Efficacy of Soy Product, Soy Protein or Soy Isoflavone, to Hypercholesterolemia and Osteoporosis: Its Molecular Biomedical Studies in Monkeys (Part II)
1Primate Research Institute, Kyoto University, Inuyama 484-8506 2INA Research /INA Research Philippines, Nagano 399-4501
ABSTRACT
Soy products are known to protect age-related disfunctions such as
hypercholesterolemia and osteoporosis in postmenopausal women. In current study
we examined an efficiency of soy protein (β-Conglycinine; β-CG) or soy isoflavone
(Soyaflavone HG; IF), which are expected to be functional component(s) in soy
products, to these age-related disfunctions. In this current study we examined
efficacy of the soy components of β-CG and IF to hypercholesterolemia or
osteoporosis in primate model. Ovariectomy (OVX) Cynomlgus macaques were
developed for model of postmenopausal age-related hypercholesterolemia and/or
osteoporosis. β-Conglycinine revealed significantly protective and/or therapeutic
effect to hypercholesterolemia in OVX-monkeys, while Soyaflavone did not showed any
effect. In PBMC LDL-R, VLDL-R and UCP-2 genes that associate with lipid
metabolism, were up-regulated by β-Conglycinine, but not Soyaflavone. In liver, β-
Conglycinine affected to up-regulate ADH7, HSP70, PPAR-γ, and GDF15 genes and to
down-regulate HSP60, CYP3A43, CYP3A4, SULT, AGTR1, SOD2, IL-7, and IL18 genes.β-Conglycinine showed significant effect to protect bone-mineral-density (BMD) loss
in OVX-osteoporosis monkeys. However Soyaflavone had little effect to prevent BMD
loss even though its preventing effect had been reported in previous studies using nonprimate
model. These results suggest that β-Conglycinine, but not Soyaflavone, is
functionally potential component to protect both of hypercholesterolemia and
osteoporosis. These also imply that β-Conglycinine or its enriched-food is a possible candidate for alternative-complementary medicine to these age- and/or life stylerelated disorders. Soy Protein Research, Japan8, 1-7, 2005.
Key words : β-Conglycinine, Isoflavone, Hypercholesterolemia, Osteoporosis, Postmenopause, Monkey model
Improvement of Methionine Contents in Soybean Through Regulation of Metabolism
Toru FUJIWARA1 and Masao ISHIMOTO2
1Biotechnology Research Center, The University of Tokyo, Tokyo 113-8657 2National Agricultural Research Center for Hokkaido Region, Sapporo 062-8555
ABSTRACT
Methionine biosynthesis in plants is regulated mainly at the step of cystathionine-γsynthase. The exon 1 of cystathionine-γ-synthase gene Arabidopsis is known responsible for maintenance of methionine levels in Arabidopsis. Mutations in exon 1 are reported that result in overaccumulation of methionine. It is also reported that overexpression of cystathionine-γ-synthase in Arabidopsis thaliana results in overaccumulation of methionine. In the present study, to improve methionine contents in soybean, chimeric genes were generated for overexpression of Arabidpsis cystathionine-γ-synthase in soybean. The constructs were used for transformation of azuki-bean and soybean. Transgenic azuki plants were generated and introduction of foreign genes were confirmed with Southern hybridization analysis. Analysis of methionine accumulation of the transgenic plants will be analyzed in the near future. Soy Protein Research, Japan8, 8-10, 2005.
Key words : methionine, transformation, cystathionine-γ-synthase
Study on Biosynthetic Enzymes of Group-A Saponin for the Breeding of Low Unpleasant Taste Soybean Variety
Masakazu SHIRAIWA
School of Agriculture, Ibaraki University, Ibaraki 300-0393
ABSTRACT
While group-A saponin is the main substance responsible for the unpleasant taste in soybean, group-DDMP saponin has various medicinal action. Therefore, it is expected that elucidation and characterization of the enzymes which catalyze biosynthetic reaction of saponins will be useful to the breeding of the value-added soybean variety in the future. This research was aimed at cDNA cloning of the key enzymes (glucuronosyltransferase and hydroxylase) of group-A saponin biosynthesis that we had discovered, previously. Some soybean EST clones that showed high similarities to glucuronosyltransferase (PsUGT1) from pea were found in the DNA databases. An open reading frame (ORF) was generated based on the nucleotide sequences of these EST, showing a homology to PsUGT1 with 69%. We presumed that the putative sequences encords a glucuronosyltransferase in soybean. On the other hand, sixteen cDNA clones for hydroxylase were also found. Comparing the converted amino acid sequences among them, the conserved region in all was found. In addition, when group-A saponin in 18 wild relatives of soybean was analyzed, three species (G. tabacina, G. tomentella, and G. wightii) that group-A saponin is deleted was revealed. These findings suggest the possible use of plant materials for cDNA cloning of hydroxylase involved in biosynthesis of group-A saponin in soybean. Soy Protein Research, Japan8, 11-18, 2005.
Key words : BLAST search, glucuronosyltransferase, hydroxylase, wild relatives of soybean, saponin biosynthesis, soybean
1Graduate School of Agriculture, Kyoto University, Uji 611-0011 2National Agricultural Research Center for Hokkaido Region, Sapporo 062-8555
ABSTRACT
We previously reported that the 10 residues C-terminal (α': PLSSILRAFY, β: PFPSILGALY) of β-conglycinin contains a vacuolar sorting determinant (VSD). VSDs identified so far are classified into C-terminal VSDs (ctVSD) and sequence specific VSDs (ssVSD). In this study, we studied the characteristics of the VSDs ofβ-conglycinin in detail. First, we examined whether a partial sequence of the 10 residues C-terminal of theα' subunit works as a VSD. Both the 4 residues N-terminal and the 6 residues Cterminal of the 10 residues C-terminal ofα' worked as a VSD. Since the addition of contiguous Gly residues at the C-terminus inhibits the function of a ctVSD, the effect of the addition of six contiguous Gly residues was examined. The 6 residues C-terminal lost the function of a VSD by the addition of Gly, whereas the 10 residues C-terminal worked as a VSD despite the addition of Gly. Furthermore, the 10 residues C-terminal worked as a VSD at the N-terminus of the reporter protein. These results suggest that the 10 residues C-terminal contains an ssVSD. The deletion and mutation experiments showed that Ile and Leu within the 10 residues C-terminal (these residues are indicated in bold: PLSSILRAFY) are functionally important to an ssVSD. On the other hand, only three amino acids (AFY) of the C-terminal could work as a ctVSD. Thus, the 10 residues C-terminal might contain a ctVSD and an ssVSD. Finally, we examined the structural features of the ctVSD. Systematic mutation analyses showed that the existence of two residues having relatively large side chains within the 4 residues C-terminal are important for the function of the ctVSD. Soy Protein Research, Japan8, 19-23, 2005.
Key words : Vacuolar sorting determinant, β-Conglycinin, seed storage protein
Expression Analysis of Genes Encoding Enzymes Involved in the Biosynthesis of Isoflavones in Soybean
Tomoyoshi AKASHI, Toshio AOKI and Shin-ichi AYABE
College of Bioresource Sciences, Nihon University, Fujisawa 252-8510
ABSTRACT
Isoflavones of leguminous plants are biosynthesized from the general flavonoid precursors (flavanones) by the action of 2-hydroxyisoflavanone synthase (IFS) and 2-hydroxyisoflavanone dehydratase (HID). In soybean, IFS cDNAs (IFS1 and IFS2) have already been identified, but genes/enzymes of HID have not been characterized. In this study, a cDNA encoding HID of soybean was cloned, and the relationship between production of isoflavones and expression of the genes of the pathway was analyzed. Using the sequence information of cDNA of an HID producing 4'-methoxylated isoflavone (formononetin) of licorice (Glycyrhhiza echinata, Fabaceae), an HID cDNA was cloned from soybean seedlings by reverse transcription (RT)-PCR. Recombinant soybean HID protein expressed in E. coli had a broad specificity and produced both 4'-hydroxylated and 4'-methoxylated isoflavones from 2-hydroxyisoflavanones. Expression of genes encoding enzymes involved in isoflavone biosynthesis was also analyzed by RT-PCR. IFS1, IFS2 and HID mRNAs were expressed in soybean seedlings. In seedlings, accumulation of isoflavones, genistein, daidzein glycitein, and expression of IFS1 and HID mRNAs were coordinately elevated. Breeding of soybeans producing high levels of isoflavones based on the gene expression information will be expected in the future. Soy Protein Research, Japan8, 24-29, 2005.
Regulation of Gene Expression of Soybean Flavonoid Biosynthetic Enzymes by cGMP
Kenji SUITA and Hiroshi YAMAGATA
Graduate School of Science and Technology and Faculty of Agriculture,
Kobe University, Kobe 657-8501
ABSTRACT
Anthocyanin content in soybean photomixotrophic cell culture (SB-P) was increased with 8-Br-cGMP as well as UV-B illumination. Several cDNAs encoding enzymes involved in flavonoid biosynthetic pathway were cloned from soybean cDNA library. Northern hybridization analyses revealed that the expressions of most genes except for an anthocyanidin synthase gene were induced by cGMP and light. Using PCR-based subtraction techniques, several genes were isolated as cGMP-induced genes. Northern analysis confirmed that genes encoding chalcone reductase (chr) and isoflavone reductase that are involved in phytoalexin synthesis were clearly induced by cGMP. The promoter of chr was cloned by inversed PCR. The analysis of transient expression of chr-GUS using particle bombardment indicated that upper region in the promoter from -998 to -576 bp from the possible transcription start is likely to be responsible for the induction of the gene expression by cGMP. Soy Protein Research, Japan8, 30-34, 2005.
Key words : soybean, flavonoid, gene expression, cGMP, phototransduction
Improvement of Taste of Soy Sauce by the Addition of Salt-Tolerant γ-Glutamyltranspeptidase as Glutaminase During its Fermentation
Hideyuki SUZUKI and Kyoko KIJIMA
Graduate School of Biostudies, Kyoto University, Kyoto 606-8502
ABSTRACT
Glutaminase of Asperigillus oryzae is not salt-tolerant and there is almost no remaining activity in the presence of 18% salt. Therefore, not all glutamine released from soy protein during soy sauce fermentation is converted to glutamic acid which is the critical amino acid for its taste, but some are converted to tasteless pyroglutamic acid spontaneously. γ-Glutamyltranspeptidase (GGT) has glutaminase activity and GGT from Bacillus subtilis is quite salt-tolerant. The effect of the addition of B. subtilis GGT to soy sauce fermentation was investigated. Without the addition of GGT, 70 mM glutamic acid was accumulated after 4 months of fermentation, while 95 mM glutamic acid was found by the addition of 1 U/1.4 L GGT. Taste of soy sauce was evaluated after 6 months of fermentation. It was found that difference of 25 mM glutamic acid in soy sauce was difficult to distinguish and that more than 50 mM difference was required. Soy
Protein Research, Japan8, 35-38, 2005.
Key words : γ-glutamyltranspeptidase, glutaminase, salt-tolerant, soy sauce
Increased Solubility of Soybean Protein by Dried Egg White and Its Functional Property
Makoto SHIMOYAMADA1,2, Aya IWASAKI1 and Ryo YAMAUCHI1
1Faculty of Applied Biological Sciences, Gifu University, Gifu 501-1193 2School of Food, Agricultural and Environmental Sciences, Miyagi University, Sendai 982-0215
ABSTRACT
Dry-heating, namely heat treatment under very low water content, was reported to improve functional properties of egg white. In this study we applied dry-heating to soybean proteins. However, acid precipitated protein (APP) of soybean was insolubilized by dry-heating. In the previous papers, egg white ovalbumin was reported to allow ovotransferrin to solubilize even after the dry-heating. So we investigated the solubilization of APP with egg white. When APP, which was extracted from defatted soybean seeds with distilled water at 30°C, was mixed with egg white, it was highly solubilized even after the dry-heating. From SDS-PAGE patterns, especially α- and α'-subunits of β-conglycinin of the APP were partially hydrolyzed. Chymotryptic hydrolysis of APP showed comparable solubility after the dry-heating. From gel filtration chromatograms of the dry-heated APP, partial hydrolysis of APP suppressed aggregate formation and resulted in insolubilization. Further, the dry-heated APP, which was previously hydrolyzed by chymotrypsin and mixed with egg white was subjected to heat-induced gel formation. The resulting gel was a harder gel compared to non-treated APP gel and softer than dry-heated APP gel. Soy Protein Research, Japan8, 39-43, 2005.
Key words : soy protein, dry-heating, solubility, protease, egg white
Alteration of Components and Tastes of Soybean during Low Temperature Steam Cooking
Naoko ITO, Takako YAMAZAKI, Yasuo HOTTA and Atsuko MURAYAMA
Department of Health and Nutrition, School of Health Sciences, Niigata University of Health and Welfare, Niigata 950-3198
ABSTRACT
We have raised a nutritional value of soybean in low temperature steam cooking, presenting an alteration of chemical components and improved taste through sensory tests. A major sugar in soybean is maltose and no quantitative change after steaming has been observed. The analysis of soluble proteins using SDS-PAGE pattern indicated that every protein has an individual temperature dependent hydrolytic pattern. N-terminal amino acid analysis of 50 kDa protein in 75°C steaming and 37 kDa protein in 85°C indicated that β-conglycinin and glycinin were the products among them, respectively. Their endogenous protease activity is higher at 85 °C than fresh, at 75°C, or 100°C steaming, and thus contributed to increased umami in soybean. Furthermore, the amount of remaining isoflavones after this low temperature steaming at 85°C was higher than at 100°C steaming or boiling. Rupture stress test indicated that the material steamed at 75°C presented as high value as fresh soybeans, but at 85°C it became lower after a longer time lapse. Sensory tests indicated that the steaming above 85°C were almost similar to general boiling and steaming at 100°C. We have concluded that the low-temperature steaming is useful for better method(s) of cooking to increase umami and nutritional function of soybean. Soy Protein Research, Japan8, 44-49, 2005.
Key words : low temperature steam cooking, protease, isoflavone, rupture stress, sensory test
Investigation of Phospholipase D Catalytic Functions and Its Application for Improvement of Phospholipids
Chiaki OGINO
Department of Chemistry and Chemical Engineering, Kanazawa University, Kanazawa 920-1192
ABSTRACT
Phospholipase D (PLD; EC 3.1.4.4) is an enzyme that occurs ubiquitously in various organisms including mammals, plants, yeast and bacteria. The enzyme catalyzes two types of reactions: hydrolysis of phosphatidylcholine (PC), a major substrate of PLD, to phosphatidic acid (PA) and choline (hydrolytic reaction), and transfer of polar head groups to others (transphosphatidylation reaction). By using transphosphatidylation reaction, PLD can convert to functional phospholipids from abundant phospholipid, PC. For example, PLD converts to phosphatidylserine (PS) from PC with serine molecule. Recently, PS is known to improve memory performance in patients suffering from age-associated memory impairment or Alzheimer's disease. Therefore, the transphosphatidylation activity of PLD is focused as phospholipid modification catalysis. However, PLD cannot recognize all type of substrate and it is limited some kind of substrates. Thus, the improvement of PLD substrate specificity should be necessary. In our laboratory, we have already identified the Actionmycetes strain, Streptoverticillium cinnamoneum, which showed the high PLD producing potential. Then, we have determined the gene encoding this strain's PLD, and constructed the recombinant expression system of PLD in Escherichia coli. In this research, PLD catalytic function was investigated by using this recombinant expression system. As a result, some mutants, that were substituted with serine amino acid at 215th and 216th, exhibited high transphosphatidylation activity compared to wild type PLD. Additionally, the small conformation change, which altered the catalytic properties, was observed by combining the structural prediction software and these mutants library. This portion, which consisted of one flexible loop, is very closely related to the putative catalytic center, and behaves like the lid structure of catalytic center. From these observations, it seemed that the catalytic function was dominated by the loop structure conformation. Therefore, we will try to investigate relationship between the lid structure and the catalytic function in near future. Soy Protein Research, Japan8, 50-55, 2005.
Key words : Phospholipase D (PLD), transphosphatidylation, phospholipid
Analysis of Profile of Neurotransmitter in Hypothalamus and Blood Signals for Mechanism of Decreasing Food Intake of Rats Fed Soy Isoflavones
Taro KISHIDA, Takafumi MIZUSHIGE and Kiyoshi EBIHARA
Faculty of Agriculture, Ehime University, Matsuyama 790-8566
ABSTRACT
Previously, we showed that fermented soy extract, rich in soy isoflavone, decreased food intake specifically in female rats. We have aimed to confirm the active substance and examine the mechanism. In the present study, we showed that daidzein, one of the major soy isoflavone, is just the active substance with decreasing effect on food intake. We also found that daidzein administration decrease only in female rats with or without ovariectomy, but an estradiol administration decreased food intake in male and ovariectomized rats. We speculated that the effect of daidzein could not simply be related to estrogenic effect of isoflavones. We examined the effect of daidzein on the hypothalamus gene expression of appetite-related neurotransmitter in ovariectomized rats, but there is no explanation of the decrease of food intake by daidzein administration. We have searched more sensitive experimental method for examining the hypothalamus gene expression of appetite-related neurotransmitter. Leptin is one of the major food intake decreasing signal agents in blood. But in the present study, we observed that daidzein administration rather decreased serum leptin concentration. Soy Protein Research, Japan8, 56-62, 2005.
Key words : soy isoflavone, daidzein, hypothalamus neurotransmitter, leptin, rat
Effect of Genistein on the Antigen-Specific Immune Responses in Mice
Tohru SAKAI1,2, Mari KOGISO2, Kaori MITSUYA2,
Tatsushi KOMATSU1 and Shigeru YAMAMOTO2
1Department of Clinical Nutrition, Osaka Prefecture University, Habikino 583-8555 2Institution of Health Bioscience, The University of Tokushima Graduate School, Tokushima 770-8503
ABSTRACT
The isoflavone genistein is a phytoestrogen found in high levels in soy products that has been associated with decreased incidences of breast and prostate cancers. In this study, we examined effect of genistein on the antigen-specific immune responses in ovalbumin (OVA)-immunized BALB/c mice. Groups of mice were exposed to vehicle or genistein by gavage. The doses of genistein used were 4 and 20 mg/kg body/day. OVA-specific proliferative responses, IFN-γ and IL-4 productions were decreased in genistein administered mice compared to those in control mice without decreasing responses to anti-CD3 mAb. OVA-specific IgG1, IgG2a and IgG2b productions were also decreased in genistein administered mice. However, genistein administration did not influence the TNP-Ficoll-specific IgM and IgG levels. These findings suggest that genistein suppresses antigen-specific immune responses by suppressing the induction of antigen-specific T cells. Moreover, treatment with ICI 182, 780 decreased OVA-specific proliferative responses, but genistein did not suppress these responses synergistically in ICI 182, 780 treated mice. The mechanism for the suppression of T cell induction by genistein is responsible for the competition with endogenous 17β-estradiol for estrogen receptors. Soy Protein Research, Japan8, 63-70, 2005.
Key words : genistein, mice, estrogen receptor, antigen, T cell
Molecular Mechanism of Unloading-Mediated Muscle Atrophy and Its Inhibition by Treatment with Soy Protein-Derived Oligopeptide
Yuko ONISHI, Katsuya HIRASAKA, Takeshi NIKAWA*, Jyunpei GOTOH, Kimiyo HISADA, Harumi FUROCHI and Kyoichi KISHI
Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima 770-8503
ABSTRACT
Skeletal muscle unloading during prolonged bed rest, paralysis, or spaceflight can result in debilitating skeletal muscle atrophy. Reduced muscle mass is characterized by a combination of decreased protein synthesis and increased protein degradation. Currently, there is no treatment to reverse the progression of atrophy. Here we show that Cbl-b, a RING-type member of the ubiquitin ligase family, is essential for skeletal muscle atrophy. Expression of Cbl-b in vivo induces significant atrophy in rat tibialis anterior muscle. In contrast, Cbl-b deficient mice are resistant to muscle atrophy and dysfunction induced during unloading by tail suspension. Upon tail suspension or zero gravity, Cbl-b interacts with and thereby degrades the skeletal muscle growth factor signaling intermediate IRS-1. Thus, Cbl-b activation appears to underlie the refractoriness of atrophic muscle tissue to growth factor stimulation by IGF/insulin and likely accounts for the overall decrease in protein synthesis observed during muscle wasting. These data suggest that Cbl-b may be a novel target for the development of therapeutics aimed at the preservation of muscle mass and function during a variety of muscle wasting diseases. In addition, soy protein-derived peptides inhibit ubiquitination and degradation of IRS-1 caused by Cbl-b in mouse myoblastic cells. Our results suggest that soy protein may contain bioactive factors for regulating proteolysis of IRS-1 in skeletal muscle. Soy Protein Research, Japan8, 71-75, 2005.
Key words : soybean, peptide, angiotensin I-converting enzyme inhibitor, whey protein
Effects of Soy Bean Ingredients on Lipid Metabolism Through the Activities of Nuclear Receptors
Ryuichiro SATO
Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657
ABSTRACT
Although it has been reported that soy products improve lipid metabolism, little is known about the molecular mechanism of the effects. Isoflavones having estrogen-like activities are not enough to explain the whole functions of soy products. In this study we examined the ligand activity of isoflavones to several nuclear receptors including FXR, LXR, PPAR, RXR and HNF-4, which are deeply involved in regulation of lipid metabolism. For this purpose we established a new assay system to evaluate the ligand activity using 96-well plates. The ligand-binding domain of each nuclear receptor was fused to GST, and this fusion protein was expressed in E. coli and purified. The coactivator protein fused to alkaline phosphatase was also prepared. This system takes advantage of the recruitment of the coactivator protein to the ligand-binding domain in the presence of the ligand. Using this assay system we examined the ligand-binding activity of several isoflavones to these nuclear receptors. Some isoflavones were found to be able to bind to LXR weekly. By using other nuclear receptor ligand-binding domains this new assay will enable us to reveal novel functions of isoflavones. Soy Protein Research, Japan8, 76-80, 2005.
Key words : isoflavones, nuclear receptor, lipid metabolism
Graduate School of Science, Osaka University, Toyonaka 560-0041
ABSTRACT
Membrane interaction of soyasaponins, which are reported to possess nutrient and pharmacological effects such as antioxidation, liver-protecting actions, and anti-hypercholesteremia. To obtain the molecular basis for these soyasaponin activities, the present study attempted to investigate the molecular interactions between soyasaponin (Soyhealth SA) and membrane lipids, particularly cholesterol. NMR and MS analysis of the sample soyasaponins indicated the major constituent to be soyasaponin Bb (I). Two kinds of labeled sterols, 3-deutero- and 6-fluoro-cholesterol, which were chemically synthesized from cholesterol, were reconstituted in phosphatidylcholine membrane. The broad band solid-state NMR spectra of these membrane preparations revealed that cholesterol interacted with the saponin in phosphatidylcholine bilayers on the basis of NMR signals characteristic of less oriented and flexible membrane constituents. The NMR spectra of liposomes incorporated with the fluorinated cholesterol also inferred the possibility of their interaction although the high mobility of lipids in liposome membrane obscured their molecular recognition. Further studies for evaluating the molecular interaction of soyasaponins with membrane are currently underway. Soy Protein Research, Japan8, 81-85, 2005.
Key words : soy saponins, membrane activity, solid state NMR
Effects of Soy Protein Isolate Diet on Rat Skin Proteoglycan
Yuichi OISHI
College of Bioscience and Biotechnology, Chubu University, Aichi 487-8501
ABSTRACT
Proteoglycan is a class of glycosylated protein characterized by the presence of glycosaminoglycan (GAG) as a carbohydrate component in the skin. GAGs are enzymatically epimerized and sulfated such as N-deacetylase/N-sulfotransferase (NDST1) and heparan sulfate 2-sulfotransferase (HS2ST). Syndecan 4, whose core protein is plasma membrane-spanning, is abundant in the skin. This proteoglycan combines with many factors such as growth factors, cytokines, and proteases, which regulates cell proliferation, the activity of proteins. To examine whether and how protein diet or peptide diet affects the status of skin proteoglycan, mRNA levels of Syndecan 4 core protein, NDST1 and HS2ST were analyzed in the skin of rat fed a 12% soy protein isolate (SPI, Fujipro) diet, 12% soybean peptide (Spep, Hinute D1) diet, and 12% soybean soluble peptide (SSP, Hinute D3) diet. The mRNA levels of NDST1 and HS2ST in rats fed a Spep diet for 1 week were similar to those in rats fed a 12% casein diet. When SPI diet and SSP diet had been given, these mRNA levels were reduced. The mRNA level of syndecan 4 core protein was not reduced. These results suggest that soybean insoluble peptide increases the mRNA levels of NDST1 and HS2ST and the regulation of syndecan 4 core protein is different from those of its transferases. Soy Protein Research, Japan8, 86-89, 2005.
1School of Life Studies, Sugiyama Jogakuen University, Nagoya 464-8662 2Graduate School of Agriculture, Kyoto University, Kyoto 611-0011
ABSTRACT
The aim of this study was to investigate the effects of chronic feeding dietary protein on glucose tolerance during weight gain induced by high fat diet in IRC mice. Protein sources of high fat diets were casein, soy protein isolate (SPI), egg white (EGW) and collagen hydrolysate (CLP). Oral glucose tolerance test showed that the serum glucose concentration in SPI fed mice gradually increased compared to casein fed mice and insulin concentration in casein fed mice were higher than that in SPI fed mice at 60 min after glucose administration. The concentraitions of acetoacetic acid were smaller in SPI fed mice compared to casein fed mice. Total ketone bodies, acetoacetic acid and 3-hydroxybutyric acid were smaller in EGW fed mice compared to casein fed mice. The protein expression of glucose transporter 4 in total gastrocnemius muscle was significantly higher in SPI fed mice compared to CLP fed mice. These results indicate that chronic feeding of SPI in high fat diet delayed the absorption of orally administered glucose and may prevent development of insulin resistance in high fat fed mice. Soy Protein Research, Japan8, 90-96, 2005.
Key words : glucose absorption, high fat diet, soybean
Isolation and Application of Physiologically Active Peptides from Soybean Whey and Okara Proteins (Part III)
Masami YONEKURA, Taeko ICHIMURA and Miki NISHIKAWA
School of Agriculture, Ibaraki University, Ibaraki 300-0393
ABSTRACT
In this study, angiotensin I-converting enzyme inhibitory peptides were isolated from protease S digests of soybean whey proteins by ethanol fractionation, gel filtration on Sephadex G-25, and repeated reverse phase HPLC. In addition the amino acid sequences of the peptides were determined. Antihypertensive effects of three tripeptides, VAP, VKP and VTP were investigated in spontaneously hypertensive rat (SHR). Single oral administration (100 mg/kg of body weight) of these peptides resulted in a significant reduction of systolic blood pressures in SHR. Moreover, antioxidative activities of 6 digests by thermolysin, papain, pepsin, trypsin and protease S and N were determined by inhibitory activities against the linoleic acid peroxidation and the oxidative stress-induced cell death of human monocytic leukemia U937 cell line. The trypsin, protease N and thermolysin digests showed potent antioxidative activities against lipid peroxidation, and protease N and thermolysin digests reduced the cell death induced by oxidative stress. These results suggest that the soybean whey peptide preparation containing VAP, VKP and VTP prepared by protease S digestion and the protease digests of soybean whey proteins may be promising materials for the development of a functional food with antihypertensive activity or antioxidative activity. Soy Protein Research, Japan8, 97102, 2005.
1Faculty of Human and Cultural Studies, Tezukayama Gakuin University, Sakai 590-0113 2Fuji Oil Co., LTD., Izumisano 598-8540
ABSTRACT
(1) Regulation of the gene expressions of uncoupling protein (UCP)-1, 2, 3 was investigated in the white and brown adipose tissues of rats fed diets containing various fats (hydrogenated fat or corn oil) and proteins (casein or soy protein isolate, SPI) for 2 wk. The mRNA and protein concentrations of UCP-1, 2, 3 were elevated in the white adipose tissues of the rats fed SPI compared to those fed casein, and those of UCP-2 were particularly elevated by corn oil/SPI diet. Moreover, in the fasted-refed experiment of rats, the UCP-2 induction in the white adipose tissues reached maximal levels at 2 h. The UCPs were rapidly induced. (2) In cultured white adipocytes, the mRNA expressions of UCPs were stimulated by the hydrophilic soy peptides (MW<1,000). (3) By giving triiodothyronine treatment to rat, the UCP-2 mRNA and protein were induced in the white adipose tissue and the UCP-1, in the brown adipose tissue. The thyroid hormone receptor (TRβ1) protein concentrations were increased in the liver and adipose tissues of rats fed SPI. Thus, SPI appeared to stimulate the energy consumption by UCPs induction. The correlation in stimulation mechanisms of UCPs expression by peptides and by triiodothyronine has not been elucidated. Soy Protein Research, Japan8, 103-107, 2005.
Key words : UCP-1, 2, 3 mRNA, protein induction, soy protein isolate, triiodothyronine, thyroid hormone receptor, rats
Effects of Soy Protein and Peptide on Intestinal Fat Metabolism in Genetically Obese Diabetic Rats Lacking Cholecystokinin-A Receptor and on Gallstone Formation in the Receptor Gene Knockout Mice
Kyoko MIYASAKA, Setsuko KANAI, Minoru OHTA and Ayako SEKIME
Department of Clinical Physiology, Tokyo Metropolitan Institute of Gerontology, Tokyo 173-0015
ABSTRACT
Otsuka Long-Evans Tokushima Fatty (OLETF) rats were developed as a model for noninsulin-dependent diabetes mellitus (NIDDM). OLETF rats are characterized by late onset of hyperglycemia, at 18 weeks of age, followed by insulin dependent diabetes mellitus at 65 weeks of age. At this age, lymphatic triglyceride (TG) transport from the intestinal lumen to the lymph was enhanced in the rats. Soybeans and their products are classical Japanese foods and are generally believed to be good for health. We examined whether the effect of a special diet consisting of soy-protein normalizes enhanced lymphatic TG transport in OLETF rats. Male 12-week old rats of OLETF strains were fed this experimental diet in parallel with CRF-1 (control). A mesenteric lyphph fistula was prepared. The intestinal fat absorption was not influenced by long-term administration of a soy-protein diet. However, simultaneous infusion of an isolated soy protein (FUJI-PURO) with fat into the small intestinal lumen decreased intestinal fat absorption. Thus, ingestion of soy-protein with fat may prevent fat absorption and hyperlipidemia. On the other hand, gallstones were usually associated with obesity and/or hyperlipidemia. However, the etiology of gallstones is multifactorial, involving interactions between genes and the environment. We generated cholecystokinin (CCK)-A receptor (R)-deficient (KO) mice and found that gallstone formation was significantly higher in CCK-ARKO mice than in wild-type mice. In the present study, we examined whether a soy-protein diet could prevent gallstone formation in CCK-ARKO mice. No gallstone formation was observed at 6 month-old regardless genotype. At 12 month of age, gallstone formation appeared in CCK-ARKO mice treated with CRF-1 and in both wild-type and CCK-ARKO mice treated with a soy-protein diet. Taken together, a long-term administration of a soy-protein diet did not show a beneficial effects on hyperlipiedemia and gallstone formation, unexpectedly, although an isolated soyprotein, when administered with fat, could inhibit fat absorption. Soy Protein Research, Japan8, 108-112, 2005.
Chemoprevention of Bowman-Birk Inhibitor from Soy Bean Against Cancers Based on Restoration of Connexin 43-Dependent Tumor-Suppressive Effect
Tomohiro YANO1, Kazuyuki SUZUKI2 and Ryuzi ASANO2
1National Institute of Health and Nutrition, Tokyo 162-8636 2College of Bioresorce Sciences, Nihon University, Fujisawa 252-8510
ABSTRACT
As a promising strategy for cancer chemoprevention, the restoration in expression and function of tumor suppressor gene has been notable. Of tumor suppressor genes, connexin (Cx) gene, a member of gap junction (GJ), acts as a tumor suppressor by maintaining electrical and metabolic homeostatsis via GJ-driven cell-cell communication, and the tumor-suppressive effect is cell- and tissue- specific. Thus, the restoration and/or maintenance of the Cx gene contribute to establishment of tissue-specific cancer prevention. On the other hand, it has been known that Bowman-Birk inhibitor (BBI) from soy bean acts as a potential chemopreventive agent, and part of the chemopreventive effect may depend on the restoration of Cx43 gene expression. In this study, we examined if BBI could be a promising chemopreventive agent against cancers caused by the down-regulation of Cx43 gene. In a mouse xenograft model, BBI treatment reduced the relative tumor weight as well as proliferating cell nuclear antigen (a marker of cell proliferation) in a dose-dependent manner. In contrast, the BBI treatment restored the level of Cx43 mRNA in the dose-dependent manner. These results suggest that the anti-carcinogenic effects of BBI induced negative growth control caused by the expression of Cx43 gene in mouse xenograft model and that the restoration of Cx43 is one of the important factors for cancer prevention by BBI. Soy Protein Research, Japan8, 113-116, 2005.
Key words : Bowman-Birk inhibitor, cancer chemoprevention, connexin 43, negative growth control
Department of Orthopaedic Surgery1, Biostatistics Center2, Department of Immunology3 and Research Center of Innovative Cancer Therapy, Kurume University School of Medicine, Kurume 830-0011
ABSTRACT
Japan is a country with the world longest longevity. One of the possible contributing factors which attract attention of scientists are Japanese foods, including soybean. On the other hand, the occurrence and morbidity from carcinoma in Japanese emigrants in USA resembles that of the resident Americans. Isoflavone of a soybean origin has a structure which resembles estrogen. Since isoflavone acts as anti-estrogen, it is expected that it has an effect on prevention and treatment of prostatic cancer. As human genome analysis is completed, one could do analysis of proteins of interest. We performed experimental studies to investigate the effect of isoflavone on prostatic cancer cell line. The effects of isoflavone in vivo were investigated by proteomics analysis. The results suggest that isoflavone prevents onset of prostatic cancer. Soy Protein Research, Japan8, 117-120, 2005.
Key words : proteomics, bioinformatics, biostatistics, proteolinks, MALDI-TOF-MS
New Cholesterol-Lowering Peptides with Bile Acids Binding Ability Derived from Soybean Glycinin
Satoshi NAGAOKA
Department of Applied Life Science, Faculty of Applied Biological Sciences, Gifu University, Gifu 501-119
ABSTRACT
In this study, we investigated the effects of VAWWMY peptide that is the most hydrophobic region in soy glycinin A1a subunit sequences, and SPH-sup (soy protein peptic hydrolysate supernatant) on cholesterol metabolism. VAWWMY peptide had a significantly greater bile acid-binding capacity in vitro compared to casein tryptic hydrolysate (CTH). In addition, it significantly lowered the micellar solubility of cholesterol in vitro and significantly suppressed cholesterol absorption in vivo compared to CTH. SPH-sup without bile acid-binding capacity has significantly lower micellar solubility of cholesterol in vitro compared to CTH. In HepG2 cells, SPH-sup significantly increased cholesterol 7α-hydroxylase mRNA level compared to CTH. Thus, we first found the peptide (VAWWMY, we designated as soystatin) derived from soybean glycinin exhibited inhibitory action of cholesterol absorption in vivo. Our results suggest that both hydrophobic peptide, for example, soystatin and water-soluble peptide contribute to the hypocholesterolemic action induced by soy protein. Soy Protein Research, Japan8, 121-126, 2005.
Key words : soystatin, glycinin, VAWWMY, cholesterol, cholesterol 7α-hydroxylase
Analysis of Tumor-Suppressive Effects of Soy Isoflavones on Estrogen-Independent Mammary Carcinoma Cells
Yoichiro MATSUOKA and Reiko TSUKAMOTO
Second Department of Pathology, Kansai Medical University, Moriguchi 570-8506
ABSTRACT
In vivo and in vitro studies have indicated that the isoflavones including genistein in soybeans may be responsible for mammary tumor suppressive effects. We confirmed the tumor suppressive effects of soy isoflavones using c-Ha-ras transgenic rats which develop estrogen-independent as well as estrogen-dependent mammary carcinomas. Three estrogen receptor positive, cell lines C3, C11 and C17 (estrogen receptor β positive) among seven lines established from the tumors in this transgenic rat were tested for estrogen responsiveness and revealed as estrogen-independent cells. Genistein at >=50μM of concentration suppressed DNA synthesis of these rat cell lines as well as MCF-7 human cells even in the presence of estrogen. In contrast, the inhibitory effect of genistein was only observed on estrogen-independent C3 and C11 cells in the absence of estrogen. Cell cycle was arrested at G2/M phase and apoptosis was induced in C11 cells after 72 hr incubation with 10μM genistein. A number of genes encoding transcription factors and apoptosis-related factors were included in the genes that showed >2-fold change in their expression levels after the treatment of C11 cells. These results suggest that genistein suppresses mammary carcinomas mainly due to cell cycle arrest at G2/M phase and an apoptosis-inducing effect in an estrogen receptor-independent manner. Soy Protein Research, Japan8, 127-132, 2005.
Key words : estrogen, genistein, isoflavone, mammary carcinoma
Health Effects and Regional Difference in Soy Isoflavone Intake and Dried Blood Spot Concentration
Melissa K. Melby and Shaw Watanabe
1Tokyo University of Agriculture 2National Institute of Health and Nutrition
ABSTRACT
Japanese women living in Japan have the longest life expectancy in the world, and relatively low rates of reproductive cancers compared to women in other developed countries. However, Japanese women living in the US often show similar rates of cancer and other diseases to those of other Americans, suggesting that environmental lifestyle factors such as diet influence these health outcomes. Soy, and particularly isoflavones, is hypothesized to play a role in the better health of Japanese women following a traditional Japanese diet. Thus, isoflavone (IF) intake and exposure were examined in this study to assess whether women with higher IF intake and/or exposure have lower prevalence of health problems. Furthermore, we hypothesized that equol producers would have lower prevalence of health problems. Japanese women of 138, aged 45-55 years, living in Kyoto and Fukushima prefectures participated in a 6 month protocol involving collection of health history, anthropometric data, weekly finger-prick dried blood spot samples and dietary records for the 24 hour period preceding blood collection. A novel HPLC-CEAD method was developed for use with dried blood spots (DBS). Participants were generally healthy, with an average BMI of 23-24. More than half were perimenopausal, and slightly greater than one quarter were premenopausal on entrance to the study. Intake of tofu was higher in Kyoto, but intakes of natto, miso and edamame were higher in Fukushima. Isoflavone intake estimated from our FFF database showed similar results. Genistein concentrations showed no significant differences, but daidzein and equol were significantly higher in Fukushima, where approximately 2/3 of individuals were identified as equol-producers compared to only 1/3 in Kyoto. Correlations between dietary intake IF estimates and DBS IF concentrations were high. Assignment of equol producer status is complicated by pharmacokinetics of IF metabolism and equol production. Logistic regression identified odds ratios of less than 1 for genistein and allergies, skin allergies and ulcers and for equol and menopausal syndrome. Although logistic regression does not permit assignment of causation, these results suggest that women with higher IF blood concentrations may have lower risk for several health problems. Soy Protein Research, Japan8, 133-137, 2005.
Soy Protein and Isoflavone Intake and Colorectal Cancer: Anatomic Site-specific Associations
Chisato NAGATA
Gifu University Graduate School of Medicine, Gifu 501-1194
ABSTRACT
The present study examined the relationship between soy protein intake and risk of colorectal cancer by subsite in a population-based cohort of Japanese men and women. Diet including soy intake was determined in 13,893 men and 16,331 women using a validated food-frequency questionnaire. The subsequent risk of colon cancer during 8 years of follow-up was inversely associated with soy intake in women. Risk decrease was observed for proximal colon cancer. There was no association between soy intake and rectal cancer in men and women. These data suggest that soy intake may perturb bile-acid profiles, thereby, reducing the risk of proximal colon cancer. Isoflavone metabolites in first-void urines were correlated with soy intake estimated from the food-frequency questionnaire as well as the 3-day diet records. Isoflavone measures in spot urines were subject to short-term effect from diet. Soy Protein Research, Japan8, 138-140, 2005.
Key words : soy protein, isoflavones, colon cancer, rectal cancer, prospective study