Studies on Effect of Soy Products to Menopause-Associated Disorders in
Monkey Model (III):
Immune Response, Intestinal Bacteria Flora and Brain-Nerve Function in
OVX-Menopause Monkeys
Shin NAKAMURA1, Fusako MITSUNAGA1, Hiroyuki HASHIMOTO1, Sanae DEI1, Takashi HAYASHI2 and Joseph GABRIEL2
1Primate Research Institute, Kyoto University Inuyama 484-8506 2INA Research /INA Research Philippines Nagano 399-4501
ABSTRACT
Soy is accepted to be a functional food to protect age-related deteriorations in
postmenopausal women, even though enough evidences on its protective action are
not accumulated yet. Most pre-clinical studies on efficiency of functional foods
and/or their components were performed in conventional animals, such as rat or
mouse, because studies using these rodent models are of low cost and/or easy to
perform. However, biomedical characteristics of rodents are markedly different from
those of humans to elucidate detailed mechanisms of the functional foods. Macaque
monkeys, Cynomolgus, Rhesus or Japanese macaque, are closely-related primates to
humans and their genomic differences are just in 2-3% to shear whole biomedical
nature with humans. This is a merit for us to perform pre-clinical studies using
monkey on efficiency and/or safety of functional foods, if examination cost could be
down to a reasonable level. In a series of this study, we developed an ovariectomy
(OVX) Cynomlgus macaque model to examine postmenopausal age-related
deteriorations and effect of soy products on them. We have found that soy protein
(β-Conglycinin; β-CG), but not soy isoflavone (Soyaflavone HG; IF), was functionally
potential component to protect both hypercholesterolemia and osteroporosis in the
postmenopausal monkeys (1-3). In this study, we examined efficacy of the soy
components of β-CG and IF on immune system, especially on Th1/Th2 response,intestinal bacteria flora, and cerebral-nerve function in postmenopausal primate
model on the level of functional genes. We also examined antigenicity of both
components to monitor production of specific IgE. Soy Protein Research, Japan9, 1-8, 2006.
Clinico-Epidemiological Studies on Soy Intake and Allergic Disorders
Chisato NAGATA1, Naoyoshi TAKATSUKA1, Noriyuki TAKEDA2 and Hiroyuki SHIMIZU3
1Gifu University Graduate School of Medicine Gifu 501-1194 2Asahi University School of Dentistry Gifu 501-0296 3Sakihai Institute Gifu 500-8842
ABSTRACT
The present study examined the relationship between soy intake and past history of
allergic diseases in 13,355 men and 15,724 women. Dietary intake, including soy
intake, was determined using a validated 169-item food-frequency questionnaire.
The mean soy intake was significantly lower in men who reported to have had
diagnosis of allergic disease than that in those who had had no allergic disease.
Similar tendency was observed in women, but the association was not statistically
significant. Longitudinal study on maternal soy intake and allergic disorders in the
offspring is being undertaken. Maternal soy intake was estimated by 5-day diet
records. Maternal soy intake, but not urinary and serum isoflavone metabolites, a
significant positive association with umbilical cord IgE level in women who gave
birth to a singleton female baby. The mean maternal soy intake was significantly
lower in the offspring who were reported to have allergic rhinitis than that in those
who had no allergic rhinitis around 2-3 years of age. Soy Protein Research, Japan9, 9-12, 2006.
Key words :soy, isoflavones, allergic disease, prospective study
Improvement of Methionine Content in Soybean through Metabolic Regulation
Toru FUJIWARA1 and Masao ISHIMOTO2
1Biotechnology Research Center, The University of Tokyo Tokyo 113-8657 2National Agricultural Research Center for Hokkaido Region
ABSTRACT
To increase the methionine content, we introduced Arabidopsis CgS gene under
control of the glycinin promoter to soybean and azuki bean. Several independent
transgenic plants from both crops were recovered and the integration of the
transgenes confirmed. The transgenic seeds of soybean and azuki bean accumulate
up to 70-fold more free methionine than non-transgenic plants. Soy Protein Research, Japan9, 13-16, 2006.
Key words : methionine, transgenic soybean, transgenic azuki bean
Study on Hydroxylase Involved in Biosynthesis of Group-A Saponin for the
Breeding of Low Unpleasant Taste Soybean Variety
Masakazu SHIRAIWA and Jun HASHIURA
School of Agriculture, Ibaraki University Ibaraki 300-0393
ABSTRACT
In the soybean seed, group-A and -DDMP saponins exist as intact saponins. Group-A
saponin is the main cause substance for the unpleasant taste in a soybean. On the
other hand, group-DDMP saponin has various pharmacodynamic properties.
Therefore, it is expected that the elucidation and characterization of the enzymes
which catalyze biosynthetic reactions 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 hydroxylases, which are the key enzymes of group-A saponin biosynthesis. From
the content of group-A saponin and the activities of gurucuronosyltransferase and
hydroxylases involved in group-A saponin biosyntheses in soybean seeds during
development, it has been understood that biosyntheses of soyasapogenol A, which is
an aglycon of group-A saponin, are most actively done at the R6 stage. When RT-PCR
was done by using degenerate primers for single-stranded cDNA made from soybean
seed hypocotyls at the R6 stage, some PCR products were obtained. Sequence
analysis will be done for these PCR products . Homology with the known CYPs based
on sequence information will be examined for the obtained sequence, the transcript
characteristics will be compared by the northern analysis, and the hydoroxylase
involved only in group-A saponin biosyntheses will be specified. Soy Protein Research, Japan9, 17-23, 2006.
Key words : soybean, saponin biosynthesis, glucuronosyltransferase, hydroxylase
Structural Features of Vacuolar Sorting Determinants of Soybean Glycinin
Nobuyuki MARUYAMA and Shigeru UTSUMI
Graduate School of Agriculture, Kyoto University Uji 611-0011
ABSTRACT
The sorting determinants of glycinin, a soybean 11S globulin, which mediates protein
targeting to the protein storage vacuole (PSV), were investigated in maturing
soybean cotyledons by transient expression assays. A C-terminal stretch of 10 amino
acids of A1aB1b, a glycinin group I subunit, was sufficient to direct green fluorescent
protein (GFP) to the PSV. This peptide may correspond to a C-terminal vacuolar
sorting determinant (ctVSD). Since functional inhibition of this putative ctVSD of
A1aB1b did not block PSV sorting of A1aB1b, we used the three-dimensional
structure of A1aB1b to identify candidates for a sequence-specific determinant
(ssVSD). We found that the sequence downstream of disordered region 4 could direct
GFP to the PSV and that Ile297 is critical for sorting. However, functional inhibition
of the ctVSD, combined with the Ile297Gly mutation did not abolish vacuolar sorting
of A1aB1b, suggesting that A1aB1b has the sorting determinant which does not
belong in ctVSD and ssVSD in addition to ctVSD. Soy Protein Research, Japan9, 24-29, 2006.
Key words : glycinin, protein storage vacuole, vacuolar sorting determinant
Analysis of the Gene Expression Control of Soybean Seed Storage Protein β-Conglycinin α subunit
Atsushi NAGAMATSU and Akira KANAZA
Graduate School of Agriculture, Hokkaido University Sapporo 060-8589
ABSTRACT
Theβ-conglycinin, a major component of seed storage proteins in soybean, comprises
three subunits, α, α' and β. The genes for these proteins appear to be transcribed in
a coordinated but not identical manner during seed development. We have previously
identified the nucleotide sequence of the β-conglycinin α subunit gene. In order to
identify regulatory elements of transcription in seeds, reporter gene constructs
comprising the upstream sequence up to -1,357 or a series of its 5'-deleted
derivatives of the α subunit gene and the β-glucuronidase (GUS) gene were made.
These reporter gene constructs were introduced into Arabidopsis thaliana plants via
Agrobacterium-mediated gene transfer. Prominent GUS activity was detected in
embryos in developing seeds of the T3 generation when 245 bp or longer sequences of
the upstream region were fused to the GUS gene, whereas no GUS activity was
detected in vegetative tissues. RY sequences with the consensus sequence CATGCA
(C/T) are widely distributed in seed-specific gene promoters. Six RY sequences were
detected upstream of the α subunit gene. We found a clear association of decreased
GUS activity with a stepwise deletion of a region containing the RY sequence from
the original reporter construct. This result is consistent with the notion that the RY
sequences are involved in the seed-specific transcriptional activation of the β-conglycininαsubunit gene in soybean. Soy Protein Research, Japan9, 30-35, 2006.
Key words : β-conglycinin, promoter, RY sequence, seed storage protein, soybean
Proteins Catalyzing the Formation of Disulfide Bonds for Correct Folding of
Seed Storage Proteins in the Endoplasmic Reticulum of Soybean Cotyledon
Reiko URADE
Graduate School of Agriculture, Kyoto University Uji 611-0011
ABSTRACT
Soybean storage proteins are synthesized in the endoplasmic reticulum (ER). Many
proteins synthesized in the ER are folded accompanying the formation of inner
molecular disulfide bonds with the aid of protein disulfide isomerase (PDI) and the
related proteins, which are characterized by thioredoxin motifs within the primary
structure. However, the identification and characterization of plant PDI family
proteins have hardly been performed and their physiological roles remain unknown.
In this study, we isolated ten kinds of cDNA clones of soybean PDI family proteins.
We succeeded in the construction of E. coli protein expression systems for GmPDIS-1b, GmPDIS-2, GmPDIM, GmPDIL-1, GmPDIL-2, GmPDIL-3a and GmPDIL-3b. Their
domain structures were analyzed by peptide mapping of the recombinant proteins
with trypsin and V8 proteinase. GmPDIS-1b, GmPDIS-2, GmPDIM, GmPDIL-1 and
GmPDIL-2 were shown to accelerate the oxidative refolding of reduced and
denatured ribonuclease A. GmPDIS-1, GmPDIS-2, GmPDIM, GmPDIL-1, GmPDIL-2
and GmPDIL-3b were detected in cotyledons, leaves, stems, roots and flowers on
Western blotting. GmPDIS-1, GmPDIM, GmPDIL-1 and GmPDIL-3b were increased
in cotyledons, when glycinin was accumulated during the development of seeds. This
regulation was unlikely to be transcriptional, since the expression of their mRNAs did
not vary with their levels. Soy
Protein Research, Japan9, 36-41, 2006.
Key words : soybean, endoplasmic reticulum, glycinin, molecular chaperone, protein
disulfide isomerase
Development of a Method to Improve the Taste of Soy Sauce by the Addition of γ-Glutamyltranspeptidase as Glutaminase During Its Fermentation (II)
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, 65 mM glutamic acid was accumulated after 98 days of
fermentation, while 115 mM glutamic acid was found by the addition of 4.1 U-GGT
/190 g-moromi. Nine out of ten panel members could distinguish the taste of these
and answered that they prefer the soy sauce fermented by the addition of GGT. Soy Protein Research, Japan9, 42-46, 2006.
Department of Food Science, Niigata University of Pharmacy and
Applied Life Sciences Niigata 956-8603
ABSTRACT
The effect of high-pressure treatment on soybean as a cellular biological material was
investigated from the viewpoints of the cell structure, mass transfer, and enzyme reaction
system. The cell structure of soybean was evaluated with the dielectric properties
measured at frequencies of 50 Hz-5 MHz. Cole-Cole arc, which indicates the existence of
cell structure, was observed in soybean samples, and its radius was decreased due to the
high pressure. The result suggested that the structure of soybean was damaged by highpressure
treatment. The hydration process of soybean was selected as one of mass
transfer phenomena, and analyzed on the basis of scaling concept. The change in water
content during hydration process was described with the scaling law with time. The
hydration process of soybean was found to be limited with diffusion inside, because the
estimated value of the exponent was close to the theoretical one. The effect of highpressure
treatment on the hydration process of soybean was not obvious. The production
rate of free amino acids in soybean during preservation at 25Ž was accelerated by highpressure
treatment of 200 MPa. The high-pressure treatment also made the enzymatic
production of gamma-aminobutyric acid (GABA) in the soybean, which enriched sodium
glutamate, increasing. From these results, the high-pressure treatment induced
destruction of cell structure and accelerated biochemical reaction in soybean. In other
words, 'high-pressure induced transformation (Hi-Pit)' had occurred in soybean. Soy Protein Research, Japan9, 47-52, 2006.
Key words : soybean, high-pressure, dielectric properties, hydration, gammaaminobutyric
acid
Effects of Polysaccharides Containing Galacturonic Acids on
the Dispersion Stability of Soy Proteins
Yasuki MATSUMURA1 and Jun LI2
1Graduate School of Agriculture, Kyoto University Uji 611-0011 2Institute of Feed Research, China Science Academy of Agriculture
ABSTRACT
Effects of pectin and soluble soybean polysaccharides (SSPS) on the dispersion state
of 11S globulin were investigated. The stability of mixture dispersions of 11S globulin
(1%) and SSPS (0-1%) or pectin (0-1%) were tested at various pHs from 3.5 to 6.5.
SSPS increased amounts of dispersed protein in dose-dependent manner at all pH
conditions. Especially, even at pH 5.5 which is close to isoelectric point of 11S
globulin, more than 60% protein could be dispersed. On the other hand, only
approximately 40% protein was stabilized by pectin at pH 5.5 or 4.5. Although 11S
globulin was well-dispersed at pH 3.5 and 6.5 without polysaccharides, pectin
decreased the dispersion stability of the protein at both pH conditions. SSPS was
shown to have the ability to suppress the precipitation of 11S globulin by addition of
calcium or magnesium. These results indicated that SSPS could disperse 11S globulin
even under the severe conditions which normally induce the precipitation of the
protein. Therefore, the use of SSPS could be promising for the design and production
of the acidic or calcium-fortified beverages including soy proteins. Soy Protein Research, Japan9, 53-57, 2006.
Key words : pectin, soluble soybean polysaccharides, dispersion state
Comparison between Soyprotein 7S and 11S Globulins and Involvement of
the Allergen Gly m Bd 30K in the Proteolysis and Coagulation of
Soyprotein Induced by Subtilisin Carlsberg
Kuniyo INOUYE
Graduate School of Agriculture, Kyoto University Kyoto 606-8502
ABSTRACT
We have previously reported that coagula were formed uniquely during the hydrolysis
of SPI (Fujipro R) by subtilisin Carlsberg, and examined the reaction mechanism of
this coagulation. In this study, we found that the heat-treatment of SPI at 70-96Ž
for 30 min was needed for the coagulation. The highest coagulation rate and the
highest amount of the coagula were obtained by the treatment at 80Ž. 7S and 11S
globulins also coagulate only when they were treated at > 70Ž before the SC
hydrolysis. It is suggested that 7S and 11S globulins (and/or their proteolytic
fragments) are cooperatively involved in the SPI coagulation. The allergen Gly m Bd
30K must be hydrolyzed by SC promptly and completely into small peptides, and
immunoreactive fragments of the allergen are not detected in the coagula and soluble
fraction of the SC-treated SPI. Accordingly, it is considered that the allergen cannot
be involved in the coagulation. Soy Protein Research, Japan9, 58-61, 2006.
Key words : allergen, coagulation, proteolysis, soyprotein, subtilisin Carlsberg
1Graduate School of Agricultural Science, Tohoku University Sendai 981-8555 2Fuji Oil Co., Ltd Izumi-sano 598-8540 3Ishinomaki-Senshu University Ishinomaki 986-8580
ABSTRACT
Astringency perceived in the mouth upon ingestion of tannic acid and other substances are generally regarded as dry, puckering sensations, which seem more closely allied to the tactile than to the gustatory sense. On the other hand, perceptual assessments of astringency may be closely linked to bitterness in human tasting study, and most of electrophysiological studies of the experimental animals showed that the chorda tympani and glossopharyngeal nerves transmit the astringency signal. Therefore, we attempted to show the possible astringency sensitive neuron profile in the chorda tympani nerve (taste nerve) fibers in adult female SD rats. Astringent compounds (tannic acid, other typical astringent food constituents, and acidified glycinin, pH 3.5) were applied to the tongue of the rat. According to the cluster analysis of 77 (Exp. 1) or 80 chorda tympani (CT) nerve fibers (Exp. 2), the astringency-sensitive fibers (0.01 M tannic acid) were closely related to the response to sour taste, however, the responded profiles of various astringent compounds were not necessarily paralleled well with that of HCl (sour). The CT nerve fiber responses to soy isoflavon, soy saponin, whey protein, glycinin and tannic acid solution were assessed, and soy isoflavon showed no response and soy saponin responded slightly. These results suggest isoflavons or saponins that coexisted in the soyprotein isolate are not key substances for the astringent sensation, but the acidified glycinin protein itself may be responsible for the astringency formation. Soy Protein Research, Japan9, 62-67, 2006.
Key words : astringency, soy protein, rats, taste nerve, glycinin
Inhibitory Effects of Soy Protein-Derived Peptides on IRS-1 Degradation
Takeshi NIKAWA, Katsuya HIRASAKA, Harumi FUROCHI, Jumpei GOTO, Reiko NAKAO and Kyoichi KISHI
Department of Nutrition, The University of Tokushima School of Medicine Tokushima 770-8503
ABSTRACT
We previously demonstrated that ubiquitin ligase Cbl-b stimulates ubiquitination and degradation of IRS-1 in skeletal muscle and bone during unloaing. In this study, we examined inhibitory effects of soy protein-derived peptides, such as glynicine and β- conglynicine on Cbl-b-mediated ubiquitination of IRS-1. Using a cell-free ubiquitination system, we found that a kind of soy protein-derived peptides inhibit IRS-1 ubiquitination mediated by Cbl-b. Our results suggest that soy protein may contain bioactive peptides for regulating proteolysis in skeletal muscle. Soy Protein Research, Japan9, 68-70, 2006.
Key words : IRS-1, skeletal muscle, soy protein-derived peptides, ubiquitination
Changes in Translation Initiation Activities for Rat Tissue Protein Synthesis by Feeding Small Peptides Derived from Soybean Protein
Hiroshi HARA, Masashi MATSUMOTO and Nanako SHIMA
Laboratory of Nutritional Biochemistry, Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University Sapporo 060-8589
ABSTRACT
Dietary peptides efficiently supply amino acids into the body and promote tissue protein synthesis. We examined effects of soybean small peptides on protein translation initiation activities with phosphorylation of eIF4E-binding protein 1 (4EBP1) in the pancreas, liver and gastrocnemius muscle, and increases in amino acid concentrations in the portal blood compared with soybean protein isolate (SPI) and amino acid mixture simulating SPI (AA). Rats were fed 20% or 50% SPI, peptide or AA diet for 14 days with an 8-hr meal feeding, and killed before and 1 and 2 hr after feeding under anesthesia on the last day. Portal absorption of amino acids was much higher in the peptide and AA groups than in the SPI group. Highly phosphorylated 4E-BP1 (γ-form) were increased after feeding test diets, and pancreatic 4E-BP1 γ- form was the highest in the peptide group, followed by the AA and then SPI groups in
20% nitrogen diets. Hepatic and muscle γ-form in the peptide and AA groups were similarly higher than that in the SPI group. In rats fed 50% nitrogen diets, γ-form of the pancreas and muscle, but not the liver, were higher in the peptide group than in the other two groups 2 hr after feeding. These results indicate that absorbed amino acids also contribute to the promotion of translation initiation activity in the pancreas the same as in the liver and muscle after feeding a diet. Soy Protein Research, Japan9, 71-76, 2006.
Key words : soybean small peptides, translation initiation, eIF4E-binding protein 1, pancreas, skeletal muscle
Prevention against Intestinal Infection by Soy Protein Hydrolysate
Makoto HATTORI
Division of Agriscience and Bioscience, Tokyo University of Agriculture and Technology Fuchu 183-8509
ABSTRACT
The preventive effects of soy protein hydrolysate (SPH) against intestinal infection were investigated. We prepared SPH by the following procedure: 1) soy protein isolate (SPI) was solubilized by the treatment with 6 M guanidine hydrochloride, 2) solubilized sample was dialyzed against 0.03 M Tris-HCl buffer (pH 8), 3) dialyzed sample was digested by trypsin, and 4) the digested sample was submitted to sizeexclusion chromatography. The binding ability of SPH to intestinal pathogenic bacteria was evaluated by a binding assay with biotinylated bacteria. SPH showed the ability to bind to Salmonella enteritidis and enterohemorrhagic Escherichia coli O157:H7. The preventive effect of SPH on the adhesion of S.enteritidis to Caco-2 cells was also investigated. SPH showed an inhibitory effect on the adhesion of S.enteritidis. Our results indicate SPH to be a promising agent for preventing intestinal infection. Soy Protein Research, Japan9, 77-81, 2006.
Key words : intestinal infection, soy protein hydrolysate, prevention of infection, Salmonella enteritidis, Escherichia coli O157:H7
Graduate School of Science, Osaka University Toyonaka 560-0041
ABSTRACT
Soyasaponins are reported to possess pharmacological or health-promoting effects such as a ntioxidation, liver-protecting actions, and anti-hypercholestelemia. To gain a better understanding of the molecular basis for these biological activities, we attempted to investigate the molecular interactions between soyasaponin (Soyhealth SA) and membrane lipids. In a previous study, the broad band solid-state NMR spectra of these membrane preparations revealed that cholesterol becomes mobile by the saponin in phosphatidylcholine bilayers on the basis of NMR signals characteristic of less oriented and flexible membrane entities. To examine the membrane permeabilizing effects by saponins, we carried out the NMR-based ion flux
experiments. In K+ flux assays based upon 31P NMR chemical shifts, soyasaponin Bb(1) revealed a marginal increase of the ion flux through egg-phospahtidylcholine large unilamellar vesicles (eggPC LUV). This partial permeabilizing activity was not affected by the addition of cholesterol. In Mn2+ flux experiments, the soyasaponin did not virtually show any activity, whereas quillajasaponin revealing the significant ion flux at 1 mol% in the PC membrane. Further studies for evaluating the molecular interaction of soyasaponins with unsaturated phospholipid bilayers are currently underway. Soy Protein Research, Japan9, 82-86, 2006.
Key words : soyasaponins, membrane permeabilizing activity, solid state NMR, ion flux through membranes
Female Specific Appetite Regulation of Daidzein in Rats
Taro KISHIDA, Asami IWAHARA and Kiyoshi EBIHARA
Faculty of Agriculture, Ehime University Matsuyama 790-8566
ABSTRACT
Previously, we showed that soy isoflavone daidzein decreased food intake specifically in female rats. Although we have evaluated hypothalamus gene expression of appetiterelated neurotransmitter to investigate the mechanism of decreasing food intake by daidzein, we have failed to acquire useful information, because of variation in the meal patterns of individual rats. In the present study, we examined hypothalamus gene expression of appetite-related neurotransmitter by synchronized meal pattern by means of meal feeding. After 10 days of training, the rats did not adapt meal feeding (two times) and daily food intake of rats in meal feeding is about 80% of rats in ad libitum feeding. Daidzein did not decrease the food intake of meal feeding rats (Experiment 1). After 12 days of training, the rats well adapted meal feeding (three times) and daily food intake of rats in meal feeding is well matched with rats in ad libitum feeding. Daidzein decreased the food intake of the meal feeding rats the same as in the ad libitum feeding rats (Experiment 2). In both experiments, we observed potential response of these gene expressions to decreasing food intake by daidzein after 14 days of test feeding but there is no explanation on the decrease of food intake by daidzein administration. Examination of more short-term meal feeding (three times) might be needed. Soy Protein Research, Japan9, 87-95, 2006.
Key words : soy isoflavone, daidzein, hypothalamus neurotransmitter, meal feeding, rat
Studies on Anti-Obesity Effects of Soy Isoflavones with Anti-Oxidant and Phytoestrogenic Activity
Naohito AOKI, Erina ARAKAWA and Miyuki ITO
Department of Life Science, Graduate School of Bioresources, Mie University Tsu 514-8507
ABSTRACT
C57BL/6J mice (male, 4wk old) were fed low fat diet (LF), high fat diet (HF), or HF supplemented with 1.0% soy isoflavone mixture (HF+Iso) for 10 wks. As expected, final body weight and adipose tissue weight were increased in HF group over LF one, but no significant difference was observed between HF and HF+Iso groups. Plasma triglyceride and TBARS were decreased in HF+Iso group as compared to HF one, whereas plasma cholesterol and glucose were indistinguishable. Gene expression and plasma level of adiponectin decreased by HF was upregulated by soy isoflavone, while no obvious effect of soy isoflavone upon resistin and leptin expression was observed. Further, expressions of macrophage markers CD68 and F4/80, and NADPH oxidase in adipose tissue induced by high fat diet were significantly reduced by soy isoflavone. Glycosylated soy isoflavones (genistin, glycitin, daizin) reduced ROS production in 3T3-L1 adipocyte and potentiated adiponectin secretion into cell culture medium. Unexpectedly, resistin secretion of 3T3-L1 adipocytes were also increased by glycosylated soy isoflavones. Thus, it is suggested that soy isoflavone modulates physiological functions of adipocyte/adipose tissue through its anti-oxidant activity. Soy Protein Research, Japan9, 96-101, 2006.
Key words : soy isoflavone, adipocyte, 3T3-L1, ROS, obesity
Nutrigenomics Study of Soy Protein Function in Light of Dietary Life
Hisanori KATO and Shinnosuke TSUJI
Graduate School of Agricultural and Life Sciences, The University of Tokyo Tokyo 113-8657
ABSTRACT
Amid the expansion of nutrigenomics research area, transcriptomics analysis is becoming one of the primary choices for the efficient analysis of the functions of foods. This technology has already been applied to the evaluation of the function of soy protein, and these studies have revealed some beneficial effects of soy protein. However, it is not clear at present that such effects can be maintained in the presence of other dietary proteins. The present study was conducted to evaluate the effect of soy protein isolate on gene expression profile when it is consumed with other proteins. Rats were fed on a 20% casein diet (C), a 20% gluten diet (G), a 20% soy protein diet (S), a 10% casein plus 10% soy protein diet (C+S), or a 10% gluten plus 10% soy protein diet (G+S) for two weeks. Only modest changes in the cholesterol and triglyceride levels in the liver and serum were observed. Liver RNA was subjected to DNA microarray analysis. Principal component analysis revealed that gene expression patterns of C+S and G+S groups were closer to S group than to C and G groups. S diet caused upregulation of the genes for cholesterol metabolism, fatty acid oxidation, antioxidation, and alcohol metabolism as well as downregulation of the genes for fatty acid synthesis. These effects were consistently observed in rats fed on the mixed protein diets. The results suggest that soy protein retain at least part of its beneficial effects even when it is ingested with other proteins. Soy Protein Research, Japan9, 102-107, 2006.
Key words : nutrigenomics, DNA microarray, lipid metabolism
Dietary Soy Protein Increases mRNA Expression of ABCG5 and ABCG8 in Liver and Biliary Output of Cholesterol in Rats
Ikuo IKEDA1, Mariko KUDO2, Koji NAGAO3, Tadateru HAMADA2, Yoko OSHIRO1, Takashi SUGAWARA1, Takashi YAMAHIRA1, Katsumi IMAIZUMI4 and Teruyoshi YANAGITA3
1Graduate School of Agricultural Science, Tohoku University Sendai 981-8555 2Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University 3Faculty of Agriculture, Saga University 4Faculty of Agriculture, Graduate School of Kyushu University
ABSTRACT
Dietary soy protein isolate (SPI) and undigested high molecular fraction of SPI exhaustively digested with proteases (HMF), compared with casein (CAS), increased mRNA expression of ATP-binding cassette transporter (ABC) G5 and ABCG8 in rat liver, but not in the intestine. Biliary excretion of cholesterol was significantly higher in rats fed SPI and HMF than in those fed CAS. Fecal excretion of acidic steroids was higher in rats fed SPI and HMF. The mRNA expression of hepatic cholesterol 7α- hydroxylase (CYP7A1) in the HMF group was significantly higher and that in the SPI group tended to be higher than in the CAS group. In contrast, mRNA expression of hepatic small heterodimer partner (SHP) was significantly lower in the feeding of SPI and HMF. It has been known that the reduction of SHP activates liver receptor homologue (LRH)-1, which binds to the promoter of CYP7A1 and increases its mRNA expression. Since it was recently reported that LRH-1 activates the promoter of ABCG5 and ABCG8, it is thought that the increase of their mRNA expression in the liver was induced by the activation of LRH-1. Soy Protein Research, Japan9, 108-112, 2006.
Key words : ATP-binding cassette transporter, Liver receptor homologue-1, rat, small heterodimer partner, soy protein
Development of Molecular Targeted Anti-Metastasis Agent Derived from Soybean
Hiroshi KOBAYASHI and Junko TSUJI
Department of Obstetrics and Gynecology, Nara Medical University Kashihara 634-8522
ABSTRACT
Human bikunin, a Kunitz-type trypsin inhibitor, inhibits inflammation by downregulating the expression of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) in tumor cells and inflammatory cells. We analyzed the effect of a soybean-derived Kunitz Trypsin Inhibitor (KTI) on TNF-α production in human gingival fibroblasts stimulated by lipopolysaccharide (LPS), an inflammatory inducer. Mitogen-activated protein (MAP) kinase activation and cytokine levels were monitored using Western blot and a specific ELISA. Here, we show 1) a soybean KTI abrogates LPS-induced up-regulation of TNF-α mRNA and protein expression in a dose-dependent manner in gingival fibroblasts; 2) KTI also blocks the induction of TNF-αtarget molecules, interleukin-1β(IL-1β) and IL-6 proteins; 3) inhibition by KTI of TNF-α induction correlates with the suppressive capacity of ERK1/2 and p38 signaling pathways, implicating repressed ERK1/2 and p38 signalings in the inhibition; and 4) pretreatment of cells with KTI blocked LPS-induced NFκB activation. Our results indicate that KTI inhibits LPS-induced up-regulation of cytokine expression possibly through suppression of ERK1/2 and p38 kinase-mediated NFκB activation. These findings may identify anti-inflammatory properties of KTI at the level of gingival fibroblasts and may be relevant to the use of KTI in modulating inflammation, including periodontal disease. Soy Protein Research, Japan9, 113-119, 2006.
Key words :gingival fibroblasts, kunitz trypsin inhibitor (KTI), signal transduction, soybean, TNF-α
Chemoprevention of Bowman-Birk Inhibitor from Soy Bean Against Cancers Based on Restoration of Connexin 43-Dependent Tumor-Suppressive Effect
Tomohiro YANO1, Teruyoshi SAITO1, 2, Kazuyuki SUZUKI2 and Ryuzi ASANO2
1Project for Complementary Factors, National Institute of Health and Nutrition Tokyo 162-8636 2Department of Veterinary Medicine, Nihon University Fujisawa 252-8510
ABSTRACT
In a previous study, we have reported that restoration of connexin (Cx)43, a tumor suppressor gene and a member of gap junctions, is an important factor for cancer prevention by Bowman-Birk inhibitor (BBI). In this study, we tried to clarify a mechanism of BBI-dependent restoration of Cx43 in cancer cells. In a mouse xenograft model, we observed that the expression of Cx43 in tumors preceded occurrence of tumor necrosis, indicating that Cx43 is a key molecule to induce tumor necrosis in tumor-bearing mice treated with BBI. In an in vitro study, we found that
BBI induced Cx43 mRNA as well as stabilization of Cx43 protein in cancer cells. Especially, the stabilization of Cx43 protein by BBI depended on inhibition of chymotrypsin-like activity in proteasome. Finally, we confirmed that BBI-dependent induction of Cx43 is closely associated with negative growth control of cancer cells. Overall, it is concluded that the main chemopreventive effect of BBI is based on both the induction of Cx43 mRNA and the stabilization of Cx43 protein. Soy Protein Research, Japan9, 120-125, 2006.
Key words : Bowman-Birk inhibitor, cancer prevention, connexin 43, proteasome inhibiton
1Faculty of Health and Science, Kio University Nara 635-0832 2Health Center of Ikaruga-cho Ikoma-gun Nara 636-0114 3Fujioka Clinic Nara 636-0103 4Orthopedics of Hirai Hospital Nara 634-0045 5Nara Prefecture Medical College Nara 634-8521
ABSTRACT
In this study, aiming at health promotion, the effects of soymilk on the serum cholesterol and bone density were examined. Soymilk (200 mL per day) was administered to slight hypercholesterolemiac women (N=34, 50-56 years old) for ten months. Serum total cholesterol (TC) in the high TC group (over 240 mg/100mL) decreased significantly at the fifth month after the start of the examination. Although TC showed a slight increase at the tenth month, TC level was lower than its levels at the start. The increase of the TC level after the fifth month may be
influenced by the seasonal variation of TC and by the confounding factors such as intake of nutrients. The changes were not observed in the low TC group though the HDL-cholesterol (HDL-C) and the LDL-cholesterol (LDL-C) decreased significantly in the high TC group at the fifth month. Bone mineral density increased slightly, and the rate of increase in the group of women over five years after menopause was higher than the group less than five years after menopause. Serum concentrations of bone formation marker, bone-specific alkaline phosphatase (BAP), increased significantly and urinary concentrations of bone absorption marker, cross-linked Ntelopeptides of type I collagen (NTX), did not change significantly. These results seem to show that age-dependent decrease in bone density may be delayed by soymilk. Soy Protein Research, Japan9, 126-132, 2006.
Key words :soymilk, cholesterol, bone density, markers of bone turnover, postmenopausal women
Study on Anti-Allergic Effects of Soybean Components on High Affinity IgE Receptor Expression
Hirofumi TACHIBANA, Satomi YANO and Koji YAMADA
Faculty of Agriculture, Kyushu University Fukuoka 812-8581
ABSTRACT
Mast cells and basophils express the high affinity IgE receptor FcεRI on their cell surface, and the cross-linking of the allergen-specific IgE bound to FcεRI leads to the release of inflammatory mediators. In this study, we examined the effect of soybean components, isoflavones and equol on the cell surface expression of FcεRI in human basophilic KU812 cells. Genistein, daidzein and equol suppressed the cell surface expression of FcεRI. Immunoblot analysis revealed that the total cellular expression of the FcεRIαchain was decreased upon treatment with these compounds. However, there were no effects of these compounds on mRNA levels of FcεRIαandγchains. In our previous work, the reduction of extracellular signal-regulated kinase1/2 (ERK1/2) phosphorylation was involved in the downregulation of the FcεRI expression and it was f-und that (-)-epigallocatechin-3-O-gallate (EGCG) suppressed the receptor expression through the reduction of ERK1/2 phosphorylation levels. As opposed to EGCG, ERK1/2 phosphorylation level was not affected by the two isoflavones and equol. These results suggest that isoflavones and equol suppressed FcεRI expression through the mechanism that differs from EGCG action. Soy Protein Research, Japan9, 133-137, 2006.
Intake of Phytochemicals by Japanese and Its Health Effects
Melissa Melby, Kyohko TAKU and Shaw WATANABE
National Institute of Health and Nutrition Tokyo 156-8502
ABSTRACT
Effects of phytochemicals on human health are suggested from various animal experiments, but human study is quite a few. We have made a composition table of various phytochemicals (polyphenols, carotenoids, sulpher compounds) and tried to estimate the amount of intake among Japanese population. The data was analyzed in relation to the various biomarkers, especially of plasma lipids. The subjects were volunteers (16 males and 63 females, averagely aged 71 and 61, respectively) in Iwate city. Average BMI was 23 in both sex. Number of hypercholesterolemia more than 220 mg/100mL was 42, and hypertriglyceridemia more than 150 mg/100mL was 12. Intake of 36 phytochemicals were calculated from one-day dietary record of all intake, by multiplying concentration of each phytochemical in foods. Phytochemicals at least taken more than 10μmole per day were catechin, isoflavones, isothiocyanate, ferulic acid, quercetin, cinnamic acid and chlorogenic acid. Chief component analysis yielded 12 factors (80%). Only a few factors showed negative association with total cholesterol and LDL cholesterol. Many factors showed positive relationship with liver function and TG. Application of these phytochemicals as supplements should be prudent to avoid adverse health effect. Soy Protein Research, Japan9, 138-146, 2006.
Key words : phytochemical, flavonoids, human, epidemiology
Suppressive Regulation of Salt-sensitive Hypertension by Flavonids -Abnormal Regulation of Aldosterone-induced ENaC Gene Expression-
Naomi NIISATO and Yoshinori MARUNAKA
Graduate School of Medical Science, Kyoto Prefectural University of Medicine Kyoto 602-8566
ABSTRACT
Renal Na+ reabsorption has a crucial role in controlling blood pressure through regulation of mineral balance in our body. We have demonstrated that high salt diet causes hypertension through induction of renal epithelial Na+ channel (ENaC) gene expression in Dahl-salt sensitive (DS) but not in Dahl salt-resistant (DR) rats, and that one of flavonids, quercetin, suppresses high salt diet-induced ENaC gene expression. To clarify the regulatory mechanism of salt-sensitive hypertension and the quercetin action, we assessed regulation of ENaC mRNA expression in the kidney of DS rats by aldosterone and quercetin. Chronic high-salt-diet caused hypertension by increasing α-ENaC mRNA expression in DS rats with a reduction of plasma aldosterone. Simultaneous quercetin intake suppressed the high-salt-diet-induced hypertension in DS rats by reducing α-ENaC mRNA expression. Acute application of aldosterone to adrenalectomized rats reduced mRNA expression of β- and γ-ENaC in DS rats unlike DR rats, whereas aldosterone increased α-ENaC mRNA expression in both DS and DR rats. Furthermore, we found that aldosterone increased serum and glucocorticoid-regulated kinase 1 (SGK1) mRNA expression in DR rats but not in DS rats. Taken together, it is suggested that, in saltsensitive hypertension, regulation of ENaC and SGK1 gene expression by aldosterone might be disturbed and that abnormal aldosterone regulation of Na+ reabsorption in the kidney might be one of the factors to cause salt-sensitive hypertension by elevating plasma volume. Soy Protein Research, Japan9, 147-152, 2006.
Molecular and Pharmacological Analysis of Genistein, Daidzein and Its Metabolite Equol, and Their Clinical Significance
Takeshi USUI
National Hospital Organization Kyoto Medical Center, Clinical Research Institute Kyoto 612-8555
ABSTRACT
Genistein and Daidzein are well characterized phytoestrogens rich in soy or soy derived foods. These two isoflavones are considered to play an important role for various soy related human health benefits such as prevention of osteoporosis or arteriosclerosis. Daidzein is metabolized to equol in most of the animals and some human, and the equol is reported to be a stronger estrogenic compound. Individuals who can metabolize daidzein to equol (equol producers) might be more sensitive to soy intake than equol non-producers. Here we characterized the estrogenic properties of these three phytoestrogens, genistein, daidzein, and equol. All of these three phytoestrogens showed strong estrogenic activity in reporter gene assay system. Daidzein showed super agonistic action to estrogen beta mediated target gene expression. Interestingly, the estrogenic activities of genistein and daidzein were reversed by, ICI182720, estrogen pure antagonist, in estrogen receptor alpha mediated target gene expression but not to estrogen receptor beta mediated target gene expression. In contrast to genistein and daidzein, estrogenic activity of equol was reversed by ICI182720 on both estrogen receptor alpha and beta mediated target gene expression. In vitro binding experiment showed that these three phytoestrogens bind to both estrogen receptor alpha and beta. Medaka vitellogenin assay and MCF-7 cell proliferation assay showed that these three phytoestrogens have estrogenic activity. Soy Protein Research, Japan9, 153-157, 2006.
Key words : phytoestrogen, isoflavone, equol, estrogen receptor