Functional Association between Storage Protein Mobilization and Redox Signaling in Narrow-Leafed Lupin (Lupinus angustifolius L.) Seed Germination and Seedling Development.

(1) Background: Seed storage mobilization, together with oxidative metabolism, with the ascorbate-glutathione (AsA-GSH) cycle as a crucial signaling and metabolic functional crossroad, is one of the main regulators of the control of cell morphogenesis and division, a fundamental physiological process driving seed germination and seedling growth. This study aims to characterize the cellular changes, composition, and patterns of the protein mobilization and ROS-dependent gene expression of redox metabolism in Lupinus angustifolius L. (narrow-leafed lupin, NLL) cotyledons during seed germination. (2) Methods: We performed gene expression analyses via RT-qPCR for conglutins α (1, 2, and 3), ß (1, 2, and 5), γ (1, 2), and δ (2 and 4), including a ubiquitin gene as a control, and for redox metabolism-related genes; GADPH was used as a control gene. A microscopic study was developed on cotyledon samples from different germination stages, including as IMB (imbibition), and 2-5, 7, 9, and 11 DAI (days after imbibition), which were processed for light microscopy. SDS-PAGE and immunocytochemistry assays were performed using an anti-ß-conglutin antibody (Agrisera), and an anti-rabbit IgG Daylight 488-conjugated secondary antibody. The controls were made while omitting primary Ab. (3) Results and Discussion: Our results showed that a large amount of seed storage protein (SSP) accumulates in protein bodies (PBs) and mobilizes during germination. Families of conglutins (ß and γ) may play important roles as functional and signaling molecules, beyond the storage function, at intermediate steps of the seed germination process. In this regard, metabolic activities are closely associated with the regulation of oxidative homeostasis through AsA-GSH activities (γ-L-Glutamyl-L-cysteine synthetase, NOS, Catalase, Cu/Zn-SOD, GPx, GR, GS, GsT) after the imbibition of NLL mature seeds, metabolism activation, and dormancy breakage, which are key molecular and regulatory signaling pathways with particular importance in morphogenesis and developmental processes. (4) Conclusions: The knowledge generated in this study provides evidence for the functional changes and cellular tightly regulated events occurring in the NLL seed cotyledon, orchestrated by the oxidative-related metabolic machinery involved in seed germination advancement.

A comprehensive dataset of the extra virgin olive oil (EVOO) proteome.

Proteins and peptides are minor components of vegetal oils. The presence of these compounds in virgin olive oil was first reported in 2001, but the nature of the olive oil proteome is still a puzzling question for food science researchers. In this paper, we have compiled for a first time a comprehensive proteomic dataset of olive fruit and fungal proteins that are present at low but measurable concentrations in a vegetable oil from a crop of great agronomical relevance as olive (Olea europaea L.). Accurate mass nLC-MS data were collected in high definition direct data analysis (HD-DDA) mode using the ion mobility separation step. Protein identification was performed using the Mascot Server v2.2.07 software (Matrix Science) against an ad hoc database made of olive protein entries. Starting from this proteomic record, the impact of these proteins on olive oil stability and quality could be tested. Moreover, the effect of olive oil proteins on human health and their potential use as functional food components could be also evaluated. In addition, this dataset provides a resource for use in further functional comparisons across other vegetable oils, and also expands the proteomic resources to non-model species, thus also allowing further comparative inter-species studies. The data presented here are related to the research article of Castro et al. [1].

Identification of seed storage proteins as the major constituents of the extra virgin olive oil proteome.

Proteins are minor components of extra virgin olive oil (EVOO), but the nature of the olive oil proteome is still elusive. In this paper, we have uncovered the EVOO proteome for the first time. Seed storage proteins of globulin-type were identified as the most abundant proteins in EVOO, which also contains an active 13-lipoxygenase and several potential allergenic proteins, including the "panallergen" profilin. We validated our proteomic data by Western blotting and enzyme activity assays. Our data also demonstrated that the seed is the main source of proteins in EVOO, while the contribution of the pulp is uncertain and needs further verification. The impact of EVOO proteins on its stability and quality, and on human health is discussed.

Effects of Virgin Olive Oils Differing in Their Bioactive Compound Contents on Metabolic Syndrome and Endothelial Functional Risk Biomarkers in Healthy Adults: A Randomized Double-Blind Controlled Trial.

The aim of this study was to evaluate the effect of virgin olive oils (VOOs) enriched with phenolic compounds and triterpenes on metabolic syndrome and endothelial function biomarkers in healthy adults. The trial was a three-week randomized, crossover, controlled, double-blind, intervention study involving 58 subjects supplemented with a daily dose (30 mL) of three oils: (1) a VOO (124 ppm of phenolic compounds and 86 ppm of triterpenes); (2) an optimized VOO (OVOO) (490 ppm of phenolic compounds and 86 ppm of triterpenes); and (3) a functional olive oil (FOO) high in phenolic compounds (487 ppm) and enriched with triterpenes (389 ppm). Metabolic syndrome and endothelial function biomarkers were determined in vivo and ex vivo. Plasma high density lipoprotein cholesterol (HDLc) increased after the OVOO intake. Plasma endothelin-1 levels decreased after the intake of the three olive oils, and in blood cell cultures challenged. Daily intake of VOO enriched in phenolic compounds improved plasma HDLc, although no differences were found at the end of the three interventions, while VOO with at least 124 ppm of phenolic compounds, regardless of the triterpenes content improved the systemic endothelin-1 levels in vivo and ex vivo. No effect of triterpenes was observed after three weeks of interventions. Results need to be confirmed in subjects with metabolic syndrome and impaired endothelial function (Clinical Trials number NCT02520739).

Characterization of narrow-leaf lupin (Lupinus angustifolius L.) recombinant major allergen IgE-binding proteins and the natural ß-conglutin counterparts in sweet lupin seed species.

ß-conglutin has been identified as a major allergen for Lupinus angustifolius seeds. The aim of this study was to evaluate the binding of IgE to five recombinant ß-conglutin isoforms (rß) that we overexpressed and purified and to their natural counterparts in different lupin species and cultivars. Western blotting suggested ß-conglutins were the main proteins responsible for the IgE reactivity of the lupin species and cultivars. Newly identified polypeptides from "sweet lupin" may constitute a potential new source of primary or cross-reactive sensitization to lupin, particularly to L. albus and L. angustifolius seed proteins. Several of them exhibited qualitative and quantitative differences in IgE-binding among these species and cultivars, mainly in sera from atopic patients that react to lupin rather than peanut. IgE-binding was more consistent to recombinant ß2 than to any of the other isoforms, making this protein a potential candidate for diagnosis and immunotherapy.

S-nitroso- and nitro- proteomes in the olive (Olea europaea L.) pollen. Predictive versus experimental data by nano-LC-MS.

The data presented here are related to the research article entitled "Generation of nitric oxide by olive (Olea europaea L.) pollen during in vitro germination and assessment of the S-nitroso- and nitro-proteomes by computational predictive methods" doi:10.1016/j.niox.2017.06.005 (Jimenez-Quesada et al., 2017) [1]. Predicted cysteine S-nitrosylation and Tyr-nitration sites in proteins derived from a de novo assembled and annotated pollen transcriptome from olive tree (Olea europaea L.) were obtained after using well-established predictive tools in silico. Predictions were performed using both default and highly restrictive thresholds. Numerous gene products identified with these characteristics are listed here. An experimental validation of the data, consisting in nano-LC-MS (Liquid Chromatography-Mass Spectrometry) determination of olive pollen proteins after immunoprecipitation with antibodies to anti-S-nitrosoCys and anti-3-NT (NitroTyrosine) allowed identification of numerous proteins subjected to these two post-translational modifications, which are listed here together with information regarding their cross-presence among the predictions.

An Abnormal Nitric Oxide Metabolism Contributes to Brain Oxidative Stress in the Mouse Model for the Fragile X Syndrome, a Possible Role in Intellectual Disability.

BACKGROUND: Fragile X syndrome is the most common genetic cause of mental disability. Although many research has been performed, the mechanism underlying the pathogenesis is unclear and needs further investigation. Oxidative stress played major roles in the syndrome. The aim was to investigate the nitric oxide metabolism, protein nitration level, the expression of NOS isoforms, and furthermore the activation of the nuclear factor NF-κB-p65 subunit in different brain areas on the fragile X mouse model. METHODS: This study involved adult male Fmr1-knockout and wild-type mice as controls. We detected nitric oxide metabolism and the activation of the nuclear factor NF-κBp65 subunit, comparing the mRNA expression and protein content of the three NOS isoforms in different brain areas. RESULTS: Fmr1-KO mice showed an abnormal nitric oxide metabolism and increased levels of protein tyrosine nitrosylation. Besides that, nuclear factor NF-κB-p65 and inducible nitric oxide synthase appeared significantly increased in the Fmr1-knockout mice. mRNA and protein levels of the neuronal nitric oxide synthase appeared significantly decreased in the knockout mice. However, the epithelial nitric oxide synthase isoform displayed no significant changes. CONCLUSIONS: These data suggest the potential involvement of an abnormal nitric oxide metabolism in the pathogenesis of the fragile X syndrome.

Hypoxia-inducible factor-1 modulates the expression of vascular endothelial growth factor and endothelial nitric oxide synthase induced by eccentric exercise.

The present study investigated the effects of acute and chronic eccentric exercise on the hypoxia-inducible factor (HIF)-1α activation response and the concomitant modulation of vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) expression in rat skeletal muscle. Twenty-four male Wistar rats were randomly assigned to three experimental groups: rested control group, acutely exercised group after an intermittent downhill protocol for 90 min, and acutely exercise group with a previous eccentric training of 8 wk. HIF-1α activation, VEGF and eNOS gene expression, protein content, and promoter activation were assessed in vastus lateralis muscle biopsies. Acute eccentric exercise induced a marked activation of HIF-1α and resulted in increased VEGF and eNOS mRNA level and protein concentration. The binding of HIF-1α to the VEGF and eNOS promoters, measured by a chromatin immunoprecipitation assay, was undetectable in rested rats, whereas it was evident in acutely exercised animals. Acute exercise also increased myeloperoxidase, toll-like receptor-4, tumor necrosis factor-α, and interleukin-1ß protein content, suggesting a contribution of proinflammatory stimuli to HIF-1α activation and VEGF overexpression. All of these effects were partially abolished by training. Moreover, training resulted in an increased capillary density. In summary, our findings indicate that eccentric exercise prompts an HIF-1α response in untrained skeletal muscle that contributes to the upregulation of VEGF and eNOS gene expression and is attenuated after an eccentric training program.

Liver X receptor α-mediated regulation of lipogenesis by core and NS5A proteins contributes to HCV-induced liver steatosis and HCV replication.

Molecular mechanisms contributing to hepatitis C virus (HCV)-associated steatosis are not well established, although HCV gene expression has been shown to alter host cell cholesterol/lipid metabolism. As liver X receptors (LXRs) play a role as key modulators of metabolism signaling in the development of steatosis, we aimed to investigate in an HCV in vitro model the effect of HCV NS5A protein, core protein, and viral replication on the intracellular lipid accumulation and the LXRα-regulated expression of lipogenic genes. The effects of LXRα siRNA or agonist GW3965 treatment on lipogenesis and HCV replication capacity in our HCV replicon system were also examined. NS5A- and core-expressing cells and replicon-containing cells exhibited an increase of lipid accumulation by inducing the gene expression and the transcriptional activity of LXRα, and leading to an increased expression of its lipogenic target genes sterol regulatory element binding protein-1c, peroxisome proliferator-activated receptor-γ, and fatty acid synthase. Transcriptional induction by NS5A protein, core protein, and viral replication occurred via LXR response element activation in the lipogenic gene promoter. No physical association between HCV proteins and LXRα was observed, whereas NS5A and core proteins indirectly upregulated LXRα through the phosphatidylinositol 3-kinase pathway. Finally, it was found that LXRα knockdown or agonist-mediated LXRα induction directly regulated HCV-induced lipogenesis and HCV replication efficiency in replicon-containing cells. Combined, our data suggest that LXRα-mediated regulation of lipogenesis by core and NS5A proteins may contribute to HCV-induced liver steatosis and to the efficient replication of HCV.

Hepatic fatty acid translocase CD36 upregulation is associated with insulin resistance, hyperinsulinaemia and increased steatosis in non-alcoholic steatohepatitis and chronic hepatitis C.

BACKGROUND: Fatty acid translocase CD36 (FAT/CD36) mediates uptake and intracellular transport of long-chain fatty acids in diverse cell types. While the pathogenic role of FAT/CD36 in hepatic steatosis in rodents is well-defined, little is known about its significance in human liver diseases. OBJECTIVE: To examine the expression of FAT/CD36 and its cellular and subcellular distribution within the liver of patients with non-alcoholic fatty liver disease (NAFLD) and chronic hepatitis C virus (HCV) infection. PATIENTS: 34 patients with non-alcoholic steatosis (NAS), 30 with non-alcoholic steatohepatitis (NASH), 66 with HCV genotype 1 (HCV G1) and 32 with non-diseased liver (NL). METHODS: Real-time PCR and western blot analysis were used to assess hepatic FAT/CD36 expression. Computational image analysis of immunostained liver biopsy sections was performed to determine subcellular distribution and FAT/CD36 expression index. RESULTS: Compared with NL, hepatic mRNA and protein levels of FAT/CD36 were significantly higher in patients with NAS (median fold increase 0.84 (range 0.15-1.61) and 0.66 (range 0.33-1.06), respectively); NASH (0.91 (0.22-1.81) and 0.81 (0.38-0.92), respectively); HCV G1 without steatosis (0.30 (0.17-1.59) and 0.33 (0.29-0.52), respectively); and HCV G1 with steatosis (0.85 (0.15-1.98) and 0.87 (0.52-1.26), respectively). In contrast to NL, FAT/CD36 was predominantly located at the plasma membrane of hepatocytes in patients with NAFLD and HCV G1 with steatosis. A significant correlation was observed between hepatic FAT/CD36 expression index and plasma insulin levels, insulin resistance (HOMA-IR) and histological grade of steatosis in patients with NASH (r=0.663, r=0.735 and r=0.711, respectively) and those with HCV G1 with steatosis (r=0.723, r=0.769 and r=0.648, respectively). CONCLUSIONS: Hepatic FAT/CD36 upregulation is significantly associated with insulin resistance, hyperinsulinaemia and increased steatosis in patients with NASH and HCV G1 with fatty liver. Translocation of this fatty acid transporter to the plasma membrane of hepatocytes may contribute to liver fat accumulation in patients with NAFLD and HCV.

Enhanced expression of pro-inflammatory mediators and liver X-receptor-regulated lipogenic genes in non-alcoholic fatty liver disease and hepatitis C.

NAFLD (non-alcoholic fatty liver disease) is one of the most frequent chronic liver diseases worldwide. The metabolic factors associated with NAFLD are also determinants of liver disease progression in chronic HCV (hepatitis C virus) infection. It has been reported that, besides inducing hepatic fatty acid biosynthesis, LXR (liver X receptor) regulates a set of inflammatory genes. We aimed to evaluate the hepatic expression of LXRα and its lipogenic and inflammatory targets in 43 patients with NAFLD, 44 with chronic HCV infection and in 22 with histologically normal liver. Real-time PCR and Western blot analysis were used to determine hepatic expression levels of LXRα and related lipogenic and inflammatory mediators in the study population. We found that the LXRα gene and its lipogenic targets PPAR-γ (peroxisome-proliferator-activated receptor-γ), SREBP (sterol-regulatory-element-binding protein)-1c, SREBP-2 and FAS (fatty acid synthase) were overexpressed in the liver of NAFLD and HCV patients who had steatosis. Moreover, up-regulation of inflammatory genes, such as TNF (tumour necrosis factor)-α, IL (interleukin)-6, OPN (osteopontin), iNOS (inducible NO synthase), COX (cyclo-oxygenase)-2 and SOCS (suppressors of cytokine signalling)-3, was observed in NAFLD and HCV patients. Interestingly, TNF-α, IL-6 and osteopontin gene expression was lower in patients with steatohepatitis than in those with steatosis. In conclusion, hepatic expression of LXRα and its related lipogenic and inflammatory genes is abnormally increased in NAFLD and HCV patients with steatosis, suggesting a potential role of LXRα in the pathogenesis of hepatic steatosis in these chronic liver diseases.