The activity of Pleurotus ostreatus extracts against pathogenic fusaria

A P. ostreatus strain, appreciated as food and for the production of nutraceuticals, was grown on a commercial substrate, dried at low temperature (<40\ub0C) and grinded in order to produce a mushroom powder. The bioactivity of the water extract conserved at 4\ub0C in the dark was then assessed on F. graminearum, F. culmorum and F. musae at different time points from production (4 hrs, 40 days, 4 months). Moreover, the effect of the extracts on trichothecene type B production was measured exploiting a F. graminearum isolate expressing GFP-tagged trichodiene synthase. This allowed to monitor the first step of toxin production using a microplate fluorimeter. While mycelial growth of F. graminearum and F. culmorum was completely blocked at 3 mg/ml, mycelial growth of F. musae was inhibited at 90%. MIC50 was measured for F. graminearum and F. culmorum at 300 micrograms/mL. A loss of the bioactivity of P. ostreaus water extract on fungal growth was observed at 40 days (-30%) and of a further -30% at 4 months. A preliminary study on the biological activities of the extract identified a strong protease activity associated to low molecular weight proteins. Their bioactivity decreased over storage time in accordance with a decreased proteolytic activity. The P. ostreatus extract modulates trichothecene production independently from the protease activity, even at concentration where no mycelium inhibition was observed (down to 0.75 micrograms/mL). Studies on the genetic determinants of the protease activity as well as the compounds able to modulate trichothecene production are ongoing

Extracellular vesicles released in the apoplast of germinating seeds contain almost exclusively a fungal glucanase inhibitor

Extracellular vesicles (EVs) are small, membrane-enclosed structures released from a cell into the surrounding environment. EVs play an important role in intercellular communication by transporting proteins and RNA. Plant EVs seem to mediate the transport of proteins to the extracellular compartment (apoplast) and could account for the so-called unconventional secretion pathway, where proteins lacking a signal peptide secreted in extracellular fluids Thus, EVs participate in defense responses, acting as carriers of active proteins through the apoplast (Regente et al., 2017). \u3b3-Conglutin from the leguminous plant Lupinus albus has been considered for a long time a storage protein, but recent studies definitely ruled out this function evidencing a multifaceted involvement in plant defence mechanisms triggered by pathogens attacks (Scarafoni et al. , 2010). \u3b3-Conglutin shares structural similarities with members of a large family of glucanase inhibitors widely distributed across the plant kingdom, such as the xyloglucan-specific endo-\uf062-1,4-glucanase inhibitors (XEGIPs) (Qin et al., 2003, Scarafoni et al., 2016). \u3b3-Conglutin was localized in the apoplasts of germinating seeds while the storage vacuoles appeared void of the protein. How \u3b3-conglutin reaches the extracellular regions is still matter of debate. The present work aimed to investigate if \u3b3-conglutin is secreted outside the cotyledonary cells during the very first moments of seed germination is conveyed by EVs. The results obtained by a combination of vacuum infiltration, 1D- and 2D-gel electrophoresis, ultracentrifugation fractionation and immunoblotting techniques, indicate that after 16 hours from the germination onset \u3b3-conglutin is the most represented protein in apoplast and that it is confined in vesicular material. The hypothesis that the seed-stored protein is released outside the cells through an unconventional secretory vesicles-based pathway, as it occurs for sunflower seed agglutinin 1 and other seed proteins, is suggestive and needs further focused investigations

Grape skin phenolics as inhibitors of mammalian α-glucosidase and α-amylase – effect of food matrix and processing on efficacy

Inhibition of mammalian α-amylase and α-glucosidase was studied for white grape skin samples recovered from wineries and found to be higher than that of the drug acarbose

Characterization of chenopodin isoforms from quinoa seeds and assessment of their potential anti-inflammatory activity in Caco-2 cells

Several food-derived molecules, including proteins and peptides, can show bioactivities toward the promotion of well-being and disease prevention in humans. There is still a lack of information about the potential effects on immune and inflammatory responses in mammalian cells following the ingestion of seed storage proteins. This study, for the first time, describes the potential immunomodulation capacity of chenopodin, the major protein component of quinoa seeds. After characterizing the molecular features of the purified protein, we were able to separate two different forms of chenopodin, indicated as LcC (Low charge Chenopodin, 30% of total chenopodin) and HcC (High charge Chenopodin, 70% of total chenopodin). The biological effects of LcC and HcC were investigated by measuring NF-\u3baB activation and IL-8 expression studies in undifferentiated Caco-2 cells. Inflammation was elicited using IL-1\u3b2. The results indicate that LcC and HcC show potential anti-inflammatory activities in an intestinal cell model, and that the proteins can act differently, depending on their structural features. Furthermore, the molecular mechanisms of action and the structural/functional relationships of the protein at the basis of the observed bioactivity were investigated using in silico analyses and structural predictions

Comparative 1D- and 2D-electrophoretic protein profiles of ancestral and modern buckwheat seeds grown in the italian alpine region

Buckwheat is an old crop whose seeds are under-utilized. The protein composition of these seeds, however, makes them suitable as much needed ingredients for the production of gluten-free products. Several buckwheat species and local cultivars are known worldwide. In this work, 1D and 2D electrophoresis were used to characterize and compare the seed protein profiles of two buckwheat species (Fagopyrum esculentum and Fagopyrum tataricum). The two analyzed cultivars of F. esculentum represent authentic landraces of an Italian Alpine valley, named Valtellina. The protein profiles of F. tataricum and the two F. esculentum cultivars did not show major differences. However, narrow but significant differences were present between these two landraces, allowing their discrimination at protein level. This work represents a molecular-based approach to the designation of origin and authenticity of local buckwheat varieties and their tracing in flours for human food

Cysteine-containing peptides are produced by sequential clipping, but not released, from lupin 11S storage globulin during early germination

The digestion of the seed storage proteins is a finely regulated process operated by several proteases whose action is influenced by the exposure of specific regions, which became progressively available upon their action. We focused our study on the initial stages of germination, where more subtle modifications to the storage proteins are expected. Small-size peptides containing cysteine residues and other possible metalbinding regions are de facto produced but are not released from the \u201cparental\u201d protein since they remain bound trough disulphide bridges. The meaning of these findings is discussed

Arbuscular mycorrhizal symbiosis affects the grain proteome of Zea mays: a field study

Maize is one of the most important crops worldwide and is strongly dependent on arbuscular mycorrhiza (AM) fungi, organisms that form a mutualistic association with land plants. In maize, AM symbiosis enhances spike dry weight, spike length, spike circumference, and the dry weight and dimensions of the grain. Notwithstanding its ubiquitous nature, the detailed relationship between AM fungal colonization and plant development is not completely understood. To facilitate a better understanding of the effects of AM fungi on plants, the work reported here assessed the effects of a consortium of AM fungi on the kernel proteome of maize, cultivated in open-field conditions. To our knowledge, this is the first report of the modulation of a plant seed proteome following AM fungal inoculation in the field. Here, it was found that AM fungi modify the maize seed proteome by up-regulating enzymes involved in energetic metabolism, embryo development, nucleotide metabolism, seed storage and stress responses

TgaA, a VirB1-like component belonging to a putative type IV secretion system of Bifidobacterium bifidum MIMBb75

Bifidobacterium bifidum MIMBb75 is a human intestinal isolate demonstrated to be interactive with the host and efficacious as a probiotic. However, the molecular biology of this microorganism is yet largely unknown. For this reason, we undertook wholegenome sequencing of B. bifidum MIMBb75 to identify potential genetic factors that would explain the metabolic and probiotic attributes of this bacterium. Comparative genomic analysis revealed a 45-kb chromosomal region that comprises 19 putative genes coding for a potential type IV secretion system (T4SS). Thus, we undertook the initial characterization of this genetic region by studying the putative virB1-like gene, named tgaA. Gene tgaA encodes a peptidoglycan lytic enzyme containing two active domains: lytic murein transglycosylase (LT, cd00254.3) and cysteine- and histidine-dependent amidohydrolase/peptidase (CHAP, pfam05257.4). By means of several in vitro assays, we experimentally confirmed that protein TgaA, consistent with its computationally assigned role, has peptidoglycan lytic activity, which is principally associated to the LT domain. Furthermore, immunofluorescence and immunogold labeling showed that the protein TgaA is abundantly expressed on the cell surface of B. bifidum MIMBb75. According to the literature, the T4SSs, which have not been characterized before in bifidobacteria, can have important implications for bacterial cell-to-cell communication as well as cross talk with host cells, justifying the interest for further studies aimed at the investigation of this genetic region

Murein lytic enzyme TgaA of bifidobacterium bifidum MIMBb75 modulates dendritic cell maturation through its cysteine- and histidine-dependent amidohydrolase/peptidase (CHAP) amidase domain

Bifidobacteria are Gram-positive inhabitants of the human gastrointestinal tract that have evolved close interaction with their host and especially with the host's immune system. The molecular mechanisms underlying such interactions, however, are largely unidentified. In this study, we investigated the immunomodulatory potential of Bifidobacterium bifidum MIMBb75, a bacterium of human intestinal origin commercially used as a probiotic. Particularly, we focused our attention on TgaA, a protein expressed on the outer surface of MIMBb75's cells and homologous to other known bacterial immunoactive proteins. TgaA is a peptidoglycan lytic enzyme containing two active domains: lytic murein transglycosylase (LT) and cysteine- and histidine-dependent amidohydrolase/peptidase (CHAP). We ran immunological experiments stimulating dendritic cells (DCs) with the B. bifidum MIMBb75 and TgaA, with the result that both the bacterium and the protein activated DCs and triggered interleukin-2 (IL-2) production. In addition, we observed that the heterologous expression of TgaA in Bifidobacterium longum transferred to the bacterium the ability to induce IL-2. Subsequently, immunological experiments performed using two purified recombinant proteins corresponding to the single domains LT and CHAP demonstrated that the CHAP domain is the immune-reactive region of TgaA. Finally, we also showed that TgaA-dependent activation of DCs requires the protein CD14, marginally involves TRIF, and is independent of Toll-like receptor 4 (TLR4) and MyD88. In conclusion, our study suggests that the bacterial CHAP domain is a novel microbe-associated molecular pattern actively participating in the cross talk mechanisms between bifidobacteria and the host's immune system

Analysis of conglutin seed storage proteins across lupin species using transcriptomic, protein and comparative genomic approaches

Background - The major proteins in lupin seeds are conglutins that have primary roles in supplying carbon, sulphur and nitrogen and energy for the germinating seedling. They fall into four families; α, β, γ and δ. Interest in these conglutins is growing as family members have been shown to have beneficial nutritional and pharmaceutical properties. Results - An in-depth transcriptome and draft genome from the narrow-leafed lupin (NLL; Lupinus angustifolius) variety, Tanjil, were examined and 16 conglutin genes were identified. Using RNAseq data sets, the structure and expression of these 16 conglutin genes were analysed across eight lupin varieties from five lupin species. Phylogenic analysis suggest that the α and γ conglutins diverged prior to lupin speciation while β and δ members diverged both prior and after speciation. A comparison of the expression of the 16 conglutin genes was performed, and in general the conglutin genes showed similar levels of RNA expression among varieties within species, but quite distinct expression patterns between lupin species. Antibodies were generated against the specific conglutin families and immunoblot analyses were used to compare the levels of conglutin proteins in various tissues and during different stages of seed development in NLL, Tanjil, confirming the expression in the seed. This analysis showed that the conglutins were expressed highly at the mature seed stage, in all lupin species, and a range of polypeptide sizes were observed for each conglutin family. Conclusions - This study has provided substantial information on the complexity of the four conglutin families in a range of lupin species in terms of their gene structure, phylogenetic relationships as well as their relative RNA and protein abundance during seed development. The results demonstrate that the majority of the heterogeneity of conglutin polypeptides is likely to arise from post-translational modification from a limited number of precursor polypeptides rather than a large number of different genes. Overall, the results demonstrate a high degree of plasticity for conglutin expression during seed development in different lupin species