English Title of the S.P.R.C Report Vol.29 Details

Soy Protein Research(Japan)

29-1

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 cross-sectional relationship between soy intake and several biomarkers related to immunological functions and oxidative and inflammatory status in participants in a health-check up program. Diet including soy intake was determined with a use of a validated 169-item food-frequency questionnaire. The existence of cedar pollinosis was assessed by the questionnaire reported by Endo. Women with cedar pollinosis had higher percentages of eosinophils and monocytes in peripheral blood than those without cedar pollinosis. Soy intake was positively associated with the percentage of monocytes. Other biomarkers measured such as white blood cell counts, uric acids, and reactive oxygen species were unrelated to soy intake among women. However, in men, white cell count was inversely associated with soy intake. Longitudinal study on maternal soy intake and allergic disorders in the offspring is undergoing. Maternal soy intake was estimated by 5-day diet records during pregnancy. About five years of follow-up has been completed. The allergic rhinitis reported before age 2-3 years but not later was inversely associated with maternal soy intake. There were no significant associations between maternal soy and the developmennts of asthma, atopic dermatitis, and food allergy.
Soy Protein Research, Japan 11, 1-5, 2008.

Key words : soy, isoflavones, allergic disease, monocytes

29-2

Systematic Review and Verification of Preventive Effects of
Soy Constituents on Lifestyle-related Diseases:
Effects of Extracted Soy Isoflavones Alone on Blood Lipid Profile:
Meta-analysis of 13 Randomized Controlled Trials (Part I)

Yoshiko ISHIMI¹, Kyoko TAKU², Keizo UMEGAKI² and Jun YAMAUCHI¹

¹Project for Bio-index, Nutritional Epidemiology Program,
²Project for information Network of Health Food, Information Center, National Institute of Health and Nutrition, Tokyo 162-8636

ABSTRACT
Recent growing interest in health and diet has led to an increased assessment of the health effects of soy foods and their functional components. However, a consensus regarding its effectiveness has not been achieved thus far. In order to propose the appropriate amount of the functional components present in soy foods, we conducted this study with the aim of (1) examining the effects of the functional components of soy foods on the prevention of lifestyle-related diseases via a systematic review and meta-analysis of the related articles, and (2) investigating the labeling and contents of the functional components of soy in health foods. In this year, we focused on soybean isoflavones. When provided concurrently with soy protein for 1-3 months, soy isoflavones would have synergistic or additive effects on cholesterol lowering. This meta-analysis was performed to evaluate effects on blood lipid profile of extracted soy isoflavones alone (not ingested concurrently with soy protein). MEDLINE were searched for published randomized controlled trials of English-, Japanese, or Chineselanguage that reported effects of soy isoflavones in humans. Thirteen trials were finally selected for the meta-analysis using REVMAN. Averaged 75 mg soy isoflavones/d (25-132 mg, as aglycone form) did not significantly changed totalcholesterol [-0.03 mmol/L (95% CI: -0.21, 0.16); P = 0.79], LDL-cholesterol [0.04mmol/L (95% CI: -0.08, 0.16); P = 0.49], and HDL-cholesterol [0.03 mmol/L (95% CI: -0.03, 0.09); P = 0.31] and triacylglycerol [-0.06 mmol/L (95% CI: -0.12, 0); P = 0.07] in menopausal women. Results of 10 high-quality trials were similar. We found that daily ingestion of approximately 75 mg of extracted soy isoflavones/d alone for 1- 3 months did not improve lipid profile in menopausal women. When the labeling and isoflavone content of 20 commercially available health foods were assessed, a wide variety of isoflavone components and contents were noted. On the basis of these findings, we believe that the health effects of isoflavones in soy foods were assessed from the differences in the bioavailability and biological activity that are caused by the existence of other ingredients and a variety of isoflavone components in soy foods.
Soy Protein Research, Japan 11, 6-14, 2008.

Key words :extracted soy isoflavones, cholesterol, triacylglycerol, health foods, labeling

29-3

Absorption Rate and Requirement of TOFU Ca in Post-Menopausal Women

Chie TARUMIZU¹, Hien Vu Thi THU¹, ², Nobuko SARUKURA¹ and Shigeru YAMAMOTO¹

¹International Nutrition, Ochanomizu University Graduate School, Tokyo 112-8610
²National Institute of Nutrition, Hanoi, Vietnam

ABSTRACT
Ca concentration of TOFU is as high as that of cow's milk, while bioavailability of the former is believed to be lower than that of the latter. It is based on a report from an old study in Japan, however, the studies from all over the world showed rather similar absorption rates of them. The purpose of this study was to find the absorption rates and requirements of Ca in TOFU and skimmed milk by balance studies. Twelve postmenopausal women were recruited on a voluntary basis and two nutrition studies were conducted. In the first study subjects were randomly allocated to 15 d on basal diets (300 mg Ca) with TOFU (300 mg Ca) or skimmed milk (300 mg Ca) separated by a 15 d washout interval in a crossover design. In the second study, only basal diets (300 mg Ca) were given for 15 d. Basal diets were 3 cycle menus. Feces and urine of the last 5 d were collected in both studies and Ca concentrations were analyzed. Apparent absorption rates of TOFU and skimmed milk were 33±32 and 21±31%, respectively. Average Ca requirements of TOFU and skimmed milk estimated by the balance study were 502±84 and 625±89 mg/d, respectively. The results suggest that TOFU is a good Ca source.
Soy Protein Research, Japan 11, 15-19, 2008.

Key words :tofu, skimmed milk, Ca balance, post-menopausal women

29-4

Regulation of Biosynthesis of Soybean Seed Components by Gene Silencing

Masao ISHIMOTO and Keito NISHIZAWA

National Agricultural Research Center for Hokkaido Region, Sapporo 062-8555

ABSTRACT
Soybean [Glycine max (L.) Merrill] is one of the world's most important crops. Elucidation of biosynthetic mechanisms of seed components at the molecular level would provide valuable information for improvement of soybean. In this study, we evaluated the application of RNA interference (RNAi) to study biosynthetic mechanism of seed components. An RNAi vector targeting the gene encoding α' subunit of β-conglycinin was introduced in soybean somatic embryos, and the substantial reduction of the α' subunit together with the other two subunits (α and β subunits), which share high sequence homology with the α' subunit, was observed in the mature transgenic somatic embryos. The expression levels of α' subunit mRNA in the mature somatic embryos was less than 3% of that in those transformed with the empty control vector, indicating that the accumulation of β-conglycinin in the mature somatic embryos was reduced by sequence dependent manner of gene silencing. Accumulation of the α' subunit and the other two subunits was also remarkably reduced in the T₁ seeds obtained from the regenerated plant from the transgenic somatic embryos. Thus RNAi is applicable to study gene function in biosynthesis of seed components, and somatic embryos will be competent to accelerate the studies as a model system of seed.
Soy Protein Research, Japan 11, 20- 25, 2008.

Key words : RNA interference, seed component, somatic embryo, gene silencing, seed storage protein

29-5

Modification of Soybean Components through Gene Expression Control by the Function of RNA

Atsushi NAGAMATSU and Akira KANAZAWA

Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589

ABSTRACT
In view of the present circumstances in which considerable amount of the information of soybean genomics has been accumulated, there is an increasing need for developing a system suitable for analyzing gene function in soybean. RNA silencing is a high-throughput tool for suppressing gene expression in a sequencespecific manner, either through RNA degradation or transcriptional repression. Here we developed a system of RNA silencing in soybean, which allows rapid analysis of gene function. We have established an RNA silencing system using a virus vector in plants. In order to determine whether our vector system mediates RNA silencing in soybean, a portion of the sf3'h1 gene, a putative gene encoding flavonoid 3'-hydroxylase (F3'H), was inserted into the cloning site of the vector, and the resulting construct was used for infection of young soybean plants. After infection with the recombinant virus, the sf3'h1 mRNA level markedly decreased and short interfering RNA accumulated in the upper leaves, which indicate that sequence-specific degradation of sf3'h1 mRNA was induced. In the flavonoid synthesis pathway, F3'H catalyzes several steps including kaempferol to quercetin. This experiment also resulted in a decrease in the content of quercetin relative to kaempferol, which indicates that downregulation of the sf3'h1 gene affects the production of quercetin from kaempferol. These results provide successful demonstration of the application of the vector system for induction of RNA silencing in soybean and indicate that the sf3'h1 gene encodes the F3'H protein.
Soy Protein Research, Japan 11, 26-31, 2008.

Key words : flavonoid biosynthesis, gene expression control, reverse genetics, RNA silencing, soybean

29-6

Identification of Genes Controlling the Contents of Seed Storage Proteins in Soybean

Takanori YOSHIKAWA¹, Sigeru UTSUMI², Takako FUKUDA2, Yuu OKUMOTO¹,
Takashi SAYAMA¹ and Takatoshi TANISAKA¹

¹Graduate School of Agriculture, Plant Breeding, Kyoto University,
²Graduate School of Agriculture, Food Quality Design and Development,
Kyoto University, Kyoto 606-8502

ABSTRACT
96 recombinant inbred lines (RILs), which were derived from the cross of two soybean varieties, Peking and Tamahomare, were planted along with their parents in three locations, Kyoto, Osaka and Nagano. QTL (quantitative trait locus) analysis using SSR (simple sequence repeat) markers suggested that one QTL named qPro1 on linkage group I (LG I) simultaneously governed α- and α'-subunit content in all the locations: the Tamahomare-type allele at qPro1 increased α-subunit content and decreasedα'-subunit content, whereas the Peking-type allele decreasedα-subunit and increased α'-subunit content. The mean value of α-subunit content of the RILs carrying the Peking-type allele in the homozygous state at qPro1 was almost equal to the value of Peking, whereas that of RILs carrying the Tamahomare-type allele was equivalent with the value of Tamahomare. Thus it was found thatα-subunit content is chiefly governed by qPro1. Two QTLs controlling both of β-conglycinin and glycinin content were detected on linkage group I (qPro1) and O (qPro7 ), and the Tamahomare-type alleles at these two loci increased β-conglycinin content and decreased glycinin content. For total protein content, two QTLs were detected on linkage group A2 (qPro8 ) and C2 (qPro5 ) in Osaka and Nagano. In these two QTL regions, many QTLs forβ-conglycinin and/or glycinin contents were detected. These results indicate that two types of genetic factors regulate the contents of soybean seed storage proteins: one (qPro5 and qPro8 ) increases the contents of seed protein(s), and the other (qPro1 and qPro7 ) determines their ratios.
Soy Protein Research, Japan 11, 32-39, 2008.

Key words :soybean, seed storage protein, QTL, qPro, recombinant inbred line

29-7

Basic Analysis for the Defense of Soybean Cyst Nematodes by Using Synthetic Peptides

Shinichiro SAWA

Graduate school of Science, University of Tokyo, Tokyo 113-0033

ABSTRACT
Because of the agricultural importance, the soybean cyst nematode Heterodera glycines that infect soybean (Glycine max) has emerged as primary research models to understand the signaling, perception, and response events during plant-nematode interactions. The recent advances in our understanding of the cellular, physiological, and molecular basis of legume-nematode interactions are merging at the crossroads of plant-microbe interactions and plant developmental biology. On the other hand, Intercellular communication is a fundamental mechanism for coordinating the development of complex bodies of multicellular organisms such as plants and animals. In plant morphogenesis, CLAVATA (CLV ) genes are key players for meristem formation. CLV1, CLV2, and CLV3 encode a leucine-rich repeat receptor-like kinase (LRR-RLK), a LRR receptor-like protein without the kinase domain, and a putative peptide, respectively. CLV3 belongs to the CLV3/ESR-related (CLE) gene family that shares significant homology in 14 amino acids at the C-terminal region and has 31 CLE members in the Arabidopsis genome. CLV3 and other CLE peptides are suggested to function in plant morphogenesis as intercellular signaling molecules. We have identified that tracheary element differentiation inhibitory factor (TDIF) and CLV3 encode dodecapeptides with two hydroxy proline residues, regulating vascular development and meristem formation, respectively by using in situ MULDITOF- MS method. Chemically synthesized TDIF and CLV3 peptides also function in our in vitro bioassay systems. Next, we chemically synthesized various length of peptide by using putative CLE domain sequence of nematode gene. As a results, some of the peptides induced short root phenotype, and some of the others induced long root phenotype. We will improve the peptide sequence, and will try to develop effective chemicals for the defence of nematode infection to crops.
Soy Protein Research, Japan 11, 40-43, 2008.

Key words : nematode, soybean, CLE, peptide

29-8

Biochemical Characterization of High Lutein Trait of Wild Soybean
(Glycine soja Sieb. et Zucc.) and Expression Analysis of Gene Encoding Enzymes
Involved in Lutein Biosynthesis for the Breeding of Lutein Rich Soybean

Kyohei KANAMARU, Tetsuya YAMADA and Keisuke KITAMURA

Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589

ABSTRACT
Soybean seeds contain low levels of lutein which is beneficial to human health. We characterized the carotenoid composition of wild soybean seed with high lutein. The peaks of xanthophylls such as neoxanthin, violaxanthin and antheraxanthin in addition to the major peak of lutein were separated by high performance liquid chromatography (HPLC). These xanthophylls were mainly originated from the cotyledon of the seed. These xanthophylls identified were positively correlated with the lutein content in the progeny populations derived from the interspecific crosses between soybean and wild soybean, suggesting that the high lutein traits of wild soybean strains might be implicated in the biosynthesis and/or accumulation of xanthophylls during the seed filling. Expression of genes encoding enzymes involved in lutein and the other xanthophylls biosynthesis was analyzed by quantitative RT-PCR. There existed no difference between the expression levels of xanthophylls biosynthetic genes in RIL with high lutein and RIL with low lutein during the seed development. Whereas, the difference between the reduction of lutein in RIL with high lutein and RIL with low lutein during the seed development was observed. These results suggested that the high lutein trait of the wild soybean strains might be related in the catabolism of lutein and xanthophylls during the seed development. Further studies are in progress for characterization of the genetic mechanism that regulates the lutein content in wild soybean seeds for the breeding of lutein rich soybean.
Soy Protein Research, Japan 11, 44-50, 2008.

Key words :soybean, wild soybean, lutein, xanthophyll, lutein biosynthesis

29-9

Analysis of Interactions between Vacuolar Sorting Determinants of Soybean Seed Storage Proteins and Receptors

Nobuyuki MARUYAMA and Shigeru UTSUMI

Graduate School of Agriculture, Kyoto University, Uji 611-0011

ABSTRACT
Glycine max homologs of VSR and RMR were cloned from mRNA of soybean developing seeds. We constructed insect cell expression systems for their luminal domains (GmVSR and GmRMR) and examined an interaction between vacuolar sorting determinants of soybean seed storage proteins and their luminal domains. Glutathione S-transferase fused to 7S α' or 11S A1aB1b C-terminal 10 residues (GST+α'CT10 or GST+A1aB1bCT10) interacted with GmVSR. Kinetic analysis exhibited that GmVSR has a high affinity with GST+α'CT10 or GST+A1aB1bCT10 sufficient to function as a VSD. On the other hand, an addition of six glycine residues at the C-terminus of GST+α'CT10 or GST+A1aB1bCT10 abolished the interaction. GmVSR did not interact with recombinant A3B4 and recombinant A1aB1b fused to six glycine residues at its C-terminus expressed in E. coli (ProA3B4 and ProA1aB1b+6G), whereas it could bind to recombinant A1aB1b (ProA1aB1b). These results suggest that GmVSR can interact with ctVSD, but not ssVSD and psVSD. On the other hand, GmRMR did not interact with all types of VSD. Therefore, molecules except VSR and RMR might exist as receptors for ssVSD and psVSD. Further, we examined the interaction between GmVSR and ctVSD in detail. GST fused to Cterminal three residues of 7S α' (GST+GAFY) had a low affinity with GmVSR, whereas GST fused to C-terminal four residues (GST+RAFY) had a high affinity. However, GST fused to three residues within C-terminal four residues (GST+RGFY, GST+RAGY and GST+RAFG) except C-terminal three residues (GST+GAFY) exhibited a high affinity with GmVSR. These results suggest that a basic residue in addition to a hydrophobic residue can contribute to the interaction between ctVSD and GmVSR.
Soy Protein Research, Japan11, 51-55, 2008.

Key words : receptor, protein storage vacuole, vacuolar sorting determinant

29-10

High-Pressure Induced Transformation of Soybean

Tomoyuki FUJII

Department of Biotechnology, Maebashi Institute of Technology, Maebashi 371-0816

ABSTRACT
The effect of high-pressure treatment on soybean as a cellular biological material was investigated from the viewpoints of the cell structure, and enzyme reaction system. The cell structure of soybean was evaluated with the dielectric properties. The dielectric properties of soybean were 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 decrease depending on high pressure. The result suggested that the structure of soybean was damaged by high-pressure treatment. The production rate of free amino acids in soybean, which swelled in water, during preservation at 25℃ was accelerated by high-pressure treatment of 200 MPa, 10 min. The high-pressure treatment also made the enzymatic production of gammaaminobutyric acid (GABA) in the soybean, which swelled in sodium glutamate solution, increasing. From these results, the high-pressure treatment induced the destruction of cell structure without denaturing enzymes and the accelerated biochemical reaction in soybean. In other words, these events are 'high-pressure induced transformation (Hi-Pit)' of soybean.
Soy Protein Research, Japan 11, 56-60, 2008.

Key words : soybean, high-pressure, dielectric properties, free amino acid, gammaaminobutyric acid

29-11

Quality Improvement of Microorganisms by Soy Peptides

Shingo IZAWA

Graduate School of Agriculture, Kyoto University, Uji 611-0011

ABSTRACT
The physiological functionality of soy peptides in mammal has been well investigated. It has been reported that soy peptides show various potencies such as scavenging oxidative stress, reducing serum lipid levels, and anti-fatigue effects in mammalian cells. Therefore, soy peptides have been attracting worldwide attention as functional food ingredients. In contrast to the recent progress in the study on mammalian physiology, effects of soy peptides on industrial microorganisms remain to be solved. To promote more effective use of soy peptides in different ways, we examined their validity as ingredients of culture media to improve the quality of food microorganisms. In this study, we show that the cultivation of yeast cells in media containing soy peptides can improve tolerance to freeze-thaw stress, indicating that soy peptides are suitable ingredients of culture media to provide high-quality yeast cells for frozen-dough technology. The baked bread using yeast cells cultured with soy peptides was of better quality in terms of texture and volume than the bread using cells with casein peptone. We further investigated the mechanisms of the improved tolerance to freeze-thaw stress by soy peptides. Compared with yeast cells cultured with casein peptone, cells cultured with soy peptides showed higher levels of intracellular proline, valine, and aspartic acid. Contrary, the cultivation with soy peptides resulted in decreased levels of intracellular alanine and arginine. Additionally, yeast cells cultured with soy peptides showed the decreased number and size of lipid particles.
Soy Protein Research, Japan 11, 61-66, 2008.

Key words : soy peptides, baker's yeast, freeze-thaw stress, frozen-dough technology, lipid particle

29-12

Screening of Aldehyde Dehydrogenase Which Degrades n-Hexanal, the Causative Substance of Soy Bean Smell

Hideyuki SUZUKI¹ and Daisuke TOMIYAMA²

¹Graduate School of Science and Technology, Kyoto Institute of Technology, Kyoto 606-8585
²Faculty of Agriculture, Kyoto University, Kyoto 606-8502

ABSTRACT
n-Hexanal, the causative substance of soy bean smell, is a big problem when we use soy products in food industry. n-Hexanal is produced through the oxidation of linoleic acid whose content is very high in soy beans. Aldehyde dehydrogenases, which may have high activity for n-hexanal, were screened in silico and we chose Bradyrhizobium japonicum from soy nodules. The gene was cloned and overexpressed in Escherichia coli. It was shown that the cell-free extracts of the overexpressed strain had very high activity against n-hexanal.
Soy Protein Research, Japan 11, 67-70, 2008.

Key words : n-hexanal, aldehyde dehydrogenase, root nodule bacteria

29-13

Neurophysiological Clarification of the Mechanism Involved in the Astringent Taste Formation by Acidified Soy Protein

Michio KOMAI¹, Sho MACHIDA¹, Tsutomu SAITO², Tomoko GOTO¹ and Hitoshi SHIRAKAWA¹

¹Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555
²Fuji Oil Co., Ltd, Izumi-Sano 598-8540

ABSTRACT
It has been reported that perceptual assessments of astringency may be closely linked to bitterness in human tasting study, however, human evaluation study suggested that astringency may be categorized as somatic sensation. On the other hand, most of the electrophysiological studies by experimental animals have shown that the chorda tympani and glossopharyngeal taste nerves transmit the astringency signal. Actually, we observed the chorda tympani nerve responses to acidified glycinin (isolated soy protein) in the rat, and the fiber analyses of this response showed the similarity to astringent sensation as tannic acid, and gallic acid, rather than acid taste sensation. Therefore, we attempted to show the role of salivary component participation in the occurrence of the astringency of acidified glycinin solution by recording from the rat taste nerve response. Adult female SD rats were used for the chorda tympani nerve responses to various stimuli. We found out that especially the NaHCO₃ had most effective component in increasing the taste nerve activity derived from acidified glycinin solution instead of the precipitation formation itself, and that the acidic preparation under pH 3.5 was another key condition to cause the taste nerve sensation. Further detailed clarification of the mechanism involved in the stimulant structure-activity relationship from the viewpoint of molecular level must be required.
Soy Protein Research, Japan 11, 71-74, 2008.

Key words : astringency, acidified soy protein, rats, taste nerve, NaHCO₃

29-14

Physiological Function and Its Mechanism of Soybean Lipophilic Protein as a Functional Food for Prevention of Metabolic Syndrome

Ryuhei KANAMOTO, Marie INAI and Wakako MIYAKI

Graduate School of Life and Environmental Science, Kyoto Prefectural University,
Kyoto 606-8522

ABSTRACT
We investigated whether Soybean lipophilic proteins (LP) and its enzymatic hydrolysate, LP-peptide (LP-Pep) were effective for prevention of a metabolic syndrome in mouse which was maintained on high fat diet for 28 and 70 days to induce obesity. Feeding of LP and LP-Pep decreased plasma concentrations of triacylglycerol (TG) and total bile acid (TBA) concomitant with the increase of fecal excretion of TBA. Although LP-Pep was more effective than LP itself, the effects of LP-Pep were less than those of HMF (high-molecular-weight fraction of soy protein digest) and SPI (soy protein isolate). Unlike previous many reports, these dietary protein and peptide did not decrease a plasma concentration of cholesterol though they increased fecal excretion of TBA in C57BL/6J mouse maintained on high fat diet. LP-Pep and HMF slightly reduced the accumulation of body fat caused by high fat diet. The expression of Cholesterol 7α-hydroxylase (CYP7A1), HMG-CoA reductase and Acetyl-CoA carboxylase (ACC) mRNA in liver were changed by experimental diets. However, the expression did not correlated with the plasma concentrations of TG, TCHO and TBA.
Soy Protein Research, Japan 11, 75-81, 2008.

Key words :lipophilic proteins, metabolic syndrome, bile acids, triacylglycerol, cholesterol

29-15

Development of Soybean-derived Molecular Targeted Anti-metastasis Agent KTI

Hiroshi KOBAYASHI and Junko TSUJI

Department of Obstetrics and Gynecology, Nara Medical University, Kashihara 634-8522

ABSTRACT
Objective: 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. Materials and Methods: To investigate the molecular mechanism of this inhibition, we analyzed the effect of bikunin on proinflammatory cytokine production and nuclear factor-kappaB (NF-kB) activation in neutrophils stimulated by lipopolysaccharide (LPS), an inflammatory inducer. Results: Here, we show 1) bikunin blocks LPS-induced secretion of proinflammatory cytokines, including TNFalpha 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 repressed at least two different signalings 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. Conclusion: 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, Japan 11, 82-87, 2008.

Key words : bikunin, lipopolysaccharide, proinflammatory cytokines, neutrophils, NFkappaB

29-16

The Role of Genistein in Breast and Prostate Cancer Prevention through the Protein Modification and Degradation Pathway

Tomohiko URANO

Department of Anti-aging Medicine, 22nd Century Medical and Research Center,
Graduate School of Medicine, University of Tokyo, Tokyo 113-8655

ABSTRACT
Estrogen-responsive finger protein (Efp) is one of the estrogen responsive genes and belongs to a family of tripartite motif (TRIM) proteins. Here, we have shown that genistein, one of the predominant soy isoflavones, repressed the expression of Efp. We also have shown that Efp functions as a ubiquitin E3 ligase and plays crucial roles in the regulation of breast and prostate cancer cell growth. We have shown that Efp targets ubiquitin mediated proteolysis of 14-3-3σ, a negative cell cycle regulator that causes cell cycle arrest. Next, we performed immunohistochemical analysis for breast and prostate malignant tissues, and investigated the association with patients' clinicopathological data. Positive Efp immunoreactivity significantly correlated with poor cancer-specific survival both in prostate and breast cancer patients, suggesting that Efp could play an important role in the tumorigenesis of breast and prostate cancer. The present data suggest that genistein could play an important role in the cancer prevention through the repression of Efp. We have demonstrate that Efp also targets ubiquitination of Retinoic-acid-inducible gene-I (RIG-I) is a cytosolic viral RNA receptor that induce type I interferon-mediated host protective innate immunity against viral infection. Furthermore, gene targeting demonstrates that Efp is essential not only for RIG-I ubiquitination but also for RIG-I-mediated interferon- production and antiviral activity in response to RNA virus infection. Thus, we demonstrate that Efp, genistein regulated gene, induces the ubiquitination of RIG-I, which is crucial for the cytosolic RIG-I signalling pathway to elicit host antiviral innate immunity.
Soy Protein Research, Japan 11, 88-94, 2008.

Key words : Estrogen, Genistein, Prostate cancer, Breast cancer, Innate immunity, Interferon, Efp

29-17

Analysis of Target Genes of Isoflavones and Clinical Significance of Equol Producers

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 metabolite daidzein to equol (equol producers) might be more sensitive to soy intake than equol non-producers. To address the molecular mechanisms of isoflavone on human health, we performed DNA microarray analysis. And we also study the clinical parameters for equol producers and equol non-producers. Our data suggest that equol producers are favorable for metabolic syndrome.
Soy Protein Research, Japan 11, 95-98, 2008.

Key words : phytoestrogen, isoflavone, equol, DNA microarray

29-18

Screening of Soy Protein-Derived Hypolipidemic Peptides Using in vitro and in vivo Systems

Koji NAGAO¹, Nao INOUE¹, Kotaro SAKATA¹, Naomi YAMANO¹,
Toshiro MATSUI² and Teruyoshi YANAGITA¹

¹Laboratory of Nutrition Biochemistry, Faculty of Agriculture, Saga University, Saga 840-8502
²Laboratory of Food Analysis, Faculty of Agriculture, Graduate School of Kyushu University, Fukuoka 812-8581

ABSTRACT
Soy protein is one of the vegetable proteins examined extensively for lipid lowering effect in humans and in experimental animals. Although soy protein isolate contains certain amount of bioactive peptides which have distinct physiological activities in lipid metabolism, it is not clear that which peptides are responsible for these effects. In the present study we have investigated the effect of soy protein-derived peptides on lipid metabolism in HepG2 cells and obese OLETF rats. In the first experiment, we found that soy crude peptide (SCP-LD3), which is made by hydrolysation of soy protein isolate with endo-type protease, showed hypolipidemic effects in HepG2 cells and in OLETF rats. In the second experiment, we found that hydrophilic peptides, separated from SCP-LD3 with hydrophobic synthetic absorbent, revealed lipid lowering effect in HepG2 cells and in OLETF rats. In the third experiment, we found that F4 peptides, fractionated from hydrophilic peptides by GPC-HPLC, significantly reduced TG synthesis and apoB secretion in HepG2 cells. In the forth experiment, we found that 0% CH₃CN/0.1% TFA-fraction, isolated from F4 peptides by ODS column chromatography, showed hypolipidemic effect in HepG2 cells. In conclusion, we could have isolated active peptide fraction of lipid lowering effects from soy protein.
Soy Protein Research, Japan 11, 99-104, 2008.

Key words : soy peptide, OLETF rat, HepG2 cell, triglyceride, apolipoprotein B-100

29-19

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. Both 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 JNK activation which is involved with adiponectin expression. The administration of soy isoflavone significantly reduced blood glucose concentration in type 2 diabetic mice.
Soy Protein Research, Japan 11, 105-109, 2008.

Key words : isoflavone, adipocyte, adiponectin, diabetes

29-20

Effects of Soy Peptides on Metabolism of Isoflavone by Fecal Flora from Mice

Motoi TAMURA¹, Atsuko SHINOHARA¹ and Tomoka FUKATSU¹

¹National Food Research Institute, Tsukuba 305-8642

ABSTRACT
We investigated the effects of soy peptides on the in vitro and in vivo equol production from daidzein by fecal flora in mice. In fecal suspension from the mice, equol concentrations were lower in the control fecal suspension than in the fecal suspension supplemented with 2 mg soy peptides HI-NUTE DH per 200μL. Male 5- week-old mice were fed a soy peptides-daidzein diet or a casein-daidzein diet for 20 days. In fecal suspension from the mice fed an experimental diet, equol concentrations were significantly higher in the fecal suspension from the mice fed the soy peptides-daidzein diet for 7 and 14 days than in the fecal suspension from the mice fed the casein-daidzein diet for 7 and 14 days. In vitro equol productivity from daidzein by fecal flora in mice fed the soy peptides-daidzein diet has increased as the days go by. The plasma equol concentration was significantly higher in the soy peptides-daidzein group. The cecal concentration of equol present as aglycone was significantly greater in the soy peptides-daidzein group. We demonstrate that the ingestion of soy peptides may enhance equol production by having an impact on the metabolic activity of the intestinal microflora.
Soy Protein Research, Japan 11, 110- 115, 2008.

Key words : soy peptides, daidzein, equol, mouse

29-21

Effect of Feeding Soy Protein Isolate on the Production of Adipocytokines such as Adiponectin and MCP-1

Fumihiko HORIO

Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601

ABSTRACT
We examined the effect of soy protein isolate (SPI) and casein feeding on adipocytokine production and diabetic phenotype in KK-Ay mice, a model for type 2 diabetes. Five-week-old male KK-A^y mice were fed a diet containing 20% SPI (soy group) or 20% casein (casein group) for 18 days. SPI feeding suppressed the development of hyperglycemia and hyperinsulinemia compared with casein feeding. Adiponectin and MCP-1 mRNA levels in epididymal adipose tissue, and serum concentrations of these adipocytokines were not different between the soy and casein groups. Hepatic triglyceride content in the soy group was significantly lower than that in the casein group. Hepatic levels of SREBP-1, fatty acid synthase and acetyl CoA carboxylase mRNA in the soy group were significantly lower than those in the casein group. These results indicate that SPI feeding prevent the development of hyperglycemia and fatty liver in KK-A^y mice, and suggest that SPI feeding improves insulin resistance and suppresses hepatic fatty acid synthesis in KK-A^y mice.
Soy Protein Research, Japan 11, 116-120, 2008.

Key words : type 2 diabetes, adiponectin, MCP-1, soy protein isolate, fatty liver

29-22

Blood Glucose Regulation by a Soybean Isoflavone, Genistein: Studies in
Cultured Cells and Type 2 Diabetic Mice

Kazumi YAGASAKI, Masato NAGAOKA and Yutaka MIURA

Division of Agriscience and Bioscience, Institute of Symbiotic Science and Technology,
Tokyo University of Agriculture and Technology, Fuchu 183-8509

ABSTRACT
Effect of a soybean isoflavone, genistein, on (1) glucose uptake by rat-derived L6 myotubes and (2) insulin secretion from rat-derived RIN-5F pancreatic β-cells was studied in vitro. Then, (3) that on blood glucose levels in type 2 diabetic KK-Ay/Ta mice was studied in vivo. Genistein dose-dependently and significantly stimulated glucose uptake by L6 myotubes and insulin secretion from RIN-5F cells in culture at 10-100μM, where genistein exerted no influence on the viability of RIN-5F cells. Feeding 0.1% genistein in 20% casein diet (20C) for 5 weeks to KK-Ay/Ta mice suppressed increases in blood glucose levels and the suppressive effect was significant at 1^st and 5^th week of feeding as compared with 20C-fed diabetic control mice. Genistein also suppressed urinary glucose excretion as compared with control mice during the feeding period. These effects of genistein were not due to reduction in food intake. From these results, genistein was strongly suggested to suppress the increases in blood glucose level in type 2 diabetic mice through stimulating glucose uptake and/or insulin secretion.
Soy Protein Research, Japan 11, 121-126, 2008.

Key words : soybean genistein, muscle cells, pancreatic β-cell, glucose uptake, insulin secretion, type 2 diabetes

29-23

Skin Permeation of Soy Derived Peptides as Cosmeceuticals

Takashi SEKINE¹, Hiroaki TODO¹, Yoshiko YOKOTE² and Kenji SUGIBAYASHI¹

¹Graduate School of Pharmaceutical Sciences, Josai University, Sakado 350-0295
²Faculty of Sciences, Josai University, Sakado 350-0295

ABSTRACT
Skin permeation of soy derived peptides (SP) and their production promotion effects of type I collagen were evaluated to examine the usefulness of SP as a cosmeteutical. Enzyme-linked immunosorbent assay (ELISA) to examine a type I collagen production promotion effect showed a marked increase in type I collagen by SP application group and a further increase by simultaneous application with ascorbic acid phosphate magnesium (VC-PMg). In addition, the effect was dependent on the application concentration of SP. Amino acids such as glutamine (GLN) and glutamic acid (GLU) entrapped in SP were easily permeated through skin, whereas peptides (GLU-GLU and GLU-GLU-GLU) were seldom permeated through skin. However, adjustment of pH to pI of amino acids and/or addition of l-menthol markedly increased their skin permeation.
Soy Protein Research, Japan 11, 127-131, 2008.

Key words : soy derived peptides, skin permeation, cosmeceutical, collagen production promotion

29-24

Studies on Bioactive Peptides Derived from Soy Protein Preventing Lifestyle-related Disease

Kousaku OHINATA, Masashi IWASAKI, Kentaro KANEKO,
Norimasa KANEGAWA and Masaaki YOSHIKAWA

Graduate School of Agriculture, Kyoto University, Uji 611-0011

ABSTRACT
Mental stress and food intake disorder are known to be involved in onset of obesity, which increase the risk of lifestyle-related disease. We investigated effect of low molecular weight peptides derived from soy protein on the central nervous system, and found bioactive peptide with both anxiolytic and anorexigenic activity. Soymorphin-5 (Tyr-Pro-Phe-Val-Val) is an opioid peptide derived form β-conglycininβ subunit. It has affinity for μopioid receptor and active in opioid assay system using guinea pig ileum. We found that soymorphin-5 increases rate of time spent in open arm in the elevated plus-maze after intraperitoneal (i.p.) or oral administrarion, suggesting that soymorphin-5 has anxiolytic activity. The anxiolytic activity after i.p. administration of soymorphin-5 was completely blocked by opioid antagonist naloxone. These results suggest that soymprhin-5 shows anxiolytic activity viaμ opioid receptor. In general, opioid peptides may stimulate food intake, however; soymorphin-5 suppressed food intake after intracerebroventricular or oral administration.
Soy Protein Research, Japan 11, 132-137, 2008.

Key words :β-conglycinin, soymorphin, casomorphin, anxiolytic activity, food intake regulation

29-25

Study on the Induced Expression of LDL Receptor by Soy Isoflavones through Function of the Estrogen Receptor

Ryuichiro SATO

Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657

ABSTRACT
Although it has been reported that estrogen and isoflavones increase LDL receptor expression and prevent atherosclerosis, little is known about the molecular mechanism of the effects. Based on our previous finding that LDL receptor mRNA is quite unstable, in this study we investigated whether estrogen and isoflavones affect its stability. Our previous study also revealed that activation of the MAPK pathway prolongs the mRNA half-life. Estradiol treatment to MCF7 cells induced the ERK phosphorylation, suggesting that activation of the estrogen receptor (ER) might exert a stimulating effect on the LDL receptor mRNA stability. Indeed, estradiol treatment to mouse primary hepatocytes elevated the endogenous LDL receptor mRNA level, and slightly affected the mRNA stability. Reporter gene assays using the promoter of the human LDL receptor gene revealed that isoflavones as well as estradiol directly augment the promoter activity only when ERα was transiently expressed. These results indicate that isoflavones as well as estradiol stimulate LDL receptor gene expression through ER.
Soy Protein Research, Japan 11, 138-141, 2008.

Key words :estrogen, isoflavone, LDL receptor, estrogen receptor, MAPK

29-26

Preventive Effects of Isoflavone on the Animal Model of Parkinson's Disease

Shinichi KYUHOU

Department of Physiology, Kansai Medical University, Moriguchi 570-8506

ABSTRACT
Parkinson's disease (PD) is a neurodegenerative disease characterized by resting tremor, rigidity and bradykinesia. In patients with PD, dopaminergic neurons in the substantia nigra pars compacta (SNc) degenerate. Women with low estrogen exposure developed PD earlier than did women with high exposure. Although estrogen has many positive effects on the treatment of PD, estrogen has many risks such as cancers. As a natural alternative to estrogen replacement after menopause, isoflavone is a potential candidate for treatment. However, it is not clear whether isoflavone is acting like estrogen in the brain to provide neuroprotection. The present animal study aims to investigate the preventive effects of isoflavone on the motor dysfunctions of PD. In ovariectomized rats, unilateral injection of 6- hydorxydopamine (6-OHDA) into the medial forebrain bundle (MFB) induced motor dysfunctions in the forelimbs on the side contralateral to the lesions assessed by cylinder and step tests. Pretreatment of genistein, an isoflavone, significantly preserved the motor dysfunctions. Morphologically, tyrosine hydroxylase (TH) positive neurons in the SNc were markedly reduced after injection of 6-OHDA into the MFB. Treatment of genistein significantly prevented the reduction of the TH positive neurons. TH positive nerve terminals and dopamine transporter expression in the striatum were also preserved after genistein administration. Taken together, isoflavone is suggested to serve to prevent PD in the post-menopausal women.
Soy Protein Research, Japan 11, 142-146, 2008.

Key words :isoflavone, estrogen, Parkinson's disease, dopamine, female

29-27

Protective Effects of Soybean Phospholipids and N-glycans from Soybean Lectin
on Endoplasmic Reticulum Induced Neuronal Cell Death

Kaoru NAGAI

Department of Epigenetic Medicine, Interdisciplinary graduate school of Medicine and
Engineering, University of Yamanashi, Chuo 409-3898

ABSTRACT
In many neurodegenerative diseases, neuronal cell death is caused by endoplasmic reticulum (ER) stress. ER stress is mainly induced by the accumulation of unfolded or misfolded proteins. In this study, we examined whether soybean phospholipids reduce ER stress induced neuronal cell death, and high-mannose type N-glycan from soybean lectin inhibits the amyloidogenesis of amyloid-β peptide which causes Alzheimer disease. First, we analyzed the effect of total phospholipids extracted from soybean on tunicamycin induced ER stress dependent mouse neuroblastoma Neuro2a cell death. More than 1μg/mL of the phospholipids significantly reduced tunicamycin induced Neuro2a cell death. We then analyzed structure-function relationships of phospholipids on ER stress induced Neuro2a cell death. ER stress was induced by tunicamycin or thapsigargin, and the cell viability was evaluated by MTT assay. Dilinoleoylphosphatidylethanolamine (DLPE) and soybean phosphatidylserine (DLPS) significantly protected the Neuro2a cells from ER stress induced cell death, while dilinoleoylphosphatidylcholine (DLPC) did not. With regard to the acyl chains, DLPE and dioleoylphosphatidylethanolamine (DOPE) showed significant protective effects, while dipalmitoylphosphatidylethanolamine (DPPE) did not. These data suggest that phosphatidylethanolamine and phosphatidylserine with unsaturated acyl chains have protective effects on ER stress induced neuronal cell death. Next, we analyzed anti-amyloidogenesis effect of N-glycan from soybean lectin. The Man₉GlcNAc₂ oligosaccharide from soybean lectin inhibited amyloidogenesis of amyloid-β 1-42 peptide dose dependently. Smaller oligosaccharide mannopentaose inhibited the amyloidogenesis, while mannotriose and mannose did not. These data indicates that sugar chains from soybean glycoproteins reduce the amyloidogenesis. Our data suggest that consumption of phospholipids or sugar chains from soybean may reduce the risk of neurodegenerative diseases.
Soy Protein Research, Japan 11, 147-152, 2008.

Key words :Neurodegenerative disease, ER stress, phospholipid, mannose oligosaccharides, amyloidogenesis

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