Discovery of Transfer Factors in Plant-Derived Proteins and an In Vitro Assessment of Their Immunological Activities.

Repeated exposure to pathogens leads to evolutionary selection of adaptive traits. Many species transfer immunological memory to their offspring to counteract future immune challenges. Transfer factors such as those found in the colostrum are among the many mechanisms where transfer of immunologic memory from one generation to the next can be achieved for an enhanced immune response. Here, a library of 100 plants with high protein contents was screened to find plant-based proteins that behave like a transfer factor moiety to boost human immunity. Aqueous extracts from candidate plants were tested in a human peripheral blood mononuclear cell (PBMC) cytotoxicity assay using human cancerous lymphoblast cells-with K562 cells as a target and natural killer cells as an effector. Plant extracts that caused PBMCs to exhibit enhanced killing beyond the capability of the colostrum-based transfer factor were considered hits. Primary screening yielded an 11% hit rate. The protein contents of these hits were tested via a Bradford assay and Coomassie-stained SDS-PAGE, where three extracts were confirmed to have high protein contents. Plants with high protein contents underwent C18 column fractionation using methanol gradients followed by membrane ultrafiltration to isolate protein fractions with molecular weights of <3 kDa, 3-30 kDa, and >30 kDa. It was found that the 3-30 kDa and >30 kDa fractions had high activity in the PBMC cytotoxicity assay. The 3-30 kDa ultrafiltrates from the top two hits, seeds from Raphanus sativus and Brassica juncea, were then selected for protein identification by mass spectrometry. The majority of the proteins in the fractions were found to be seed storage proteins, with a low abundance of proteins involved in plant defense and stress response. These findings suggest that Raphanus sativus or Brassica juncea extracts could be considered for further characterization and immune functional exploration with a possibility of supplemental use to bolster recipients' immune response.

Comparative Assessment of the Acute Effects of Whey, Rice and Potato Protein Isolate Intake on Markers of Glycaemic Regulation and Appetite in Healthy Males Using a Randomised Study Design.

Global protein consumption has been increasing for decades due to changes in demographics and consumer shifts towards higher protein intake to gain health benefits in performance nutrition and appetite regulation. Plant-derived proteins may provide a more environmentally sustainable alternative to animal-derived proteins. This study, therefore, aimed to investigate, for the first time, the acute effects on glycaemic indices, gut hormones, and subjective appetite ratings of two high-quality, plant-derived protein isolates (potato and rice), in comparison to a whey protein isolate in a single-blind, triple-crossover design study with nine male participants (30.8 ± 9.3 yrs). Following a 12 h overnight fast, participants consumed an equal volume of the three isocaloric protein shakes on different days, with at least a one-week washout period. Glycaemic indices and gut hormones were measured at baseline, then at 30, 60, 120, 180 min at each visit. Subjective palatability and appetite ratings were measured using visual analogue scales (VAS) over the 3 h, at each visit. This data showed significant differences in insulin secretion with an increase in whey (+141.8 ± 35.1 pmol/L; p = 0.011) and rice (-64.4 ± 20.9 pmol/L; p = 0.046) at 30 min compared to potato protein. A significantly larger total incremental area under the curve (iAUC) was observed with whey versus potato and rice with p < 0.001 and p = 0.010, respectively. There was no significant difference observed in average appetite perception between the different proteins. In conclusion, this study suggests that both plant-derived proteins had a lower insulinaemic response and improved glucose maintenance compared to whey protein.

In vitro assessment of the efficacy of protein exudates from seeds against Haemonchus contortus.

Nematodes develop resistance to the most common commercially available drugs. The aim of this study was to identify and evaluate the action of protein exudates from Mimosa caesalpiniifolia, Leucaena leucocephala, Acacia mangium, and Stylosanthes capitata seeds on the gastrointestinal nematode Haemonchus contortus. The exuded proteins were precipitated, dialyzed, lyophilized, and assessed for their effect on egg hatching and artificial larval exsheathment inhibition. Proteome analysis of the protein extracts was also performed. Although no egg-hatching inhibition was observed, all exudates showed efficacy in inhibiting the larval exsheathment of H. contortus larvae with an EC50 varying from 0.61 to 0.26 mg P mL-1. Proteomic analysis revealed the presence of proteases, protease inhibitors, chitinases, and lectins among other proteins in the exudates. Most of the exuded proteins belong to the oxidative stress/plant defense and energy/carbohydrate metabolism functional clusters. This study concluded that the bioactive proteins from different classes exuded by seeds of M. caesalpiniifolia, L. leucocephala, A. mangium, and S. capitata show stage-specific inhibition against H. contortus.

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-κB activation and IL-8 expression studies in undifferentiated Caco-2 cells. Inflammation was elicited using IL-1ß. 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.

Assessment of the functional properties of protein extracted from the brown seaweed Himanthalia elongata (Linnaeus) S. F. Gray.

A protein extract from the brown seaweed Himanthalia elongata (Linnaeus) S. F. Gray was prepared and its functional properties, colour and amino acid composition were assessed for its potential future use by the food industry. The total content of amino acids was determined as 54.02±0.46gaminoacids/kg dry weight, with high levels of the essential amino acids lysine and methionine. SDS-PAGE showed 5 protein bands with molecular weights of 71.6, 53.7, 43.3, 36.4 and 27.1kDa. The water holding capacity and oil holding capacity were determined as 10.27±0.09gH2O/g and 8.1±0.07goil/g respectively. Foaming activity and stability were higher at alkaline pH values. The emulsifying capacity and stability of the extract varied depending on the pH and oil used. These results demonstrate the potential use of Himanthalia elongata protein extract in the food industry.

Assessment of the protein interaction between coagulation factor XII and corn trypsin inhibitor by molecular docking and biochemical validation.

Essentials Corn Trypsin Inhibitor (CTI) is a selective inhibitor of coagulation Factor XII (FXII). Molecular modelling of the CTI-FXIIa complex suggested a canonical inhibitor binding mode. Mutagenesis revealed the CTI inhibitory loop and helices α1 and α2 mediate the interaction. This confirms that CTI inhibits FXII in canonical fashion and validates the molecular model. SUMMARY: Background Corn trypsin inhibitor (CTI) has selectivity for the serine proteases coagulation factor XII and trypsin. CTI is in widespread use as a reagent that specifically inhibits the intrinsic pathway of blood coagulation but not the extrinsic pathway. Objectives To investigate the molecular basis of FXII inhibition by CTI. Methods We performed molecular docking of CTI, using its known crystal structure, with a model of the activated FXII (FXIIa) protease domain. The interaction model was verified by use of a panel of recombinant CTI variants tested for their ability to inhibit FXIIa enzymatic activity in a substrate cleavage assay. Results The docking predicted that: (i) the CTI central inhibitory loop P1 Arg34 side chain forms a salt bridge with the FXIIa S1 pocket Asp189 side chain; (ii) Trp22 from CTI helix α1 interacts with the FXIIa S3 pocket; and (iii) Arg43 from CTI helix α2 forms a salt bridge with FXIIa H1 pocket Asp60A. CTI amino acid substitution R34A negated all inhibitory activity, whereas the G32W, L35A, W22A and R42A/R43A substitutions reduced activity by large degrees of 108-fold, 41-fold, 158-fold, and 100-fold, respectively; the R27A, W37A, W39A and R42A substitutions had no effect. Synthetic peptides spanning CTI residues 20-44 had inhibitory activity that was three-fold to 4000-fold less than that of full-length CTI. Conclusions The data confirm the validity of a canonical model of the FXIIa-CTI interaction, with helix α1 (Trp22), central inhibitory loop (Arg34) and helix α2 (Arg43) of CTI being required for effective binding by contacting the S1, S3 and H1 pockets of FXIIa, respectively.

Effects of Dietary Protein Source and Quantity during Weight Loss on Appetite, Energy Expenditure, and Cardio-Metabolic Responses.

Higher protein meals increase satiety and the thermic effect of feeding (TEF) in acute settings, but it is unclear whether these effects remain after a person becomes acclimated to energy restriction or a given protein intake. This study assessed the effects of predominant protein source (omnivorous, beef/pork vs. lacto-ovo vegetarian, soy/legume) and quantity (10%, 20%, or 30% of energy from protein) on appetite, energy expenditure, and cardio-metabolic indices during energy restriction (ER) in overweight and obese adults. Subjects were randomly assigned to one protein source and then consumed diets with different quantities of protein (4 weeks each) in a randomized crossover manner. Perceived appetite ratings (free-living and in-lab), TEF, and fasting cardio-metabolic indices were assessed at the end of each 4-week period. Protein source and quantity did not affect TEF, hunger, or desire to eat, other than a modestly higher daily composite fullness rating with 30% vs. 10% protein diet (p = 0.03). While the 20% and 30% protein diets reduced cholesterol, triacylglycerol, and APO-B vs. 10% protein (p < 0.05), protein source did not affect cardio-metabolic indices. In conclusion, diets varying in protein quantity with either beef/pork or soy/legume as the predominant source have minimal effects on appetite control, energy expenditure and cardio-metabolic risk factors during ER-induced weight loss.

Hybrid protease inhibitors for pest and pathogen control--a functional cost for the fusion partners?

Fusion proteins integrating dual pesticidal functions have been devised over the last 10 years to improve the effectiveness and potential durability of pest-resistant transgenic crops, but little attention has been paid to the impact of the fusion partners on the actual activity of the resulting hybrids. Here we assessed the ability of the rice cysteine protease inhibitor, oryzacystatin I (OCI), to retain its protease inhibitory potency when used as a template to devise hybrid inhibitors with dual activity against papain-like proteases and carboxypeptidase A (CPA). C-terminal variants of OCI were generated by fusing to its C-terminal end: (i) the primary inhibitory site of the small CPA inhibitor potato carboxypeptidase inhibitor (PCI, amino acids 35-39); or (ii) the complete sequence of PCI (a.a. 1-39). The hybrid inhibitors were expressed in E. coli and tested for their inhibitory activity against papain, CPA and digestive cysteine proteases of herbivorous and predatory arthropods. In contrast with the primary inhibitory site of PCI, the entire PCI attached to OCI was as active against CPA as free, purified PCI. The OCI-PCI hybrids also showed activity against papain, but the presence of extra amino acids at the C terminus of OCI negatively altered its inhibitory potency against cysteine proteases. This negative effect, although not preventing dual binding to papain and CPA, was correlated with an increased binding affinity for papain presumably due to non-specific interactions with the PCI domain. These results confirm the potential of OCI and PCI for the design of fusion inhibitors with dual protease inhibitory activity, but also point out the possible functional costs associated with protein domain grafting to recipient pesticidal proteins.

Assessment of the anthelmintic effect of natural plant cysteine proteinases against the gastrointestinal nematode, Heligmosomoides polygyrus, in vitro.

We examined the mechanism of action and compared the anthelmintic efficacy of cysteine proteinases from papaya, pineapple, fig, kiwi fruit and Egyptian milkweed in vitro using the rodent gastrointestinal nematode Heligmosomoides polygyrus. Within a 2 h incubation period, all the cysteine proteinases, with the exception of the kiwi fruit extract, caused marked damage to the cuticle of H. polygyrus adult male and female worms, reflected in the loss of surface cuticular layers. Efficacy was comparable for both sexes of worms, was dependent on the presence of cysteine and was completely inhibited by the cysteine proteinase inhibitor, E-64. LD50 values indicated that the purified proteinases were more efficacious than the proteinases in the crude latex, with purified ficin, papain, chymopapain, Egyptian milkweed latex extract and pineapple fruit extract containing fruit bromelain, having the most potent effect. The mechanism of action of these plant enzymes (i.e. an attack on the protective cuticle of the worm) suggests that resistance would be slow to develop in the field. The efficacy and mode of action make plant cysteine proteinases potential candidates for a novel class of anthelmintics urgently required for the treatment of humans and domestic livestock.

Direct defense or ecological costs: responses of herbivorous beetles to volatiles released by wild Lima bean (Phaseolus lunatus).

In response to feeding damage, Lima bean releases herbivore-induced plant volatiles (HIPV), which are generally assumed to attract carnivorous arthropods as an indirect defense. While many studies have focused on such tritrophic interactions, few have investigated effects of HIPV on herbivores. I used natural herbivores of wild Lima bean and studied their responses to jasmonic acid-induced plants in an olfactometer and in feeding trials. Both Cerotoma ruficornis and Gynandrobrotica guerreroensis (Chrysomelidae) significantly preferred control plants to induced ones in the olfactometer, and they avoided feeding on induced plants. In contrast, Curculionidae significantly preferred HIPV of the induced plant to those of the control in one plant pair and did not choose in the case of a second pair. In feeding trials, no choice occurred in the first plant pair, while control leaves were preferred in the second. Release of HIPV deterred Chrysomelid herbivores and, thus, acted as a direct defense. This may be an important addition to indirect defensive effects. Whether or not HIPV released by induced plants attracted herbivorous Curculionidae, thus incurring ecological costs, varied among plants. Such differences could be related to various HIPV blends released by individual plants.

Peptide mapping and assessment of cryoprotective activity of 26/27-kDa dehydrin from soybean seeds.

To characterize the molecular weight diversity of seed dehydrin among soybean cultivars, 26/27-kDa soybean dehydrins were purified and compared in peptide mapping patterns, partial amino acid sequences, and cryoprotective activity on enzyme. In reverse phase chromatograms of their trypsin digests, we detected several distinctive peaks, one of which was attributed to a part of the internal glycine-rich region. Partial amino acid sequences of peptide fragments from trypsin and S. aureus V8 protease cleavage were found to be identical to the Mat9 translation. The CP50 of purified 26/27-kDa dehydrins were estimated to be 0.30 and 0.11 microM, respectively.

Evaluation of potato proteins on the growth performance of early-weaned pigs.

We conducted five experiments to evaluate conventional and low-glycoalkaloid potato protein (CPP and LGPP, respectively) in diets for early-weaned pigs. In Exp. 1, 150 weanling pigs (initially 4.4 +/- .9 kg and 15.5 +/- 2 d of age) were fed either a control diet containing 3% spray-dried animal plasma (SDAP) or diets with additional SDAP (2.5 or 5% added; 5.5 or 8% total) or CPP (2.6% or 5.1%) substituted on a total lysine basis. From d 0 to 14 after weaning, increasing SDAP increased (linear, P < .05) ADG and ADFI, whereas increasing CCP had no effect on growth performance. In Exp. 2, 180 weanling pigs (initially 5.9 +/- 1.2 kg and 20 +/- 2 d of age) were fed diets containing a LGPP replacing 25, 50, 75, or 100% of the 7% dietary SDAP on a digestible lysine basis. From d 0 to 7 after weaning, increasing LGPP increased and then returned to control levels ADG and ADFI (quadratic, P < .01) and gain:feed ratio (quadratic, P < .05). In Exp. 3, 175 weanling pigs (initially 5.5 +/- 1.1 kg and 20 +/- 3 d of age) were fed either a control diet containing 20% dried whey, 17.5% dried skim milk, and 4% select menhaden fish meal (SMFM) or diets consisting of lactose and either 3.5 and 7.0% SDAP or 4.0 and 8.0% LGPP added at the expense of dried skim milk on a digestible lysine basis. From d 0 to 7 after weaning, ADG and ADFI increased (linear, P < .05) with increasing SDAP. With increasing LGPP, ADG and ADFI increased and then decreased (quadratic, P < .10 and P < .05, respectively). Gain:feed ratio (G/F) was not affected by SDAP and was improved (linear, P < .05) for pigs fed increasing LGPP. In Exp. 4, 270 weanling pigs (initially 6.2 +/- 1.6 kg and 20 +/- 3 d of age) were used to compare three diets that contained either 2.5% spray-dried blood meal (SDBM), 4.8% SMFM, or 3.92% CPP; test feedstuffs were substituted on a total lysine basis and diets were fed from d 7 to 28 after weaning. Pigs fed CPP had decreased (P < .05) ADG and G/F compared with those fed the other protein sources. In Exp. 5, 255 weanling pigs (initially 5.3 +/- 1.2 kg and 17 +/- 2 d of age), were used to compare five diets that contained either 2.5% SDBM, 5.51% SMFM, 4.17% CPP, 4.17% LGPP or 8.34% LGPP; feedstuffs were substituted on a digestible lysine basis and diets were fed from d 7 to 28 after weaning. No differences (P > .10) were observed in growth performance among pigs fed any of the protein sources within the experiment. However, pigs fed the LGPP had numerically greater ADG and better G/F than those fed CPP. In conclusion, these results suggest that LGPP can be an effective replacement for a portion of the SDAP in diets for weanling pigs.

Vitamin B-6 requirement and status assessment: young women fed a depletion diet followed by a plant- or animal-protein diet with graded amounts of vitamin B-6.

The vitamin B-6 requirement of young women consuming a high-protein diet (1.55 g/kg body wt) and the effect of protein quality on this requirement was studied. In addition, the response of clinical, functional, and biochemical measures of vitamin B-6 nutriture to short-term depletion and step-wise repletion of vitamin B-6 were evaluated. Eight healthy young women resided in a metabolic unit and were fed a formula depletion diet (< 0.05 mg vitamin B-6/d) for 11-28 d followed by either an animal-protein (AP) or plant-protein (PP) diet with successively increasing vitamin B-6 intakes (0.5, 1.0, 1.5, and 2.0 mg/d) for periods of 14-21 d. Animal proteins were primarily from dairy and poultry sources and plant proteins were primarily from legumes. Vitamin B-6 status measures were assessed at weekly intervals. Results showed that a PP diet does not elevate the vitamin B-6 requirement over that required for an AP diet given the high amount of dietary protein used in this study. It was also found that 0.015 mg vitamin B-6/g protein intake normalized most biochemical indexes of vitamin B-6 status (including those indicative of functional status), and that 0.020 mg/g protein normalized all biochemical measures except total urinary vitamin B-6. Adding a margin of safety to either the 0.015 or 0.020 mg/g protein intake would raise the vitamin B-6 requirement for young women above the currently recommended dietary allowance of 0.016 mg/g protein.