Crystal Structure of Agaricus bisporus Mushroom Tyrosinase: Identity of the Tetramer Subunits and Interaction with Tropolone

Tyrosinase catalyzes the conversion of phenolic compounds into their quinone derivatives, which are precursors for the formation of melanin, a ubiquitous pigment in living organisms. Because of its importance for browning reactions in the food industry, the tyrosinase from the mushroom Agaricus bisporus has been investigated in depth. In previous studies the tyrosinase enzyme complex was shown to be a H2L2 tetramer, but no clues were obtained of the identities of the subunits, their mode of association, and the 3D structure of the complex. Here we unravel this tetramer at the molecular level. Its 2.3 Å resolution crystal structure is the first structure of the full fungal tyrosinase complex. The complex comprises two H subunits of ~392 residues and two L subunits of ~150 residues. The H subunit originates from the ppo3 gene and has a fold similar to other tyrosinases, but it is ~100 residues larger. The L subunit appeared to be the product of orf239342 and has a lectin-like fold. The H subunit contains a binuclear copper-binding site in the deoxy-state, in which three histidine residues coordinate each copper ion. The side chains of these histidines have their orientation fixed by hydrogen bonds or, in the case of His85, by a thioether bridge with the side chain of Cys83. The specific tyrosinase inhibitor tropolone forms a pre-Michaelis complex with the enzyme. It binds near the binuclear copper site without directly coordinating the copper ions. The function of the ORF239342 subunits is not known. Carbohydrate binding sites identified in other lectins are not conserved in ORF239342, and the subunits are over 25 Å away from the active site, making a role in activity unlikely. The structures explain how calcium ions stabilize the tetrameric state of the enzyme.

Combined dietary supplementation of long chain inulin and Lactobacillus acidophilus W37 supports oral vaccination efficacy against Salmonella Typhimurium in piglets

Routine use of antibiotics in livestock animals strongly contributed to the creation of multidrug-resistant Salmonella Typhimurium strains (STM). Vaccination is an alternative to the use of antibiotics but often suffers from low efficacy. The present study investigated whether long-chain inulin (lcITF) and Lactobacillus acidophilus W37 (LaW37) can support vaccination efficacy against STM and if the interventions influence possible gut microbiota changes. Piglets received daily supplementation until sacrifice. Animals were vaccinated on day 25 after birth, one day after weaning, and were challenged with STM on days 52-54. Dietary intervention with lcITF/LaW37 enhanced vaccination efficacy by 2-fold during challenge and resulted in higher relative abundance of Prevotellaceae and lower relative abundance of Lactobacillaceae in faeces. Although strongest microbial effects were observed post STM challenge on day 55, transient effects of the lcITF/LaW37 intervention were also detected on day 10 after birth, and post-weaning on day 30 where increased relative abundance of faecal lactobacilli was correlated with higher faecal consistency. LcITF treatment increased post-weaning feed efficiency and faecal consistency but did not support vaccination efficacy. Vaccination in immune-immature young animals can be enhanced with functional additives which can simultaneously promote health in an ingredient-dependent fashion

Brush border enzyme hydrolysis and glycaemic effects of isomaltulose compared to other saccharides in dogs

Digestible carbohydrates differ in glycaemic response, therewith having the potential to influence metabolic conditions such as insulin resistance and diabetes mellitus. Isomaltulose has been proven to lower the glycaemic response in humans, which to date has not been studied in dogs. Therefore, the aim of the present study was to characterise the digestibility, as well as the physiological effects of isomaltulose in dogs, in comparison to other saccharides. To this end, three studies were performed. Study 1 was an in vitro study, evaluating the small intestinal hydrolysis of isomaltulose compared to other relevant carbohydrate sources. Three of these saccharides, having close and low-moderate degrees of hydrolysis by brush border enzymes, were also evaluated in vivo for their glycaemic effects by measuring plasma levels of glucose, insulin and glucagon-like peptide 1 (GLP-1) 0-180 min after administration of a single dosage after an overnight fast (i.e., isomaltulose, sucrose and maltodextrin in a 3 × 3 Latin-square design, in 9 dogs, Study 2). To understand if digestive enzymes, underlying glycaemic responses for isomaltulose and sucrose can be upregulated, we exposed dogs to these saccharides for 2 weeks and repeated the measurements after an overnight fast in 18 dogs (Study 3). Isomaltulose was hydrolysed by intestinal enzyme preparation from all three dogs, but the degrading activity was low (e.g., 3.95 ± 1.03 times lower vs. sucrose), indicating a slower rate of hydrolysis. Isomaltulose had a low glycaemic response, in line with in vitro data. In vitro hydrolysis of sucrose was comparable or even higher than maltodextrin in contrast to the more pronounced glycaemic response to maltodextrin observed in vivo. The numerically higher blood glucose response to sucrose after continuous consumption, might indicate an adaptive response. In conclusion, the current work provides valuable insights into the digestion physiology of various saccharides in dogs. Further investigations on related benefits are thus warranted

Effects of Digested Onion Extracts on Intestinal Gene Expression: An Interspecies Comparison Using Different Intestine Models.

Human intestinal tissue samples are barely accessible to study potential health benefits of nutritional compounds. Numbers of animals used in animal trials, however, need to be minimalized. Therefore, we explored the applicability of in vitro (human Caco-2 cells) and ex vivo intestine models (rat precision cut intestine slices and the pig in-situ small intestinal segment perfusion (SISP) technique) to study the effect of food compounds. In vitro digested yellow (YOd) and white onion extracts (WOd) were used as model food compounds and transcriptomics was applied to obtain more insight into which extent mode of actions depend on the model. The three intestine models shared 9,140 genes which were used to compare the responses to digested onions between the models. Unsupervised clustering analysis showed that genes up- or down-regulated by WOd in human Caco-2 cells and rat intestine slices were similarly regulated by YOd, indicating comparable modes of action for the two onion species. Highly variable responses to onion were found in the pig SISP model. By focussing only on genes with significant differential expression, in combination with a fold change > 1.5, 15 genes showed similar onion-induced expression in human Caco-2 cells and rat intestine slices and 2 overlapping genes were found between the human Caco-2 and pig SISP model. Pathway analyses revealed that mainly processes related to oxidative stress, and especially the Keap1-Nrf2 pathway, were affected by onions in all three models. Our data fit with previous in vivo studies showing that the beneficial effects of onions are mostly linked to their antioxidant properties. Taken together, our data indicate that each of the in vitro and ex vivo intestine models used in this study, taking into account their limitations, can be used to determine modes of action of nutritional compounds and can thereby reduce the number of animals used in conventional nutritional intervention studies

Identification and in silico bioinformatics analysis of PR10 proteins in cashew nut

Proteins from cashew nut can elicit mild to severe allergic reactions. Three allergenic proteins have already been identified, and it is expected that additional allergens are present in cashew nut. pathogenesis-related protein 10 (PR10) allergens from pollen have been found to elicit similar allergic reactions as those from nuts and seeds. Therefore, we investigated the presence of PR10 genes in cashew nut. Using RNA-seq analysis, we were able to identify several PR10-like transcripts in cashew nut and cl

Adjuvant Effect of Orally Applied Preparations Containing Non-Digestible Polysaccharides on Influenza Vaccination in Healthy Seniors: A Double-Blind, Randomised, Controlled Pilot Trial.

Senior individuals can suffer from immunosenescence and novel strategies to bolster the immune response could contribute to healthy ageing. In this double-blind, randomised, controlled pilot trial, we investigated the ability of non-digestible polysaccharide (NPS) preparations to enhance the immune response in a human vaccination model. In total, 239 subjects (aged 50-79 years) were randomised to consume one of five different NPS (yeast β-glucan (YBG), shiitake β-glucan (SBG), oat β-glucan (OBG), arabinoxylan (AX), bacterial exopolysaccharide (EPS)) or control (CTRL) product daily for five weeks. After two weeks of intervention, subjects were vaccinated with seasonal influenza vaccine. The post-vaccination increases in haemagglutination inhibition antibody titres and seroprotection rate against the influenza strains were non-significantly enhanced in the NPS intervention groups compared to CTRL. Specifically, a trend towards a higher mean log2 fold increase was observed in the AX group (uncorrected p = 0.074) combined with a trend for an increased seroprotection rate, AX group (48.7%) compared to CTRL (25.6%) (uncorrected p = 0.057), for the influenza A H1N1 strain. Subjects consuming AX also had a reduced incidence of common colds compared to CTRL (1 vs. 8; p = 0.029 in Fisher exact test). No adverse effects of NPS consumption were reported. The findings of this pilot study warrant further research to study AX as an oral adjuvant to support vaccine efficacy

Macrophages treated with non-digestible polysaccharides reveal a transcriptionally unique phenotype

Dietary non-digestible polysaccharides (NDPs) might promote intestinal health via immuno-modulation. Immunomodulatory effects of NDP are most likely brought about by antigen processing cells such as macrophages that populate the intestine, although the mechanisms are still poorly understood. We validated the in vitro model of M1 and M2 macrophages to mimic the intestinal inflammatory and tolerant macrophages using literature and microarray-derived gene markers. All these markers were used to characterise the macrophage phenotype following NDP stimulation. This identified an alternative subset, termed M(NDP), which commonly modulated a set of 126 genes, involved in migration, metabolic processes, cell cycle, and inflammatory immune function. This gene-based analysis for macrophage subsets provides an additional tool to characterise NDP bioactivity for their in vivo potential.</p

Immunomodulatory activity of protein hydrolysates derived from Virgibacillus halodenitrificans SK1-3-7 proteinase

Modulation of inflammation-related immune response on THP-1 macrophages of protein hydrolysates derived from tilapia mince, casein and pea protein, were investigated. The protein substrates were hydrolyzed by Virgibacillus halodenitrificans SK1-3-7 proteinase. The degree of hydrolysis (DH) of casein was observed to be the highest throughout the course of hydrolysis. When challenging THP-1 macrophages, tilapia mince hydrolysate (TMH) enhanced innate immunity through induction of IL-1β and COX-2 expression. Anti-inflammatory activity was observed in casein hydrolysate (CH) and pea protein hydrolysate (PPH) by attenuating lipopolysaccharide- (LPS) induced pro-inflammatory gene expression in THP-1 macrophages. CH suppressed IL-1β, IL-6, IL-8, TNF-α and COX-2, while PPH reduced LPS-induced IL-6 and TNF-α responses. In addition, CH and PPH showed stronger suppression of LPS-induced pro-inflammatory gene expression compared with non-hydrolyzed casein and pea protein. These results suggest that TMH, CH and PPH prepared from V. halodenitrificans SK1-3-7 proteinase are potential functional food ingredients with immunomodulatory activity.</p

Lipopolysaccharide quantification and alkali-based inactivation in polysaccharide preparations to enable in vitro immune modulatory studies

-The correct identification of immune-modulatory activity of polysaccharides is often hampered by immune-stimulatory contaminants, with pyrogens such as lipopolysaccharide (LPS) as a very potent example. In order to avoid false positive immuno-stimulatory properties to be attributed to polysaccharides, accurate quantification and inactivation of LPS in test samples is crucial. To quantify LPS in polysaccharide preparations of different origin and structure we used two different limulus amoebocyte lysate test kits in two different laboratories. We observed larger variation in detection of LPS contamination between kits than between labs. LPS quantification proved unreliable for some polysaccharide preparations as spike controls resulted in spike recoveries outside the acceptable range. We designed a cellular in vitro assay as alternative method to detect the presence of functional LPS. This HEK-Blue hTLR4 cell culture provides a reliable assay, when combined with a cell viability test, for determining functional LPS in polysaccharide preparations. Finally, to inactivate LPS in polysaccharide preparations, we setup an alkaline-ethanol-based treatment. With this assay we observed that our treatment (5 h incubation in 0.1 M NaOH) at 56 °C efficiently inactivated LPS in all polysaccharide preparations below immune-stimulatory levels. At this elevated temperature, however, we also observed minimal to severe degradation of polysaccharide preparations as determined with SEC-RI. Taken together, we describe methods and precautions to reliably detect and inactivate LPS in polysaccharide preparations to allow reliable in vitro investigations towards immune-modulatory potential of polysaccharide preparations