGE phosphoproteome analysis of PBMCs and compared two phosphoprotein enrichment methods, lanthanum chloride precipitation and affinity micro-column. Further, the different specificity for PBMCs phosphorylated proteins of each method was investigated. RESULTS: PBMCs were isolated from fresh whole blood of ten healthy donors. PBMCs phosphoproteins were enriched either by phosphoprotein precipitation using lanthanum chloride, with an overall yield of 8.9 ± 4.7%, or by using an affinity micro-column, with a lower yield of 3.2 ± 1.6% (p = 0.05). Image analysis of Sypro-stained analytical 2D-PAGE gels detected 554 ± 68 protein spots for the lanthanum pattern [inter-assay coefficient of variation (CV) = 27.0%, intra-assay CV = 10.7%] and 575 ± 35 protein spots for the micro-column pattern (inter-assay CV = 26.8%; intra-assay CV = 11.0%) (p = 0.6), with 57% match of the spots detected by the 2 approaches. 1D gel electrophoresis and western blot analyses of the supernatants suggested a better lanthanum ions capability to deplete phosphoproteins in a PBMCs protein lysate compared to the affinity micro-column. On the other hand, 1D gel electrophoresis analysis of dephosphorylated PBMCs protein lysate revealed a relatively higher unspecificity for the lanthanum ions compared to affinity micro-column. Filamin-A, coronin 1A, pyruvate kinase isozymes M1/M2 and ficolin-1 were considerably more expressed in the lanthanum phosphoprotein pattern. Interestingly, ficolin-1 had not been reported in 2DE-PBMCs proteome profiles so far. CONCLUSION: Our results describe the differences and the validity of phosphoprotein enrichment methods and provide two successful and complementary approaches for the 2DE phosphoproteome analysis of PBMCs.

Identification and functional characterization of three NoLS (nucleolar localisation signals) mutations of the CDC73 gene.

Hyperparathyroidism Jaw-Tumour Syndrome (HPT-JT) is characterized by primary hyperparathyroidism (PHPT), maxillary/mandible ossifying fibromas and by parathyroid carcinoma in 15% of cases. Inactivating mutations of the tumour suppressor CDC73/HRPT2 gene have been found in HPT-JT patients and also as genetic determinants of sporadic parathyroid carcinoma/atypical adenomas and, rarely, typical adenomas, in familial PHPT. Here we report the genetic and molecular analysis of the CDC73/HRPT2 gene in three patients affected by PHPT due to atypical and typical parathyroid adenomas, in one case belonging to familial PHPT. Flag-tagged WT and mutant CDC73/HRPT2 proteins were transiently transfected in HEK293 cells and functional assays were performed in order to investigate the effect of the variants on the whole protein expression, nuclear localization and cell overgrowth induction. We identified four CDC73/HRPT2 gene mutations, three germline (c.679_680delAG, p.Val85_Val86del and p.Glu81_Pro84del), one somatic (p.Arg77Pro). In three cases the mutation was located within the Nucleolar Localisation Signals (NoLS). The three NoLS variants led to instability either of the corresponding mutated protein or mRNA or both. When transfected in HEK293 cells, NoLS mutated proteins mislocalized with a predeliction for cytoplasmic or nucleo-cytoplasmic localization and, finally, they resulted in overgrowth, consistent with a dominant negative interfering effect in the presence of the endogenous protein.