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 prospectively examined the association between soy intake and
cedar pollen allergy, asthma, and allergic dermatitis in a community in Japan. Diet
including soy intake was determined using a validated 169-item food-frequency
questionnaire at the baseline. Information about experience with cedar pollinosis
and history of doctor-diagnosed asthma and allergic dermatitis was collected about
10 years later using a questionnaire. The risks of cedar pollen allergy, asthma, and
allergic dermatitis according to tertile of soy product or soy isoflavone intake were
estimated using Cox hazard model or logistic model including age and smoking
status at baseline as covariates. The association between soy intake and the risk of
cedar pollinosis was nearly null in both men and women. There was no significant
association between soy intake and risk of asthma. A significantly decreased risk of
allergic dermatitis was observed in the second tertile of soy intake in men, although
the trend was not statistically significant. Similar tendency was observed in women.
Soy Protein Research, Japan10, 1-5, 2007.
Key words :soy, cedar pollinosis, asthma, allergic dermatitis, prospective study
Proteins Catalyzing Folding of Seed Storage Proteins of Soybean
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 become folded with the formation of inner molecular
disulfide bonds with the aid of protein disulfide isomerase (PDI) and related proteins,
which are characterized by thioredoxin motifs within the primary structure.
However, identification and characterization of plant PDI family proteins have hardly
been performed and thus their physiological roles remain unknown. Previously, we
isolated cDNA clones of soybean PDI family proteins, GmPDIS-1, GmPDIS-2,
GmPDIM, GmPDIL-1, GmPDIL-2, GmPDIL-3a and GmPDIL-3b, and detected their
expression in soybean cotyledons. In this study, it was shown that all of them are
distributed in the ER lumen in cotyledon cells. GmPDIS-1 and GmPDIS-2 were shown
to be colocalized with storage proteins in protein storage vacuoles in addition to the
ER. Data obtained in co-immunoprecipitation and cross-linking experiments
suggested that GmPDIS-1, GmPDIM, GmPDIL-1 and GmPDIL-2 are associated with
proglycinin, a precursor of the seed-storage protein glycinin, in the cotyledons. In
addition, the association of GmPDIM, GmPDIL-1, GmPDIL-2 and BiP with β-
conglycinin α' was found. From these results, it was suggested that PDI family
proteins act not only as oxidoreductases for the formation of disulfide bonds, but also
as molecular chaperones for the folding of seed storage proteins. Soy Protein Research,
Japan10, 6-11, 2007.
Key words :soybean, endoplasmic reticulum, glycinin, molecular chaperone, protein
disulfide isomerase
Gene Expression Control of β-conglycinin in Molecular Breeding of Soybean Seed Storage Protein
Yusuke IMOTO, Atsushi NAGAMATSU and Akira KANAZAWA
Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589
ABSTRACT
Theβ-conglycinin, a major component of seed storage proteins in soybean, comprises
three subunits,α,α' andβ. The genes for these proteins appear to be transcribed in a
coordinated but not completely identical manner during seed development. We have
previously identified the nucleotide sequence of theβ-conglycininαsubunit gene. In
order to understand regulatory mechanisms of the expression of the α subunit gene
during seed development, we have made transgenic Arabidopsis thaliana plants
containing reporter gene constructs comprising the upstream sequence of theα subunit gene and the β-glucuronidase (GUS) gene. In the present study, tissuespecific
expression of the GUS gene was analyzed by staining embryos in developing
seeds of these transgenic plants. Prominent GUS activity was detected in entire
embryos in fully matured seeds when 245 bp or longer sequences of the upstream
region were fused to the GUS gene. In order to know the transition of tissue-specific
gene expression during seed development, we examined time-course dependency of
gene expression after flowering. Prominent GUS activity was detected first in
cotyledons but not in embryonic axes around 7-8 days after flowering. The
expression was detected in entire embryos including embryonic axes later in seed
development. These results suggest that the expression of the α subunit gene in
embryos was controlled according to both time and space during seed development
mainly at the transcriptional level. Soy Protein Research, Japan10, 12-17, 2007.
Key words : β-conglycinin, promoter, embryo, seed storage protein, soybean
Potential of Soybean Somatic Embryos as a Model System to Investigate the Biosynthetic Pathways of Seed Components
Masao ISHIMOTO and Keito NISHIZAWA
National Agricultural Research Center for Hokkaido Region, Sapporo 062-8555
ABSTRACT
Soybean (Glycine max [L.] Merr.) is one of the world's most important crops.
Elucidation of biosynthetic mechanisms of the seed components at a molecular level
would provide valuable information for improvement of soybean. In this study, we
focused on soybean somatic embryos as tools for analyzing seed specific traits, since
the reproducible somatic embryos are easily maintained and matured synchronously
in large-scale, and a stable transformation technique using somatic embryos has been
established. Furthermore, although it takes at least six months to obtain transgenic
seeds, it takes only three months to obtain transgenic somatic embryos. Application
of somatic embryos for analysis of biosynthetic mechanisms of seed components
would accelerate study of soybean. We characterized the developmental change of
soybean somatic embryos by examining accumulation of major seed storage proteins
(β-conglycinin and glycinin) as indicators. In developing somatic embryos, which
finally obtain partial desiccation tolerance and germination competency in 25-30
days, the seed storage proteins were gradually synthesized and stored in the protein
storage vacuoles as well as in seeds. α and α' subunits of β-conglycinin were
detected earlier than β subunit of β-conglycinin and glycinin. In addition, the α and α' subunits accumulated in both cotyledons and hypocotyls of somatic embryos,
whereas the β subunit and glycinin accumulated only in cotyledons. These
regulations in accumulation timing and the tissue specificity among the molecular
species are corresponding to those in seeds, indicating that somatic embryos develop
in similar manner to seeds. Thus somatic embryos are expected to be competent to
study seed components in soybean. Soy Protein Research, Japan10, 18-23, 2007.
Key words : somatic embryo, tissue culture, transformation, developing seed, storage
protein
Regulation of Gene Expression of Soybean Flavonoid-biosynthetic Enzymes by NO, cGMP and Light
Kenji SUITA1, Maki SAWADA1 and Hiroshi YAMAGATA2
1Graduate School of Science and Technology and 2Graduate School of Agricultural Science, Kobe University, Kobe 657-8501
ABSTRACT
Cyclic guanosine 3', 5'-monophosphate (cGMP) and nitric oxide (NO) control various
cellular functions cooperatively as second messengers in several signal transduction
pathways in animal cells. In plants, the functional involvement of cGMP in several
physiological processes such as anthocyanin biosynthesis has been reported, but their
molecular mechanisms and the functional relationships between cGMP and NO are
unknown. Using soybean photomixotrophic cell culture, we investigated the cGMPresponsiveness
of various structural genes for enzymes of legume-specific flavonoidbiosynthetic
pathway including isoflavone and phytoalexin synthesis and found that
most of these genes were induced by cGMP as well as NO. All cGMP/NO-induced
genes that we examined were also stimulated transiently by illumination of cultured
cells with white light. The NO-dependent induction of these genes were blocked by
LY83583, an inhibitor of guanylyl cyclase. Also, endogenous cGMP levels of cultured
cells were transiently increased by NO treatment. Consistent with the increase of the
transcripts for enzymes involved in flavonoid biosynthetic pathway, the accumulation
of anthocyanin in response to cGMP, NO, and white light was observed. Together
with our previous studies, these results suggest that cGMP and NO are linked
functionally and act as the second messengers in phototransduction that induces the
expression of genes for enzymes involved in flavonoid biosynthetic pathway to
produce anthocyanin in soybean.
Soy Protein Research, Japan10, 24-29, 2007.
Key words : flavonoid, gene expression, cGMP, nitric oxide, phototransduction
Elucidation of the Reason Why Glycinin Has a High Ability to Form Heat-induced Gel
Takao NAGANO
Department of Clinical Nutrition, Kawasaki University of Medical Welfare,
Kurashiki 701-0193
ABSTRACT
Heat-induced gels of glycinin and soybean protein isolate (SPI) were prepared using
magnesium chloride (MgCl2) as a coagulant. Viscoelastic properties and
microstructures of glycinin and SPI gels were investigated using dynamic viscoelastic
measurements (DVM) and confocal laser scanning microscopy (CLSM). DVM
indicated that glycinin was formed stiffer than SPI under all experimental conditions.
CLSM images showed that the structures of glycinin gels were coarser and more
heterogeneous than SPI gels in comparable conditions. The microstructures of
glycinin and SPI gels were analyzed by two parameters: the fractal dimension and the
average density of network structures. Compared to SPI, glycinin forms MgCl2-
induced gels with a lower fractal dimension and a higher density of network
structures. Soy Protein Research, Japan10, 30-33, 2007.
Effects of Soluble Soybean Polysaccharides Addition on Gelling Behavior of Soy Proteins
Yasuki MATSUMURA1, Hitomi YAMAGUCHI1 and Jun LI2
1Graduate School of Agriculture, Kyoto University, Uji 611-0011 2Institute of Feed Research, China Science Academy of Agriculture
ABSTRACT
Effects of SSPS addition on the gelation behavior of 11S globulin under the high ionic
strength condition were investigated. With the increase in SSPS concentration, the
rupture stress of 11S globulin gel decreased. It was demonstrated by DSC
measurement that the thermal stability of 11S globulin molecule was enhanced by
SSPS. SDS-PAGE analyses revealed that the polymer formation via disulfide linkage
of 11S globulin was inhibited by SSPS. The retardation of gelling of 11S globulin by
SSPS was demonstrated using dynamic oscillation measurement. The observation of
gel microstructure by scanning electron microscopy showed that the network
structure of 11S globulin gel was destroyed by SSPS addition. These results indicate
the inhibition of gelling process of 11S globulin by SSPS. However, the storage
modulus of 11S globulin gel was found to rise by SSPS addition based on the results of
dynamic oscillation measurement. Furthermore, the gigantic polymers of 11S
globulin were observed in the micrograph image of mixed gel system of 11S globulin
and SSPS instead of the typical network structure of protein gel. This means SSPS
could modify the structure and physical properties of 11S globulin gel, thereby
producing the new type gel. Soy Protein Research, Japan 10, 34-39, 2007.
Neurophysiological Study on the Mechanism Involved in the Astringent Taste Formation by Acidified Soy Protein
Michio KOMAI1, Sho MACHIDA1, Tsutomu SAITO2 and Hitoshi SHIRAKAWA1
1Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555 2Fuji Oil Co., Ltd, Izumi-Sano 598-8540
ABSTRACT
Human evaluation studies suggest that 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 the
electrophysiological studies by experimental animals have shown that the chorda
tympani and glossopharyngeal taste nerves transmit the astringency signal.
Therefore, we attempted to show the role of salivary component participation in the
occurrence of the astringency of acidified glycinin (isolated soy protein) solution by
recording from the rat taste nerve response. Adult female SD rats were used for the
chorda tympani nerve responses to various acidified glycinin solutions. We showed at
first the human saliva can be used for this analysis, and found out that especially the
NaHCO3 component had a big influence on the taste nerve activity derived from
acidified glycinin solution, instead of the precipitation formation itself by the
interaction of acidified soy protein (glycinin) and salivary component. Further
molecular level studies are required to clarify the phenomenon. Soy Protein Research, Japan10, 40-43, 2007.
Key words :astringency, acidified soy protein, rats, taste nerve
Flavor Release from Hamburger Steak Added Textured
Soy Protein During Mastication
Sachiko ODAKE
Department of Food Science and Technology, Nippon Veterinary
Life Science University, Tokyo 180-8602
ABSTRACT
Flavor release from hamburger steak, added granular type of textured soy protein,
during mimic mastication using mouth model system was studied. Elasticity and
hardness of the protein added hamburger steaks were evaluated higher than those
without protein by sensory test (p <0.05). Protein added samples showed higher
breaking force than those without protein as measured by a rheometer (p <0.01). The
total amount of released flavor from protein added samples was less than those
without protein, and this result showed consistency with the result obtained in
sensory evaluation. Sixty volatile compounds were detected from both samples with
and without protein, but only nine volatile compounds were identified only from
protein added samples. Mastication force during mimic mastication of protein added
sample was higher and the degree of breaking down was less than that in sample
without protein. This was considered to be one of the reasons for the less amount of
the released flavor from protein added hamburger steaks. Soy Protein Research, Japan10, 44-47, 2007.
Key words : textured soy protein, hamburger steak, mouth model
Effect of Soy Resistant Protein on Fermentation and Microbiota in the Large Intestine
Naomichi NISHIMURA, Yuki MITA, Maiko SAKURAI, Tatsuro YAMAMOTO and Toru OTA
Department of Nutritional Sciences, Faculty of Health and Welfare Science,
Nayoro City University, Nayoro 096-8641
ABSTRACT
To determine the mechanisms by which the supply of nitrogen into the large intestine
modifies the fermentation, we examined changes in fermentation and microbiota in
the large intestine by the administration of both pectin (PE) and soy resistant protein
(SRP). In experiment 1, PE and SRP were fermented in vitro and samples were taken
at 0, 5, 10 and 24 h, and pH change, production of short-chain fatty acid and ammonia,
and organic matter disappearance were determined. In experiment 2, rats were fed
the control, PE and PE+SRP diets for 10 d, and cecal short chain fatty acid
concentration and cecal microbiota were determined. In experiment 1, propionate
and butyrate production was increased by SRP in a dose-dependent manner,
suggesting that the supply of nitrogen by SRP plays a crucial role in an increased
propionate and butyrate production. The addition of SRP also increased ammonia
production dose-dependently, but the ammonia concentration decreased after h 10,
suggesting that ammonia produced is used as a nitrogen source by intestinal bacteria.
In experiment 2, the contents and concentrations of cecal propionate and butyrate
were significantly higher in rats fed the PE+SRP diet than in those fed the control
and the PE diets. According to PCR-DGGE analysis, the feeding of pectin resulted in
new clusters of cecal microbiota. The addition of SRP to the PE diet caused little
change in clusters of the cecal microbiota. However, a particular band, which
indicates one species of bacteria, was observed in rats fed the PE+SRP diet only. In
conclusion, when pectin is fermented by intestinal bacteria, SRP seems to increase propionate and butyrate production through the activation of particular bacterium. Soy Protein Research, Japan10, 48-54, 2007.
Key words : soy resistant protein, large intestine, fermentation, microbiota, rat
Mechanism of Decreasing Food Intake of Rats Fed Daidzein
-Approach Using Equol, Metabolite of Daidzein by Microorganis-
Taro KISHIDA, Asami IWAHARA and Kiyoshi EBIHARA
Faculty of Agriculture, Ehime University, Matsuyama 790-8566
ABSTRACT
We have previously shown that soy isoflavone daidzein decreased food intake in
female rats. In this study we aimed to confirm the active substance and mechanism.
We found that equol, a metabolite of daidzein by intestinal microorganism, is the
active substance that decreases food intake. We speculate that the effect of equol
could not simply be related to estrogenic effect of isoflavones because of the
difference of effect on food intake and estrogen receptor activation between estradiol
and equol. Soy Protein Research, Japan10, 55-58, 2007.
Key words : soy isoflavone, daidzein, equol, estrogen receptor, rat
Effect of a Soy-derived Oligopeptide Diet on Plasma Levels of Endogenous Morphine-like Analgesic Peptides in Rats
Toshiyuki SHIRAGA
1Department of Foods and Human Nutrition, Faculty of Human Life Sciences, Notre Dame
Seishin University, Okayama 700-8516
ABSTRACT
Endogenous morphine-like analgesic peptides, enkephalins, endorphins and
dynorphins, show an analgesic effect by binding to their specific receptors,
respectively. Previous pharmacological studies indicated that a subcutaneous
administration of L-arginine to animals induces an antinociceptive effect by an
increase in the brain level of kyotorphin (L-tyrosinyl-L-arginine) kyotrophin is an
endogenous analgesic peptide and an enkephalin releaser in the brain, and that an
intravenous administration of L-arginine to patients with various kinds of persistent
pain resulted in lasting analgesia. It is also reported that a diet containing protein
derived from soy had an effect on the analgesia in murine bone cancer pain model. In
addition, it is known that digested products of dietary proteins, di- and tripeptides,
are absorbed via an intestinal H+/peptide transporter PepT1 which has a high
capacity, and that soy proteins have an excellent nutritional specificity compared
with those in the other beans. From these facts, the effect of soy oligopeptides both
in the absorption and the analgesic action can be expected. The aim of this study was
to investigate the effects of a soy-derived oligopeptide diet on plasma levels of leucineenkephalin, β-endorphin and dynorphin A in rats. Plasma levels of these opioid
peptides in rats fed a diet containing either soy protein isolate or soy-derived
oligopeptides were significantly higher than those in rats fed a milk casein diet,
respectively. It is, therefore, suggested that a soy oligopeptide diet might be effective
for pain control by increasing the plasma opioids. Soy Protein Research, Japan10, 59-
62, 2007.
Effect of Dietary Soy Protein Isolate on the Production of Adipocytokines such as Adiponectin
Fumihiko HORIO
Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601
ABSTRACT
We investigated the effects of dietary soy protein isolate (SPI), casein or wheat
gluten on the development of Type 2 diabetes and serum concentrations of
adipocytokines (adiponectin and monocyte chemoattractant protein 1 (MCP-1)) in
SMXA-5 and KK-Ay mice. These two strains of mouse are animal models for Type 2
diabetes. Feeding SPI delayed the development of diabetes in KK-Ay mice compared
to feeding casein. The serum concentration of adiponectin in mice fed SPI was not
different from that in mice fed casein, but the serum concentration of MCP-1 was
significantly lower in KK-Ay mice. MCP-1 causes the insulin resistance in peripheral
tissues, such as adipose tissue and skeletal muscle, and liver. The results in this
study suggest that feeding SPI has a preventive effect of Type 2 diabetes by
reducing the production of MCP-1 in adipose tissue. Soy Protein Research, Japan10,
63-66, 2007.
Key words : type 2 diabetes, adiponectin, monocyte chemoattractant protein 1, soy
protein isolate, casein, KK-Ay mice, SMXA-5 mice
Interaction of Dietaryβ-Conglycinin and Conjugated Linoleic Acid on Body Fat Mass and Lipid Metabolism in Rats
Kazunori KOBA1, Asuka AKAHOSHI2, Kazunari TANAKA1 and Michihiro SUGANO3
1Faculty of Nursing and Nutrition, Siebold University of Nagasaki, Nagasaki 851-2195 2Faculty of Environmental and Symbiotic Sciences, Prefectural University of Kumamoto,
Kumamoto 862-8502 3Professor Emeritus, Kyushu University, Fukuoka 813-0043
ABSTRACT
The combined effect of conjugated linoleic acid (CLA) with different proteins on lipid
metabolism was examined in rats. Male rats were fed diets containing 1% CLA or
linoleic acid (LA) in combination with different proteins (casein (CAS), soy protein
isolate (SPI) or β-conglycinin (CON)) for 4 weeks. Dietary CLA, as compared with
LA, reduced the weight of epididymal and perirenal adipose tissues. Feeding SPI
and CON as compared with CAS decreased these adipose tissue weights irrespective
of dietary CLA, and the magnitude of the reduction was greater in the latter. The
concentration of serum triglyceride was also affected by dietary CLA and proteins in
the same manner. Feeding SPI, and CON decreased the activity of liver cytosolic
fatty acid synthase, irrespective of dietary CLA. The carnitine-palmitoyl transferase
activity in liver mitochondria was significantly increased in the combination of CLA
and CON more than the other groups. Moreover, CLA tended to increase serum
adiponectin concentration irrespective of dietary protein. Feeding CON
significantly increased the adiponectin concentration more than the other two
proteins, irrespective of dietary CLA. Therefore, the reduction of adipose tissue
weight and serum triglyceride concentration could be associated with decreased
fatty acid synthesis and enhanced β-oxidation in liver, and increased serum
adiponectin concentration. The results in the present study indicated that the body
fat-reducing effect of CLA was modulated by the type of dietary protein, and CON could enhance the CLA effect more than SPI. Soy Protein Research, Japan10, 67-71,
2007.
Key words : soy protein isolate,β-conglycinin, adipose tissue weight,β-oxidation, rats
Effects of Feeding on the Soy Protein Isolate Diet on Ceramides in Rat Epidermis
Yuichi OISHI
Faculty of Bio-Science, Tokyo University of Agriculture, Tokyo 156-8502
ABSTRACT
Ceramides foam extracellular lamellar membrane structures within the stratum
corneum that subserve the permeability barrier to water loss, are necessary for the
survival of mammals in a terrestrial environment. To examine whether and how soy
protein diet or soy peptide diet affects the status of stratum ceramides (SC), the
amount of SC and the mRNA levels of serine palmitoyl transferase (SPT), the first
committed step for ceramide synthesis, were analyzed in the epidermis of rat fed on a
12% soy protein isolate (SPI, Fujipro) diet, a 12% soybean peptide (SP, Hinute D1) diet,
a 12% soybean soluble peptide (SSP, Hinute D3) diet, a 12% casein diet (C), or a
protein-free diet (PF). The amount of SC in rats fed on SPI, SP, SSP, and PF diets did
not decrease in comparison with that in rats fed on a C diet. While the mRNA levels
of SPI in rats fed on the SP diet drastically decreased, the levels in rats fed on the PF
diet increased. Cholesterol is also one of the lipids in the stratum corneum. The
mRNA levels of HMG CoA reductase, an important enzyme of cholesterol synthesis,
did not significantly decrease in rats fed on the SP diet, but the levels in rats fed on a
PF diet drastically decreased. These results suggest that the SP diet for a week
affected ceramide de novo synthesis, but did not affect the amount of ceramides in
stratum corneum. Furthermore, the regulation of SPT by dietary protein might be
different from that of HMG CoA reductase in rat epidermis. Soy Protein Research,
Japan10, 72-75, 2007.
Key words : soybean, ceramide, serine palmitoyl transferase, stratum corneum
Transcriptome Analysis of the Effect of Soy Protein Intake in Cholesterol-loaded Rats
Hisanori KATO and Shinnosuke TSUJI
Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657
ABSTRACT
We have previously reported the effect of consumption of soy protein isolate (SPI) in
combination with other proteins on gene expression profile of rat liver in an attempt
to evaluate of the effect of SPI intake in dietary life. The result showed that a large
part of the action of SPI including ones on fatty acid and cholesterol metabolism was
maintained even in the presence of other dietary proteins. However the influence of
such diets on serum cholesterol level was negligible. In the present study, similar
analysis was conducted under a different experimental condition. Ten-week-old
male Wistar rats were pair-fed on experimental diets containing 0.5% cholesterol for
2 weeks. The experimental diets used were 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). The increase of serum cholesterol level was
significantly suppressed in S, G and G+S groups. Triglyceride and cholesterol levels
in the liver were lowest in the S-fed group. The result of DNA microarray analysis
showed that the total number of the genes that responded to dietary protein was
lower in the mixed protein groups as compared with S and G groups. The expression
levels of the genes for cholesterol synthesis pathway were low, and the impact of
dietary protein on them was not evident. On the other hand, the expression levels of
genes for the enzymes involved in fatty acid synthesis were relatively consistently
reduced in groups fed on S- and G-containing proteins. The result indicates that the
effects of mixed-protein feeding are less prominent under the present condition as
compared with the previous experiment and that the responses of genes for fatty
acid synthesis pathway are retained even when SPI is consumed with other proteins. Soy Protein Research, Japan 10, 76-82, 2007.
Key words : nutrigenomics, DNA microarray, lipid metabolism, cholesterol
Cholesterol Lowering Effect of Soybean Lipophilic Proteins Associated with Phospholipids in Rat
Ryuhei KANAMOTO, Shinya KIMURA and Gaku OKAMURA
Graduate School of Agriculture, Kyoto Prefectural University, Kyoto 606-8522
ABSTRACT
Recently, a group of lipophilic proteins (LP) associated with lecithin (phospholipids)
was fractionated from soy protein isolate (SPI) by newly developed methods of acidprecipitation.
LP was found a major component of SPI and comprises about 30% of
the total proteins. In this study, we examined effect of LP on plasma concentration of
cholesterol and triacylglycerol to clarify whether LP exerts hypocholesterolemic
action as observed in SPI. Five-week-old male Sprague-Dawley rats were fed diets
containing casein, LP and its partial hydrolysate, LP-peptide or SPI as a protein
source. The plasma levels of cholesterol and triacylglycerol were lower in the rats fed
either LP, LP-peptide or SPI diet than in the rat fed casein diet. The lowering effect
is greater in the rat fed LP-peptide than in the rat fed LP itself. The fecal bile acids
and total lipids excretion did not differ significantly among the groups. Therefore, it
can be considered that LP-peptide as well as LP itself modifies lipid metabolism
directly and affect the plasma cholesterol and triacylglycerol levels. However, theexpression of liver HMG-CoA reductase, Cholesterol 7α-hydroxylase (CYP7A1) and LDL receptor mRNA did not differ significantly among dietary groups. Soy Protein
Research, Japan10, 83-87, 2007.
Key words : lipophilic proteins, soy protein isolate, hypocholesterolemic action, bile
acids, triacyl glycerol, rat
Prevention of Metabolic Syndrome by Soy Isoflavone
Takanori TSUDA
College of Bioscience and biotechnology, Chubu University, Kasugai 487-8501
ABSTRACT
Adipocyte dysfunction is strongly associated with the development of obesity and
insulin resistance. It is accepted that the regulation of adipocytokine expression is
one of the most important targets for the prevention of obesity and amelioration of
insulin sensitivity. In this study, we demonstrated that soy isoflavone has the
potency of a unique pharmacological function in 3T3-L1 adipocytes. The pretreatment
of isoflavone significantly blocked downregulation of adiponectin
expression by TNF-α in 3T3-L1 adipocytes. Interestingly, isoflavone did not activate
PPARγ transcriptional activity. Genistein significantly inhibited TNF-α induced
MAPK signaling (SAPK/JNK) which is involved with adiponectin expression. These
data indicate that isoflavone blocks downregulation of adiponectin expression
through inhibition of SAPK/JNK pathway.
Soy Protein Research, Japan10, 88-92,
2007.
Key words : isoflavone, adipocyte, adiponectin, diabetes
Evaluation Study on Novel Physiological Activities of Soy Isoflavones
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 increase overall metabolism, little is
known about the molecular mechanism of the effects. Isoflavones having estrogenlike
activities are thought to contribute to it. In this study we utilized our new assay
system to evaluate the ligand-binding activity to estrogen receptor and examined the
ligand activity of isoflavones, their glycosides and their derivatives metabolized by
bacterial flora in the intestine. We further tested other food factors (~120
compounds) in the same assay. Isoflavones were found to be more potent ligands for
the receptor than other food factors. Coumestrol has the highest ligand-binding
activity among all compounds tested in the current assay. Equal, a daizein
metabolite, has a stronger ligand-binding and receptor inducible activity than daizein
does. This suggests that it is required to study the biological activities of isoflavone
metabolites in our body. Soy Protein Research, Japan10, 93-95, 2007.
Key words : estrogen, coumestrol, isoflavone, equal, daizein
Unloading-mediated Expression of a Muscle Atrophy-related Gene, Cbl-b and Its Regulation by Soy-protein-derived Peptides
Takeshi NIKAWA, Reiko NAKAO, Chiaki UENISHI, Katsuya HIRASAKA and Kyoichi KISHI
Institute of Health Biosciences,
The University of Tokushima Graduate School, Tokushima 770-8503
ABSTRACT
The ubiquitin-proteasome pathway is a primary regulator of muscle protein turnover,
providing a mechanism for selective degradation of regulatory and structural
proteins. This pathway is constitutively active in muscle fibers and mediates both
intracellular signaling events and normal muscle protein turnover. However,
conditions of decreased muscle use, so called unloading, remarkably stimulate
activity of this pathway, resulting in loss of muscle protein. In fact, we previously
reported that expression of several ubiquitin ligase genes, such as MuRF-1, Cbl-b, and
Siah-1A, which are rate-limiting enzymes of the ubiquitin-proteasome proteolytic
pathway, are significantly up-regulated in rat skeletal muscle during spaceflight.
Moreover, we found that Cbl-b-mediated ubiquitination and degradation of IRS-1, an
important intermediate of IGF-1 signal transduction, contributes to muscle atrophy
during unloading. Therefore, we hypothesized that inhibition of Cbl-b-mediated
ubiquitination and degradation of IRS-1 leads to prevention of muscle atrophy during
unloading. In this study, we aimed to evaluate oligopeptide as an inhibitor against
ubiquitination of IRS-1 by Cbl-b. We synthesized various oligopeptides that may
competitively inhibit the binding of Cbl-b to IRS-1 on the basis of their structures and
screened inhibitory effects of these synthesized oligopeptides on Cbl-b-mediated
ubiquitination of IRS-1 using in vitro ubiquitination systems. We found that two
synthetic oligopeptides with specific amino acid sequences effectively inhibited
interaction with Cbl-b and IRS-1, resulting in decreased ubiquitination and
degradation of IRS-1 (Patent pending). In contrast, we also found inhibitory activity against Cbl-b-mediated ubiquitination of IRS-1 in soy protein-derived oligopeptides,
whereas its inhibitory effect was weaker than those of synthetic oligopeptides. Our
results suggest that specific oligopeptides may be available as a functional food
against the muscle atrophy, especially through downregulation of the Cbl-b-mediated
IRS-1 degradation. Soy Protein Research, Japan10, 96-104, 2007.
Key words : muscle atrophy, ubiquitin ligase inhibitor, soy-derived peptides
Screening and Evaluation of the New Cholesterol-lowering Peptides Derived from Soybean by a New Evaluation System Including a Genetic Engineering Approach
Satoshi NAGAOKA
Faculty of Applied Biological Sciences, Gifu University, Gifu 501-1193
ABSTRACT
Although the hypocholesterolemic action of soybean protein is well known,
hypocholesterolemic peptides has not been found yet. Thus, in the present study, we
investigated the effects of soybean peptides (SPH-sup: peptide mixture with molecular
weight less than <8 KDa.) on gene expression related to cholesterol metabolism. The
SPH-sup exhibited hypocholesterolemic action in rats by increasing the mRNA level
of cholesterol 7α-hydroxylase, the rate limiting enzyme for cholesterol catabolism,
compared to casein tryptic hydrolyzate (CTH) accompanying the decrease of cellular
cholesterol. However, both HMG-CoA reductase and LDL-receptor mRNA levels
were unchanged by SPH-sup in HepG2 cells. Furthermore, when we investigated the
cholesterol absorption, excretion, and storage in Caco-2 cells using a membrane
culture system, the cholesterol excretion significantly increased accompanying the
decrease of cellular cholesterol absorption. ABCA1, NPC1L1, SR-BI and ACAT2
mRNA were unchanged by SPH-sup in Caco-2 cells. Taken all together, soypeptides
may directly affect the regulatory system related to the hepatic and intestinal
cholesterol metabolism. Soy Protein Research, Japan10, 105-110, 2007.
Development of Soybean-derived Molecular Targeted Anti-metastasis Agent by Gene Technology
Hiroshi KOBAYASHI and Junko TSUJI
Department of Obstetrics and Gynecology, Nara Medical University, Kashihara 634-8522
ABSTRACT
Activated neutrophils contribute to the development of preterm delivery. Because of its
ability to suppress inflammation, bikunin, a Kunitz-type protease inhibitor, is currently in
clinical trials. How bikunin inhibits neutrophil activation is poorly understood. To
investigate the molecular mechanism of this inhibition, we analyzed the effect of
bikunin on proinflammatory cytokine production and nuclear factor-kappa B (NF-kB)
activation in neutrophils stimulated by lipopolysaccharide (LPS), an inflammatory
inducer. Here, we show 1) bikunin blocks LPS-induced secretion of proinflammatory
cytokines, including TNF-alpha and IL-1beta, in a dose-dependent manner, reaching
65% inhibition at the highest doses of bikunin tested (5μM); 2) inhibition by bikunin of
cytokine induction correlates with the suppressive capacity of ERK1/2 and p38
signaling pathways, implicating repression of at least two different signals in the
inhibition; and 3) bikunin inhibited sequentially the LPS-induced phosphorylation of
IkB-alpha, degradation of IkB-alpha, and nuclear translocation of NF-kB. These
results demonstrate that bikunin can inhibit LPS-induced neutrophil activation and
also suggest that such effects are important in vivo to play a major contributory role
in abrogation of neutrophil-mediated inflammatory responses, such as preterm
delivery. Soy Protein Research, Japan10, 111-114, 2007.
Key words : bikunin, lipopolysaccharide, proinflammatory cytokines, neutrophils, NFkappa
B
Negative Growth Control of Bowman-Birk Inhibitor from Soy Bean Against Mesothelioma Based on Restoration of Connexin 43-Dependent
Tumor-suppressive Effect
Tomohiro YANO1, Korehito KASHIWAGI1 and Hiromi SATO1, 2
1Project for Complementary Factors, National Institute of Health and Nutrition, Tokyo 162-8636 2Graduate School of Medical and Pharmaceutical Sciences, Chiba University, Chiba 263-8522
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 Bowman-
Birk protease inhibitor (BBI)-dependent tumor-suppressive effect. In this study, we
investigated if BBI could regulate growth of mesothelioma cells via the restoration of
Cx43-dependent tumor-suppressive effect. In an in vitro tumor promotion model,
BBI prevented disruption of Cx43-depdendent gap junction intercellular
communication (GJIC) from tumor promotion stimulation. In other in vitro
mesothelioma cell culture study, we found that BBI induced Cx43 mRNA as well as
stabilization of Cx43 protein in mesothelioma cancer cells. Especially, the
stabilization of Cx43 protein by BBI depended on inhibition of chymotrypsin-like
activity in proteasome. Furthermore, we confirmed that BBI-dependent induction of
Cx43 was closely associated with negative growth control of the mesothelioma cells.
We also found that cisplatin-dependent cytotoxicity against the mesothelioma cells
was reinforced by BBI treatment, maybe via the restoration of Cx43-dependent GJIC.
Taken together, it is concluded that BBI-dependent restoration of Cx43 is effective
for prevention and therapy in mesothelioma. Soy Protein Research, Japan10, 115-119,
2007.
Key words : Bowman-Birk protease inhibitor, mesotheiloma, connexin 43,
proteasome inhibiton, prevention and therapy
Isolation and Identification of Food-derived Soy Peptides in Blood
Kenji SATO
Department of Food Sciences and Nutritional Health,
Kyoto Prefectural University, Kyoto 606-8522
ABSTRACT
An enzymatic hydrolysate of soy protein concentrate, which was prepared in an industrial
scale, was administered to rat stomach at 100 mg/kg of body weight. The portal blood
was collected before and 30 min after the administration. The blood was deproteonized by
mixing with TCA to give 5% (w/v). Peptides and amino acids in the supernatant were
captured to a cation exchanger packed in a spin column (5 mm × 5 mm, i.d.) and eluted
with 7.5 M ammonia-50% methanol solution. The clarified peptides were fractionated by
size-exclusion chromatography (SEC) and reacted with phenylisothiocyanate. The
derivatives were resolved by reversed phase HPLC. Three peaks appeared in the SEC
fractions eluted from 18 to 20 mL specifically after the ingestion, which could be foodderived
soy peptides. Soy Protein Research, Japan10, 120-123, 2007.
Key words :soy peptide, absorption, food-derived peptide
Study on the Preventive Effects of Soybean Ingredients on Inflammatory Bowel Disease
Hideo SATSU, Ja Shil HYUN, Tomohiko MIZUKAMI, Mika HAMADA, Yuto HIURA and Makoto SHIMIZU
Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657
ABSTRACT
The effect of soybean components on inflammatory bowel disease (IBD) was
investigated. As IBD has been understood to be caused by disregulation of
inflammatory cytokines, including interleukin-8 (IL-8), tumor necrosis factor-alpha
(TNF-α), interleukin-1beta (IL-1β), we especially focused on IL-8 and the effect of
soybean components on the secretion of IL-8 in intestinal epithelial cells was
examined in this study. Among soybean components, soyaflavone HG, the initial
component of which is isoflavone, suppressed TNF-α-induced IL-8 secretion in human
intestinal epithelial-like Caco-2 cells. However, soyhealth SA, the initial component of
which is saponin, or soy peptide had little or no inhibitory effect on IL-8 secretion.
The induction of IL-8 secretion by hydrogen peroxide or IL-1β was not suppressed by
soyaflavone HG, suggesting that the inhibitory effect of soyaflavone HG was specific
for TNF-α-induced regulation of IL-8. The increased expression level of IL-8 mRNA
by TNF-α was almost completely suppressed by soyaflavone HG. Further, the
transcriptional activity of human IL-8 promoter was increased by TNF-α treatment,
and soyaflavone HG suppressed its induction in a dose-dependent manner. These
results show that soyaflavone HG suppressed TNF-α-induced IL-8 production at the
transcriptional level, suggesting that soyaflavone HG is a promising soybean
component for preventing IBD. Soy Protein Research, Japan10, 124-127, 2007.
Study on the Molecules Involved in Anti-allergic Effect of Soybean Components
Hirofumi TACHIBANA1, 2, Satomi YANO1 and Koji YAMADA1
1Faculty of Agriculture, Kyushu University, Fukuoka 812-8581 2Bio-Architecture Center, Kyushu University, Fukuoka 812-8581
ABSTRACT
Genistein and daidzein are polyphenolic isoflavones contained in soy protein and intestinal
bacteria metabolize daidzein to equol. We previously reported that isoflavones and equol
suppress the expression of the high-affinity IgE receptor FcεRI in human basophilic cells.
In this study, we examined what molecules are involved in the suppressive effect of
isoflavones and equol on FcεRI expression. Isoflavones are structurally similar to estrogen
and generally bind to estrogen receptors (ERs) with high affinity. To assess whether the
suppressive effect of isoflavones and equol on FcεRI expression is mediated by ERs, we
investigated the effect of 17β-estradiol (E2) on the cell surface expression of FcεRI. We
found that E2 did not affect the FcεRI expression. Moreover, the suppressive effect of
isoflavones and equol on the FcεRI expression was not inhibited by pre-treatment with
ERs antagonist ICI 182780. On the other hand, we have reported that the FcεRI
expression is negatively regulated by the PPARγ ligand, prostaglandin D2 metabolite 15
deoxy-Δ12, 14 prostaglandin J2 (15d-PGJ2). In addition, it has been reported that some
flavonoids including genistein exhibited weak peroxisome proliferator-activated receptorγ(PPARγ) agonist activities. To explore whether the effect of isoflavones and equol are
mediated by PPARγ, RNA interference-mediated gene silencing was utilized to downregulate
PPARγ expression. The PPARγ down-regulation did not affect the suppressive
effect of isoflavones and equol on the cell surface expression of FcεRI. These results
suggest that the suppressive effect of isoflavones and equol on FcεRI expression is not
mediated by either ERs or PPARγ. Soy Protein Research, Japan10, 128-133, 2007.