Bacillus cereus Response to a Proanthocyanidin Trimer, a Transcriptional and Functional Analysis.

Proanthocyanidins are abundant in peanut skin, and in this study, the antibacterial effects of a peanut skin extract (PSE) against food-borne bacteria were investigated to find its minimum inhibitory concentration. Food-borne gram-positive bacteria, and in particular Bacillus cereus, was more sensitive to PSE. In particular, the inhibitory activity of epicatechin-(4ߠ→ 6)-epicatechin-(2ߠ→ O→7, 4ߠ→ 8)-catechin (EEC), a proanthocyanidin trimer from peanut skin, against B. cereus was stronger than that of procyanidin A1, a proanthocyanidin dimer. DNA microarray analysis of B. cereus treated with EEC was carried out, with a finding that 597 genes were significantly up-regulated. Analysis of the up-regulated genes suggested that EEC disrupted the normal condition of the cell membrane and wall of B. cereus and alter its usual nutritional metabolism. Moreover, treatment of B. cereus with EEC inhibited glucose uptake, suggesting that EEC affects the cell-surface adsorption.

Administration of a maple syrup extract to mitigate their hepatic inflammation induced by a high-fat diet: a transcriptome analysis.

Effects of the administration of maple syrup extract (MSX) on hepatic gene expression were investigated in mice fed a high-fat diet. Gene annotation enrichment analysis based on gene ontology revealed some changes in the expression of genes related to lipid metabolism and the immune response in MSX-fed mice. Detailed analysis of these data indicated that MSX ingestion mitigates hepatic inflammation.

Characterization of the quality of imbibed soybean at an early stage of pre-germination for the development of a new protein food item.

This was a pilot study carried out to develop a new protein food item from imbibed soybean before germination. It identified the significance of a short stage after imbibition and before germination, and that vitamin C production was activated in as little as 16 h from the start of imbibition, without any influence on the soy protein quality or sensory acceptability, while longer imbibition caused the imbibed soybean to activate its phytophysiological metabolism for germination. DNA microarray analysis indicated that the genes for carbohydrate metabolism were up-regulated prior to 16 h, and that the expression rates of genes responsible for environmental factors were down-regulated. Thereafter, the expression rates of the genes associated with lipid metabolism and secondary metabolite production were changed. This information should contribute to a better understanding of how to develop a new soy protein item in pre-germination before active physiological processes begin.

Peanut-skin polyphenols, procyanidin A1 and epicatechin-(4 ß â†’ 6)-epicatechin-(2 ß â†’ O → 7, 4 ß â†’ 8)-catechin, exert cholesterol micelle-degrading activity in vitro.

We identified epicatechin-(4 ß â†’ 6)-epicatechin-(2 ß â†’ O → 7, 4 ß â†’ 8)-catechin (EEC) in the skin of the peanut (Arachis hypogaea L.). EEC (a trimer) showed more potent cholesterol micelle-degrading activity than procyanidin A1 (a dimer) did in vitro. The hypercholesterolemia suppressing effect of a peanut skin polyphenol on rats fed high-cholesterol diet in our preceding experiments might thus have been due primarily to a micelle degrading effect in the intestine.

Serum cholesterol reduction by feeding a high-cholesterol diet containing a lower-molecular-weight polyphenol fraction from peanut skin.

Feeding a high-cholesterol diet with a water-soluble peanut skin polyphenol fraction to rats reduced their plasma cholesterol level, with an increase in fecal cholesterol excretion. The hypocholesterolemic effect was greater with the lower-molecular-weight rather than higher-molecular-weight polyphenol fraction. This effect was possibly due to some oligomeric polyphenols which reduced the solubility of dietary cholesterol in intestinal bile acid-emulsified micelles.

Novel angiotensin I-converting enzyme inhibitory peptides found in a thermolysin-treated elastin with antihypertensive activity.

Angiotensin I-converting enzyme (ACE) inhibitory activity was generated from elastin and collagen by hydrolyzing with thermolysin. The IC50 value of 531.6 µg/mL for ACE inhibition by the elastin hydrolysate was five times less than 2885.1 µg/mL by the collagen hydrolysate. We confirmed the antihypertensive activity of the elastin hydrolysate in vivo by feeding spontaneously hypertensive rats (male) on a diet containing 1% of the elastin hydrolysate for 9 weeks. About 4 week later, the systolic blood pressure of the rats in the elastin hydrolysate group had become significantly lower than that of the control group. We identified novel ACE inhibitory peptides, VGHyp, VVPG and VYPGG, in the elastin hydrolysate by using a protein sequencer and quadrupole linear ion trap (QIT)-LC/MS/MS. VYPGG had the highest IC50 value of 244 µM against ACE and may have potential use as a functional food.

Hepatic oxidoreduction-related genes are upregulated by administration of hydrogen-saturated drinking water.

The effects of the administration of molecular hydrogen-saturated drinking water (hydrogen water) on hepatic gene expression were investigated in rats. Using DNA microarrays, 548 upregulated and 695 downregulated genes were detected in the liver after 4 weeks of administration of hydrogen water. Gene Ontology analysis revealed that genes for oxidoreduction-related proteins, including hydroxymethylglutaryl CoA reductase, were significantly enriched in the upregulated genes.

Effects of peanut-skin procyanidin A1 on degranulation of RBL-2H3 cells.

Peanut skin contains large amounts of polyphenols having antiallergic effects. We found that a peanut-skin extract (PSE) inhibits the degranulation induced by antigen stimulation of rat basophilic leukemia (RBL-2H3) cells. A low-molecular-weight fraction from PSE, PSEL, also had inhibitory activity against allergic degranulation. A main polyphenol in PSEL was purified by gel chromatography and fractionated by YMC-gel ODS-AQ 120S50 column. Electrospray ionization mass spectrometry (ESI-MS) analysis of the purified polyphenol gave m/z 599 [M+Na]⁺. Based on the results of ¹H-NMR, ¹³C-NMR spectra, and optical rotation analysis, the polyphenol was identified as procyanidin A1. It inhibited the degranulation caused by antigen stimulation at the IC50 of 20.3 µM. Phorbol-12-myristate-13-acetate (PMA) and 2,5,-di(tert-butyl)-1,4-hydroquinone (DTBHQ)-induced processes of degranulation were also inhibited by procyanidin A1. These results indicate that peanut-skin procyanidin A1 inhibits degranulation downstream of protein kinase C activation or Ca²âº influx from an internal store in RBL-2H3 cells.

Ingested maple syrup evokes a possible liver-protecting effect-physiologic and genomic investigations with rats.

Rats fed a 20%-maple syrup diet (maple syrup group) for 11 d showed significantly lower values of the hepatic function markers than those fed a 20%-sugar mix syrup diet (control). The reason was suggested by a DNA microarray analysis which revealed that the expression of genes for the enzymes of ammonia formation were down-regulated in the liver of the maple syrup group.

Dietary iron-deficient anemia induces a variety of metabolic changes and even apoptosis in rat liver: a DNA microarray study.

Anemia can be induced by dietary iron deficiency, as well as by hemorrhagia. It may also be associated with changes in lipid metabolism. However, no global analysis detailing the consequences of iron deficiency in the liver has yet been conducted. Since the liver is a metabolically important organ and also a major iron-storing organ, we performed a comprehensive transcriptome analysis to determine the effects of iron deficiency on hepatic gene expression. Four-week-old rats were fed an iron-deficient diet, approximately 3 ppm iron, ad libitum for 16 days. These rats were compared with similar rats pair-fed a control diet with a normal iron level, 48 ppm iron. The 16-day iron-deficient diet apparently induced anemia. On day 17, the rats were killed under anesthesia, and their livers were dissected for DNA microarray analysis. We identified 600 upregulated and 500 downregulated probe sets that characterized the iron-deficient diet group. In the upregulated probe sets, genes involved in cholesterol, amino acid, and glucose metabolism were significantly enriched, while genes related to lipid metabolism were significantly enriched in the downregulated probe sets. We also found that genes for caspases 3 and 12, which mediate endoplasmic reticulum (ER)-specific apoptosis, were upregulated in the iron-deficient group. Combined, these results suggest that iron deficiency exerts various influences, not only on nutrient metabolism but also on apoptosis, as a consequence of ER stress in the liver.

Propagule Powder of Japanese Yam (Dioscorea Japonica) Reduces High-Fat Diet-Induced Metabolic Stress in Mice through the Regulation of Hepatic Gene Expression.

SCOPE: Japanese yam propagules are supposed to have high potential as a functional food. However, there are almost no studies examining their physiological function. This study aims to elucidate the physiological function of Japanese yam propagules that are heated, freeze-dried, and powdered. METHODS AND RESULTS: A high-fat diet with Japanese yam propagules is administered to mice for 4 weeks. High-fat loading induces a decline in respiratory quotient, and a high-fat diet with propagules reduces it more. This result suggests that propagules increase fat oxidation, indicating fat utilization. The hepatic transcriptome is analyzed using a DNA microarray. Some of the genes affected by high-fat loading are reversed by simultaneous ingestion of propagules. Such genes are mainly involved in the immune system and fat metabolism. High-fat loading induces hepatic inflammation, which is repressed by simultaneous ingestion of propagules. For lipid metabolism, propagules repress an increase in cholesterol biosynthesis and catabolism by high-fat loading. Regarding carbohydrate metabolism, propagules decrease glycolysis and glycogen synthesis and increase gluconeogenesis. Moreover, amino acids are converted into pyruvate and then used for gluconeogenesis. CONCLUSION: Propagules act to delay the occurrence of hepatic disease by suppressing carbohydrate and fat metabolism disorders in high-fat loaded mice.

Hypocholesterolemic effect of peanut skin and its fractions: a case record of rats fed on a high-cholesterol diet.

Peanut skin (PS) is characterized by almost exclusively consisting of polyphenols and fiber. We fractionated PS into a water-soluble fraction (WSF) and water-insoluble fraction (WIF), and further fractionated WSF into a soluble dietary fiber fraction (DF) and dietary fiber-free, water-soluble fraction (DFF-WSF). Male Sprague-Dawley rats were fed on high-cholesterol diets supplemented with PS and its fractions. PS, WSF, and DFF-WSF decreased the serum lipid and cholesterol levels and increased those in feces. This effect was probably due to the polyphenols that inhibited intestinal cholesterol absorption.

Serum cholesterol-decreasing effect of heat-moisture-treated high-amylose cornstarch in cholesterol-loaded rats.

Rats were fed on a diet containing cholesterol (Chol) at a level corresponding to the standard Chol intake in humans, and the influence of heat-moisture-treated high-amylose cornstarch (HHA) on their serum Chol level was investigated. HHA decreased the serum level of Chol in rats fed on the diet containing 0.1% Chol, which corresponds to a Chol intake in humans of 800 mg/d, although the liver levels of Chol increased in these rats. HHA did not influence the fecal excretion of Chol/bile acids. It is possible that the decrease in serum Chol level in the rats fed on the high-Chol diet can be attributed to the promotion of Chol uptake in the liver.

Increase of serum cholesterol levels by heat-moisture-treated high-amylose cornstarch in rats fed a high-cholesterol diet.

The effects of four cornstarches containing various contents of resistant starch on serum and liver cholesterol levels in rats fed a high-cholesterol diet were investigated. Male Sprague Dawley rats (aged 4 weeks) were divided into four groups (n = 7) and fed high-cholesterol diets containing 15% of cornstarch (CS), heat-moisture-treated CS (HCS), high-amylose CS (HA), or heat-moisture-treated HA (HHA) for 21 days. The results showed that the serum and hepatic level of total cholesterol, LDL-cholesterol, and triglyceride in rats of the HHA group and their arteriosclerosis index were significantly higher, suggesting that HHA increases the risk of arteriosclerosis under a high-cholesterol dietary condition. No significant between-group differences were noted in the levels of plasma mevalonic acid and hepatic HMG-CoA reductase mRNA, whereas fecal cholesterol excretion was significantly higher in the HHA group, indicating that the elevation of the serum and liver cholesterol levels was not due to the promotion of liver cholesterol synthesis and cholesterol absorption in the intestine.

Blood substrates and hormonal responses to increased egg white protein intake prior to a 12,000 m run in heat.

This study was undertaken to investigate the effects of isoenergetic and increased amounts of egg white protein one hour before a run on the changes in the post-exercise blood biochemistry and the rating of the perceived exertion (RPE). Twenty-four male distance runners were divided into four groups. Venous blood samples were collected at three time points: just before the experiment (Pre), just after a 12,000 m run (Post 0 h) and one hour after the run (Post 1 h). After the first blood sampling, each participant consumed one of the four isoenergetic supplements (86 kcal); 0 g, 5 g, 10 g, or 20 g of egg white protein. The blood glucose, free amino acid, and branched chain amino acid (BCAA) levels in the 0 g, 5 g, and 10 g protein groups were higher at Post 0 h than at Pre. The pre-exercise intake of the 20 g protein group showed the smallest changes in the blood biochemicals. The RPE scores were significantly higher at Post 0 h, and did not vary among the four protein groups. Accordingly, the pre-exercise carbohydrate intakes significantly altered the post-exercise blood biochemisty findings, but the pre-exercise protein intake did not. Furthermore, the changes in the RPE scores in our present study were not explained by changes in the serum free tryptophan or the BCAA levels, and an increased dietary intake of egg white protein might not prevent post-exercise increases in the RPE scores.

Gliadain, a gibberellin-inducible cysteine proteinase occurring in germinating seeds of wheat, Triticum aestivum L., specifically digests gliadin and is regulated by intrinsic cystatins.

We cloned a new cysteine proteinase of wheat seed origin, which hydrolyzed the storage protein gliadin almost specifically, and was named gliadain. Gliadain mRNA was expressed 1 day after the start of seed imbibition, and showed a gradual increase thereafter. Gliadain expression was suppressed when uniconazol, a gibberellin synthesis inhibitor, was added to germinating seeds. Histochemical detection with anti-gliadain serum indicated that gliadain was present in the aleurone layer and also that its expression intensity increased in sites nearer the embryo. The enzymological characteristics of gliadain were investigated using recombinant glutathione S-transferase (GST)-progliadain fusion protein produced in Escherichia coli. The GST-progliadain almost specifically digested gliadin into low molecular mass peptides. These results indicate that gliadain is produced via gibberellin-mediated gene activation in aleurone cells and secreted into the endosperm to digest its storage proteins. Enzymologically, the GST-progliadain hydrolyzed benzyloxycarbonyl-Phe-Arg-7-amino-4-methylcoumarin (Z-Phe-Arg-NH(2)-Mec) at K(m) = 9.5 microm, which is equivalent to the K(m) value for hydrolysis of this substrate by cathepsin L. Hydrolysis was inhibited by two wheat cystatins, WC1 and WC4, with IC(50) values of 1.7 x 10(-8) and 5.0 x 10(-8) m, respectively. These values are comparable with those found for GST-progliadain inhibition by E-64 and egg-white cystatin, and are consistent with the possibility that, in germinating wheat seeds, gliadain is under the control of intrinsic cystatins.

Crystal structure of neoculin: insights into its sweetness and taste-modifying activity.

Although the majority of sweet compounds are of low molecular mass, several proteins are known to elicit sweet taste responses in humans. The fruit of Curculigo latifolia contains a heterodimeric protein, neoculin, which has both sweetness and a taste-modifying activity that converts sourness to sweetness. Here, we report the crystal structure of neoculin at 2.76A resolution. This is the first well-defined tertiary structure of a taste-modifying protein of this kind. The overall structure is quite similar to those of monocot mannose-binding lectins. However, crucial topological differences are observed in the C-terminal regions of both subunits. In both subunits of neoculin, the C-terminal tails turn up to form loops fixed by inter-subunit disulfide bonds that are not observed in the lectins. Indeed, the corresponding regions of the lectins stretch straight over the surface of another subunit. Such a C-terminal structural feature as is observed in neoculin results in a decrease in subunit-subunit interactions. Moreover, distribution of electrostatic potential on the surface of neoculin is unique and significantly different from those of the lectins, particularly in the basic subunit (NBS). We have found that there is a large cluster composed of six basic residues on the surface of NBS, and speculate that it might be involved in the elicitation of sweetness and/or taste-modifying activity of neoculin. Molecular dynamics simulation based on the crystallography results suggests that neoculin may adopt a widely "open" conformation at acidic pH, while unprotonated neoculin at neutral pH is in a "closed" conformation. Based on these simulations and the generation of a docking model between neoculin and the sweet-taste receptor, T1R2-T1R3, we propose the hypothesis that neoculin is in dynamic equilibrium between open and closed states, and that the addition of an acid shifts the equilibrium to the open state, allowing ligand-receptor interaction.

Molecular cloning and characterization of an alpha-amylase from Pichia burtonii 15-1.

An alpha-amylase secreted by Pichia burtonii 15-1 isolated from a traditional starter murcha of Nepal, named Pichia burtonii alpha-amylase (PBA), was studied. The gene was cloned and its nucleotide sequence was determined. PBA was deduced to consist of 494 amino acid residues. It shared certain degrees of amino acid sequence identity with other homologous proteins: 60% with Schwanniomyces occidentalis alpha-amylase, 58% with Saccharomycopsis sp. alpha-amylase, and 47% with Taka-amylase A from Aspergillus oryzae. A three-dimensional structural model of PBA generated using the known three-dimensional structure of Taka-amylase A as a template suggested high structural similarity between them. Kinetic analysis revealed that the K(m) values of PBA were lower than those of Taka-amylase A for the oligosaccharides. Although the k(cat) values of PBA were lower than those of Taka-amylase A for the oligosaccharide substrates, the k(cat)/K(m) values of PBA were higher.

Quantitative deviating effects of maple syrup extract supplementation on the hepatic gene expression of mice fed a high-fat diet.

SCOPE: Maple syrup contains various polyphenols and we investigated the effects of a polyphenol-rich maple syrup extract (MSXH) on the physiology of mice fed a high-fat diet (HFD). METHODS AND RESULTS: The mice fed a low-fat diet (LFD), an HFD, or an HFD supplemented with 0.02% (002MSXH) or 0.05% MSXH (005MSXH) for 4 weeks. Global gene expression analysis of the liver was performed, and the differentially expressed genes were classified into three expression patterns; pattern A (LFD < HFD > 002MSXH = 005MSXH, LFD > HFD < 002MSXH = 005MSXH), pattern B (LFD < HFD = 002MSXH > 005MSXH, LFD > HFD = 002MSXH < 005MSXH), and pattern C (LFD < HFD > 002MSXH < 005MSXH, LFD > HFD < 002MSXH > 005MSXH). Pattern A was enriched in glycolysis, fatty acid metabolism, and folate metabolism. Pattern B was enriched in tricarboxylic acid cycle while pattern C was enriched in gluconeogenesis, cholesterol metabolism, amino acid metabolism, and endoplasmic reticulum stress-related event. CONCLUSION: Our study suggested that the effects of MSXH ingestion showed (i) dose-dependent pattern involved in energy metabolisms and (ii) reversely pattern involved in stress responses.

Neoculin, a taste-modifying protein, is recognized by human sweet taste receptor.

Neoculin, a sweet protein occurring in Curculigo latifolia, is unique in that it also has taste-modifying activity capable of converting sourness to sweetness. Calcium imaging analysis with HEK cells expressing the human sweet taste receptor, hT1R2/T1R3 demonstrated that the intracellular calcium concentration increased following the addition of 20 microM neoculin. The use of lactisole, a blocker of hT1R3, inhibited the intracellular calcium concentration increase almost completely. In sensory tests, when acetate buffers with different pH values were placed on the tongue after tasting neoculin, a higher intensity of sweetness was detected at lower pH. The sweetness was also suppressed with the addition of lactisole. These results suggest that both the sweetness and the taste-modifying activity are mediated via the human sweet taste receptor.