Phenylthiourea Binding to Human Tyrosinase-Related Protein 1

Tyrosinase-related protein 1 (TYRP1) is one of the three human melanogenic enzymes involved in the biosynthesis of melanin, a pigment responsible for the color of the skin, hair, and eyes. It shares high sequence identity with tyrosinase, but has two zinc ions in its active site rather than two copper ions as in tyrosinase. Typical tyrosinase inhibitors do not directly coordinate to the zinc ions of TYRP1. Here, we show, from an X-ray crystal structure determination, that phenylthiourea, a highly potent tyrosinase inhibitor, does neither coordinate the active site zinc ions, but binds differently from other structurally characterized TYRP1-inhibitor complexes. Its aromatic ring is directed outwards from the active site, apparently as a result from the absence of polar oxygen substituents that can take the position of water molecules bound in the active site. The compound binds via hydrophobic interactions, thereby blocking substrate access to the active site.</p

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.

Investigation into the potential of commercially available lesser mealworm (A. diaperinus) protein to serve as sources of peptides with DPP-IV inhibitory activity

Food-derived peptides are known to possess inhibitory activity against the dipeptidyl-peptidase IV (DPP-IV) enzyme, a target in the management of type 2 diabetes. While proteins from commonly consumed food commodities have been investigated as precursors of DPP-IV-inhibiting peptides, studies on novel protein sources, such as those from insects, are sparse. This research aimed to determine if DPP-IV inhibitors can be generated upon in vitro digestion or enzymatic hydrolysis of lesser mealworm protein isolate and concentrate. Treatment of the proteins with digestive enzymes and proteases generated hydrolysates with varying potency, thermolysin being the most effective at releasing active peptides (IC50= 0.63 and 0.60 mg mL−1 for the isolate and concentrate). Ultrafiltration of the thermolysin-treated hydrolysates did not significantly improve the potency. This study shows that DPP-IV inhibitors can be generated from lesser mealworm protein and provides insight on the potential of insects to serve as functional food ingredients.</p

Favourable effects of consuming a Palaeolithic-type diet on characteristics of the metabolic syndrome:a randomized controlled pilot-study

Background: The main goal of this randomized controlled single-blinded pilot study was to study whether, independent of weight loss, a Palaeolithic-type diet alters characteristics of the metabolic syndrome. Next we searched for outcome variables that might become favourably influenced by a Paleolithic-type diet and may provide new insights in the pathophysiological mechanisms underlying the metabolic syndrome. In addition, more information on feasibility and designing an innovative dietary research program on the basis of a Palaeolithic-type diet was obtained.Methods: Thirty-four subjects, with at least two characteristics of the metabolic syndrome, were randomized to a two weeks Palaeolithic-type diet (n = 18) or an isoenergetic healthy reference diet, based on the guidelines of the Dutch Health Council (n = 14). Thirty-two subjects completed the study. Measures were taken to keep bodyweight stable. As primary outcomes oral glucose tolerance and characteristics of the metabolic syndrome (abdominal circumference, blood pressure, glucose, lipids) were measured. Secondary outcomes were intestinal permeability, inflammation and salivary cortisol. Data were collected at baseline and after the intervention.Results: Subjects were 53.5 (SD9.7) year old men (n = 9) and women (n = 25) with mean BMI of 31.8 (SD5.7) kg/m(2). The Palaeolithic-type diet resulted in lower systolic blood pressure (-9.1 mmHg; P = 0.015), diastolic blood pressure (-5.2 mmHg; P = 0.038), total cholesterol (-0.52 mmol/l; P = 0.037), triglycerides (-0.89 mmol/l; P = 0.001) and higher HDL-cholesterol (+0.15 mmol/l; P = 0.013), compared to reference. The number of characteristics of the metabolic syndrome decreased with 1.07 (P = 0.010) upon the Palaeolithic-type diet, compared to reference. Despite efforts to keep bodyweight stable, it decreased in the Palaeolithic group compared to reference (-1.32 kg; P = 0.012). However, favourable effects remained after post-hoc adjustments for this unintended weight loss. No changes were observed for intestinal permeability, inflammation and salivary cortisol.Conclusions: We conclude that consuming a Palaeolithic-type diet for two weeks improved several cardiovascular risk factors compared to a healthy reference diet in subjects with the metabolic syndrome.</p

Release of Major Peanut Allergens from Their Matrix under Various pH and Simulated Saliva Conditions—Ara h2 and Ara h6 Are Readily Bio-Accessible

The oral mucosa is the first immune tissue that encounters allergens upon ingestion of food. We hypothesized that the bio-accessibility of allergens at this stage may be a key determinant for sensitization. Light roasted peanut flour was suspended at various pH in buffers mimicking saliva. Protein concentrations and allergens profiles were determined in the supernatants. Peanut protein solubility was poor in the pH range between 3 and 6, while at a low pH (1.5) and at moderately high pHs (\u3e8), it increased. In the pH range of saliva, between 6.5 and 8.5, the allergens Ara h2 and Ara h6 were readily released, whereas Ara h1 and Ara h3 were poorly released. Increasing the pH from 6.5 to 8.5 slightly increased the release of Ara h1 and Ara h3, but the recovery remained low (approximately 20%) compared to that of Ara h2 and Ara h6 (approximately 100% and 65%, respectively). This remarkable difference in the extraction kinetics suggests that Ara h2 and Ara h6 are the first allergens an individual is exposed to upon ingestion of peanut-containing food. We conclude that the peanut allergens Ara h2 and Ara h6 are quickly bio-accessible in the mouth, potentially explaining their extraordinary allergenicity

Functional differences between primary monocyte-derived and THP-1 macrophages and their response to LCPUFAs

Background: In immune cell models, macrophages are one of the most frequently used cell types. THP-1 cells are often used as model to study macrophage function, however they may act differently from primary human monocyte derived macrophages (MDMs). Methods: In this study, we investigated the intrinsic baseline differences between the human macrophage cell line THP-1 and human primary MDMs. Additionally, we studied the difference in response to treatment with long-chain polyunsaturated fatty acids (LCPUFAs): well-described immunomodulators. Results: Although the amount of cells that phagocytose were similar between the cell types, primary MDMs consumed significantly more E. coli bioparticles compared to THP-1 macrophages. In M1 macrophages, IL-12 secretion was almost fifty times higher by primary MDMs compared to THP-1 macrophages, thereby increasing the IL-12/IL-10 ratio. Despite this, the IL-12 secretion by THP-1 M1 macrophages was higher that the secretion of IL-10, thereby showing that it is still a suitable M1 type. Cytokine profiles differed between primary MDMs and THP-1 M1 and M2 macrophages. In response to LCPUFAs, primary M1 MDMs and THP-1 M1 macrophages were alike. Interestingly, primary M2 MDMs secreted less IL-10 and CCL22 when treated with LCPUFAs, whereas THP-1 M2 macrophages secreted more IL-10 when treated with LCPUFAs and showed no difference in CCL22 secretion. Conclusions: In conclusion, in an M1 setting, both THP-1 and primary MDMs are suitable models. However, when interested in M2 models, the model choice highly depends on the research question

Multicentre Double-Blind Placebo-Controlled Food Challenge Study in Children Sensitised to Cashew Nut

Few studies with a limited number of patients have provided indications that cashew-allergic patients may experience severe allergic reactions to minimal amounts of cashew nut. The objectives of this multicentre study were to assess the clinical relevance of cashew nut sensitisation, to study the clinical reaction patterns in double-blind placebo-controlled food challenge tests and to establish the amount of cashew nuts that can elicit an allergic reaction.A total of 179 children were included (median age 9.0 years; range 2-17 years) with cashew nut sensitisation and a clinical history of reactions to cashew nuts or unknown exposure. Sensitised children who could tolerate cashew nuts were excluded. The study included three clinical visits and a telephone consultation. During the first visit, the medical history was evaluated, physical examinations were conducted, blood samples were drawn and skin prick tests were performed. The children underwent a double-blind placebo-controlled food challenge test with cashew nut during the second and third visits. The study showed that 137 (76.5%) of the sensitised children suspected of allergy to cashew nut had a positive double-blind placebo-controlled food challenge test, with 46% (63) manifesting subjective symptoms to the lowest dose of 1 mg cashew nut protein and 11% (15) developing objective symptoms to the lowest dose. Children most frequently had gastro-intestinal symptoms, followed by oral allergy and skin symptoms. A total of 36% (49/137) of the children experienced an anaphylactic reaction and 6% (8/137) of the children were treated with epinephrine.This prospective study demonstrated a strikingly high percentage of clinical reactions to cashew nut in this third line population. Severe allergic reactions, including anaphylaxis requiring epinephrine, were observed. These reactions were to minimal amounts of cashew nut, demonstrated the high potency of this allergens.www.ncbi.nlm.nih.gov/pubmed NTR3572

Long Chain Polyunsaturated Fatty Acids (LCPUFAs) in the Prevention of Food Allergy

N-3 long chain polyunsaturated fatty acids (LCPUFAs) are considered to possess protective properties for human health by impacting on immunological reactions. An “inflammation-suppressive” effect appears to be the common denominator of the beneficial effects of most of these dietary components which may protect against the development of chronic immune disorders such as (food) allergy. LCPUFAs, especially n-3 LCPUFAs, have been shown to interact with both the sensitization as well as the effector phase in food allergy in pre-clinical models. In this review, we explore the anti-allergic properties of LCPUFAs by providing an overview of clinical, in vivo and in vitro studies. Furthermore, we discuss the susceptibility of LCPUFAs to lipid oxidation and possible strategies to support the efficacy of LCPUFAs in reducing the allergy risk by using additional components with anti-oxidative and anti-inflammatory capacities such as the flavonoid quercetin. Finally, we propose new strategies to prevent (food) allergy using combinations of LCPUFAs and additional nutrients in diets or supplements, and postulate to investigate the use of LCPUFAs in allergic symptom relief

IgE Cross-Reactivity of Cashew Nut Allergens

Background: Allergic sensitisation towards cashew nut often happens without a clear history of eating cashew nut. IgE cross-reactivity between cashew and pistachio nut is well described; however, the ability of cashew nut-specific IgE to cross-react to common tree nut species and other Anacardiaceae, like mango, pink peppercorn, or sumac is largely unknown. Objectives: Cashew nut allergic individuals may cross-react to foods that are phylogenetically related to cashew. We aimed to determine IgE cross-sensitisation and cross-reactivity profiles in cashew nut-sensitised subjects, towards botanically related proteins of other Anacardiaceae family members and related tree nut species. Method: Sera from children with a suspected cashew nut allergy (n = 56) were assessed for IgE sensitisation to common tree nuts, mango, pink peppercorn, and sumac using dot blot technique. Allergen cross-reactivity patterns between Anacardiaceae species were subsequently examined by SDS-PAGE and immunoblot inhibition, and IgE-reactive allergens were identified by LC-MS/MS. Results: From the 56 subjects analysed, 36 were positive on dot blot for cashew nut (63%). Of these, 50% were mono-sensitised to cashew nuts, 19% were co-sensitised to Anacardiaceae species, and 31% were co-sensitised to tree nuts. Subjects co-sensitised to Anacardiaceae species displayed a different allergen recognition pattern than subjects sensitised to common tree nuts. In pink peppercorn, putative albumin- and legumin-type seed storage proteins were found to cross-react with serum of cashew nut-sensitised subjects in vitro. In addition, a putative luminal binding protein was identified, which, among others, may be involved in cross-reactivity between several Anacardiaceae species. Conclusions: Results demonstrate the in vitro presence of IgE cross-sensitisation in children towards multiple Anacardiaceae species. In this study, putative novel allergens were identified in cashew, pistachio, and pink peppercorn, which may pose factors that underlie the observed cross-sensitivity to these species. The clinical relevance of this widespread cross-sensitisation is unknown.</p