Publications by authors named "Takahiro Eitsuka"

55 Publications

Ethanolamine Plasmalogen Suppresses Apoptosis in Human Intestinal Tract Cells by Attenuating Induced Inflammatory Stress.

ACS Omega 2021 Feb 22;6(4):3140-3148. Epub 2021 Jan 22.

Department of Life and Food Sciences, Obihiro University of Agriculture and Veterinary Medicine, Obihiro 080-8555, Japan.

Ethanolamine plasmalogen (PlsEtn) is a subtype of ethanolamine glycerophospholipids (EtnGpl). Recently, PlsEtn has attracted increasing research interest due to its beneficial effects in health and disease; however, its functional role in colonic health has not been well established. This study was conducted to determine the mechanism underlying the antiapoptotic effect of PlsEtn in human intestinal tract cells under induced inflammatory stress. Lipopolysaccharide induced apoptosis of differentiated Caco-2 cells, which was suppressed by EtnGpl in a dose-dependent manner. Cells treated with ascidian muscle EtnGpl containing high levels of PlsEtn demonstrated a lower degree of apoptosis, and downregulated TNF-α and apoptosis-related proteins compared to those treated with porcine liver EtnGpl containing low PlsEtn. This indicates that PlsEtn exerted the observed effects, which provided protection against induced inflammatory stress. Overall, our results suggest that PlsEtn with abundant vinyl ether linkages is potentially beneficial in preventing the initiation of inflammatory bowel disease and colon cancer.
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http://dx.doi.org/10.1021/acsomega.0c05545DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7860056PMC
February 2021

Structural changes of ethanolamine plasmalogen during intestinal absorption.

Food Funct 2020 Sep;11(9):8068-8076

Food and Biodynamic Chemistry Laboratory, Graduate School of Agricultural Science, Tohoku University, Sendai, Miyagi 980-8572, Japan.

Considerable attention has been paid to the absorption mechanisms of plasmalogen (Pls) because its intake has been expected to have preventive effects on brain-related diseases. Possible structural changes of Pls during absorption (i.e., preferential arachidonic acid re-esterification at the sn-2 position and base conversion of ethanolamine Pls (PE-Pls) into choline Pls (PC-Pls)) have previously been proposed. Since the physiological functions of Pls differ according to its structure, further elucidation of such structural changes during absorption is important to understand how Pls exerts its physiological effects in vivo. Hence, the absorption mechanism of Pls was investigated using the lymph-cannulation method and the everted jejunal sac model, with a focus on Pls molecular species. In the lymph-cannulation method, relatively high amounts of PE-Pls 18:0/20:4 and PC-Pls 18:0/20:4 were detected from the lymph even though these species were minor in the administered emulsion. Moreover, a significant increase of PE-Pls 18:0/20:4 and PC-Pls 18:0/20:4 in the intestinal mucosa was also confirmed by the everted jejunal sac model. Therefore, structural changes of PE-Pls in the intestinal mucosa were strongly suggested. The results of this study may provide an understanding of the relationship between intestinal absorption of Pls and exertion of its physiological functions in vivo.
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http://dx.doi.org/10.1039/d0fo01666gDOI Listing
September 2020

Intestinal Absorption and Tissue Distribution of Aza-Sugars from Mulberry Leaves and Evaluation of Their Transport by Sugar Transporters.

J Agric Food Chem 2020 Jun 5;68(24):6656-6663. Epub 2020 Jun 5.

Food and Biodynamic Chemistry Laboratory, Tohoku University Graduate School of Agricultural Science Faculty of Agriculture, Graduate School of Agricultural Science 468-1, Aramaki Aza Aoba, Aoba-ku, Sendai, Miyagi, 980-8572, Japan.

Mulberry leaves are rich in aza-sugars, particularly 1-deoxynojirimycin (DNJ), fagomine, and 2--α-d-galactopyranosyl-1-deoxynojirimycin (GAL-DNJ), which have antidiabetes and antiobesity properties. To help us understand the mechanisms of action of aza-sugars, pharmacokinetic studies are necessary. Therefore, in this study, we evaluated and compared the absorption and organ distribution of these aza-sugars in rats. Following oral intake, DNJ exhibited the highest plasma concentration followed by fagomine and GAL-DNJ. Meanwhile, similar amounts of DNJ and fagomine were present in organs, while GAL-DNJ was hardly detected, suggesting the diversity in absorption and distribution characteristics of these aza-sugars. We then investigated the role of the sodium-glucose cotransporter and the glucose transporter (GLUT) in the transport of aza-sugars and found that both are involved in DNJ transport, while transport of fagomine is solely facilitated by the GLUT. These findings provide insight into the bioavailability and bioactive mechanisms of these aza-sugars.
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http://dx.doi.org/10.1021/acs.jafc.0c03005DOI Listing
June 2020

Supplementation of Bacillus amyloliquefaciens AS385 culture broth powder containing 1-deoxynojirimycin in a high-fat diet altered the gene expressions related to lipid metabolism and insulin signaling in mice epididymal white adipose tissue.

Food Funct 2020 May 6;11(5):3926-3940. Epub 2020 May 6.

Food and Biodynamic Chemistry Laboratory, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan.

1-Deoxynojirimycin (DNJ) has been known for its functional properties, such as its anti-hyperglycemic and anti-obesity activities. Previously, we developed a sustainable procedure to produce culture broth powder (CBP) containing DNJ using Bacillus amyloliquefaciens AS385 and demonstrated its regulatory effect on the blood glucose and lipid parameters in C57BL/6J mice. The present study was aimed to determine the molecular mechanism underlying the physiological effects of CBP intake in different concentrations (low, medium and high) towards the development of high-fat diet (HFD)-induced metabolic disorders. Ten-week consumption of CBP-supplemented diets ameliorated HFD-induced adiposity, glucose intolerance, and reduced insulin sensitivity in C57BL/6J mice. To investigate how these physiological events could take place, we analyzed the expression of genes involved in lipid metabolism and insulin signaling in epididymal white adipose tissue and found that CBP had a regulatory effect on the expression of genes related to lipid metabolism (Pparγ, Srebf1c, Acc, Scd, Hsl, Lpl), adiponectin secretion (Foxo1 and Sirt1), and insulin signaling (Irs1 and Akt2). Next, we confirmed that DNJ acted as the main active component in CBP and detected the dose-dependent DNJ uptake in vital metabolic tissues, which may explain the dose-dependent alteration in the metabolic parameters and related gene expressions following the CBP intake in this study. Collectively, our results suggested that DNJ intake in the form of CBP prevented the progression of HFD-induced metabolic disorders through regulation of adipocyte gene expression involved in lipid metabolism and insulin signaling.
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http://dx.doi.org/10.1039/d0fo00271bDOI Listing
May 2020

Human Derived Immortalized Dermal Papilla Cells With a Constant Expression of Testosterone Receptor.

Front Cell Dev Biol 2020 18;8:157. Epub 2020 Mar 18.

Division of Carcinogenesis and Cancer Prevention and Department of Cell Culture Technology, National Cancer Center Research Institute, Tokyo, Japan.

Androgenetic alopecia (AGA) is the most common type of hair loss, and is mainly caused by the biological effects of testosterone on dermal papilla cells (DPCs). culturing of DPCs might be a useful tool for the screening of target molecule of AGA. However, primary DPCs cannot continuously proliferate owing to cellular senescence and cell culture stress. In this study, we introduced mutant cyclin-dependent kinase 4 (CDK4), Cyclin D1, and telomerase reverse transcriptase (TERT) into DPCs. We confirmed protein expression of CDK4 and Cyclin D1, and enzymatic activity of TERT. Furthermore, we found the established cell line was free from cellular senescence. We also introduced the androgen receptor gene using a recombinant retrovirus, to compensate the transcriptional suppressed endogenous androgen receptor in the process of cell proliferation. Furthermore, we detected the efficient nuclear translocation of androgen receptor into the nucleus after the treatment of dihydrotestosterone, indicating the functionality of our introduced receptor. Our established cell line is a useful tool to identify the downstream signaling pathway, which activated by the testosterone.
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http://dx.doi.org/10.3389/fcell.2020.00157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7109449PMC
March 2020

Primary and immortalized cell lines derived from the Amami rabbit (Pentalagus furnessi) and evolutionally conserved cell cycle control with CDK4 and Cyclin D1.

Biochem Biophys Res Commun 2020 05 13;525(4):1046-1053. Epub 2020 Mar 13.

Graduate School of Science and Engineering, Iwate University, Morioka, Japan; Soft-Path Engineering Research Center (SPERC), Iwate University, Morioka, Japan; Wildlife Genome Collaborative Research Group, National Institute for Environmental Studies, Japan. Electronic address:

The Amami rabbit (Pentagulus furnessi) is a dark brown-furred rabbit classified as an endangered species and only found in the Amami Islands of Japan. They are often called living fossils because they retain primitive characteristics of ancient rabbits that lived approximately 1 million years ago, such as short feet and hind legs and small ears. Although the ancient rabbit has disappeared due to the competition with European rabbit (Oryctolagus cuniculus) in the most of the Asian area, Amami rabbit survived since Amami Islands has isolated from Japan and Taiwan. Although Amari rabbit is one of the protected animals, their population decreases each year due to human activities, such as deforestation and roadkill. In this study, we collected roadkill samples of Amami rabbits and established primary and immortalized fibroblast cell lines. Combined expression of human-derived mutant Cyclin-dependent kinase 4, Cyclin D1, and hTERT allowed us to immortalize fibroblasts successfully in three individuals of Amami rabbits. The immortalized fibroblasts dramatically extended the cell culture period, when it was compared with the cell culture period of wild type cells. Furthermore, the immortalized cells maintained their normal chromosomal pattern (2n = 46). Our results suggest that cellular senescence which mainly regulated by p16-RB signaling pathway is conserved in animal evolution at least from 1 million years ago.
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http://dx.doi.org/10.1016/j.bbrc.2020.03.036DOI Listing
May 2020

Efficient immortalization of human dental pulp stem cells with expression of cell cycle regulators with the intact chromosomal condition.

PLoS One 2020 2;15(3):e0229996. Epub 2020 Mar 2.

Graduate School of Science and Engineering, Iwate University, Morioka, Iwate, Japan.

Clinical studies have recently demonstrated that autologous transplantation of mobilized dental pulp stem cells is a safe and efficacious potential therapy for pulp regeneration. However, some limitations need to be addressed, such as the high cost of the safety and quality control tests for isolated individual dental pulp cell products before transplantation. Therefore, more efficient in vitro culturing of human dental pulp stem cells might be useful for providing low cost and high reliability testing for pulp regeneration therapy. In this study, we established a novel immortalized dental pulp stem cell line by co-expressing a mutant cyclin-dependent kinase 4 (CDK4R24C), Cyclin D1, and telomerase reverse transcriptase (TERT). The established cell line maintained its original diploid chromosomes and stemness characteristics and exhibited an enhanced proliferation rate. In addition, we showed the immortalized human dental pulp stem cells still keeps their osteogenic and adipogenic differentiation abilities under appropriate culture conditions even though the cell proliferation was accelerated. Taken together, our established cell lines could serve as a useful in vitro tool for pulp regeneration therapy, and can contribute to reproducibility and ease of cell handling, thereby saving time and costs associated with safety and quality control tests.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0229996PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7051082PMC
June 2020

Author Correction: Definitive evidence of the presence of 24-methylenecycloartanyl ferulate and 24-methylenecycloartanyl caffeate in barley.

Sci Rep 2020 Jan 17;10(1):974. Epub 2020 Jan 17.

Food and Biodynamic Chemistry Laboratory, Graduate School of Agricultural Science, Tohoku University, Sendai, Miyagi, 980-8572, Japan.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41598-020-57690-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6965130PMC
January 2020

Analysis of oxidation products of α-tocopherol in extra virgin olive oil using liquid chromatography-tandem mass spectrometry.

Food Chem 2020 Feb 4;306:125582. Epub 2019 Oct 4.

Food and Biodynamic Chemistry Laboratory, Graduate School of Agricultural Science, Tohoku University, Sendai, Miyagi 980-8572, Japan; J-Oil Mills Innovation Laboratory, Graduate School of Agricultural Science, Tohoku University, Sendai, Miyagi 980-8572, Japan. Electronic address:

When α-tocopherol (α-Toc) exerts its antioxidative effect, a portion of α-Toc is converted to certain oxidation products. Although accumulation of such oxidation products is considered to cause a deterioration in the quality of foods, their distribution and generation in food samples have been still unknown. In this study, we tried to analyze α-Toc hydroperoxide (Toc-OOH) stereoisomers and tocopherylquinone (TQ) in extra virgin olive oil (EVOO) using liquid chromatography-tandem mass spectrometry. Photo-irradiation (5000 lx) to EVOO increased Toc-OOH stereoisomers but not TQ. In contrast, thermal oxidation (150 °C) of EVOO increased TQ but not Toc-OOH. We considered that the generation of Toc-OOH and TQ were due to the [4+2]-cycloaddition reaction and proton donation from the phenolic hydrogen, respectively. Our data and method would be helpful for understanding of α-Toc oxidation mechanisms in edible oil samples or the estimation of food quality.
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http://dx.doi.org/10.1016/j.foodchem.2019.125582DOI Listing
February 2020

Significance of Squalene in Rice Bran Oil and Perspectives on Squalene Oxidation.

J Nutr Sci Vitaminol (Tokyo) 2019 ;65(Supplement):S62-S66

Food and Biodynamic Chemistry Laboratory, Graduate School of Agricultural Science, Tohoku University.

As an intermediate metabolite during the biosynthesis of sterols, squalene is found ubiquitously in plants and animals. In rice, squalene is contained in rice bran, and consequently, squalene in rice bran oil has gained attention. Studies have shown that the intake of squalene from food sources demonstrate various physiological benefits such as the prevention of cancer and cardiovascular disease. Squalene is also known as an effective antioxidant in edible oils. However, due to its chemical structure, squalene is susceptible to oxidation, which may cause a decline in the nutraceutical and antioxidative effects of squalene in edible oils. Oxidative degradation of squalene also results in the formation of scission products (i.e., aldehydes and ketones) which may lead to off-flavor. Since the rate of squalene oxidation depends on the factors that induce its oxidation (i.e., light or heat), emphasis on oxidation mechanisms is necessary. It has been demonstrated in previous studies that the oxidation products formed by the singlet oxygen oxidation and free radical oxidation of squalene are different, and more recently, we demonstrated that different squalene monohydroperoxide isomers are formed by each oxidation mechanism. We herein discuss the significance of squalene in rice bran oil as well as the oxidative degradation of squalene in edible oils with focus on oxidation mechanisms.
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http://dx.doi.org/10.3177/jnsv.65.S62DOI Listing
March 2020

Definitive evidence of the presence of 24-methylenecycloartanyl ferulate and 24-methylenecycloartanyl caffeate in barley.

Sci Rep 2019 08 29;9(1):12572. Epub 2019 Aug 29.

Food and Biodynamic Chemistry Laboratory, Graduate School of Agricultural Science, Tohoku University, Sendai, Miyagi, 980-8572, Japan.

γ-Oryzanol (OZ), which has a lot of beneficial effects, is a mixture of ferulic acid esters of triterpene alcohols (i.e., triterpene alcohol type of OZ (TTA-OZ)) and ferulic acid esters of plant sterols (i.e., plant sterol type of OZ (PS-OZ)). Although it has been reported that OZ is found in several kinds of cereal typified by rice, TTA-OZ (e.g., 24-methylenecycloartanyl ferulate (24MCA-FA)) has been believed to be characteristic to rice and has not been found in other cereals. In this study, we isolated a compound considered as a TTA-OZ (i.e., 24MCA-FA) from barley and determined the chemical structure using by HPLC-UV-MS, high-resolution MS, and NMR. Based on these results, we proved for the first time that barley certainly contains 24MCA-FA (i.e., TTA-OZ). During the isolation and purification of 24MCA-FA from barley, we found the prospect that a compound with similar properties to OZ (compound-X) might exist. To confirm this finding, the compound-X was also isolated, determined the chemical structure, and identified as a caffeic acid ester of 24-methylenecycloartanol (24MCA-CA), which has rarely been reported before. We also quantified these compounds in various species of barley cultivars and found for the first time the existence of 24MCA-FA and 24MCA-CA in various barley. Through these findings, it opens the possibility to use barley as a new source of 24MCA-FA and 24MCA-CA.
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http://dx.doi.org/10.1038/s41598-019-48985-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6715696PMC
August 2019

Evaluation of squalene oxidation mechanisms in human skin surface lipids and shark liver oil supplements.

Ann N Y Acad Sci 2019 12 26;1457(1):158-165. Epub 2019 Aug 26.

Food and Biodynamic Chemistry Laboratory, Graduate School of Agricultural Science, Tohoku University, Sendai, Miyagi, Japan.

Squalene is a terpenoid found in human skin surface lipids (SSLs) and foods that possesses beneficial properties. However, since oxidation of squalene causes various complications, it is necessary to identify the mechanisms by which squalene is oxidized. In this study, we aimed to determine the oxidation mechanisms of squalene in SSLs and shark liver oil (SLO) supplements by the analysis of squalene monohydroperoxide (SQOOH) isomers, on the basis of our previous finding that different oxidation mechanisms yield different SQOOH isomers. Liquid chromatography-tandem mass spectrometry analysis of SQOOH isomers revealed that squalene in human SSLs was oxidized by singlet oxygen oxidation, whereas squalene in SLO was oxidized mainly by free radicals. As a result, we have presented the first evidence suggesting that the analysis of SQOOH isomers enables estimation of oxidation mechanisms. Estimating oxidation mechanisms by analyzing SQOOH isomers may provide a foundation for the prevention of skin diseases and food deterioration via regulation of squalene oxidation.
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http://dx.doi.org/10.1111/nyas.14219DOI Listing
December 2019

Establishment of immortalized primary cell from the critically endangered Bonin flying fox (Pteropus pselaphon).

PLoS One 2019 26;14(8):e0221364. Epub 2019 Aug 26.

Wild life Genome Collaborative Research Group, National Institute for Environmental Studies, Tsukuba, Ibaraki, Japan.

The Bonin flying fox (Pteropus pselaphon) is one of the most critically endangered species of animals. The number of this species is estimated to be around 150; being classified at the top rank in the list by International Union of Animal Conservation. Our group previously showed that expression of CDK4, CYCLIN D1, and telomerase reverse transcriptase (TERT) efficiently induce immortalization of human, bovine, swine, monkey, and buffalo-derived cells. In this manuscript, we successfully established the primary cells from Bonin flying fox. We introduced CDK4, CYCLIN D1, and TERT into the primary cells. The established cells showed efficient expression of introduced genes at the protein level. Furthermore, the established cells were free from senescence, indicating it reached to immortalization. Moreover, we showed that interspecies somatic cell nuclear transfer of Bonin flying fox derived cell into bovine embryo allowed the development of the embryo to 8 cell stages. Our established cell has the potential to contribute to species conservation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0221364PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6709887PMC
March 2020

Human-Derived Corneal Epithelial Cells Expressing Cell Cycle Regulators as a New Resource for Ocular Toxicity Testing.

Front Genet 2019 16;10:587. Epub 2019 Jul 16.

Division of Carcinogenesis and Cancer Prevention, National Cancer Center Research Institute, Tokyo, Japan.

The Draize test has been used on rabbits since the 1960s to evaluate the irritation caused by commercial chemicals in products such as cosmetics or hairdressing products. However, since 2003, such tests, including the Draize test for cosmetics, have been prohibited in European countries because they are considered problematic to animal welfare. For this reason, replacement of methods with the alternative methods has become an important goal. In this study, we established a corneal epithelial cell line co-expressing a mutant cyclin-dependent kinase 4 (), Cyclin D1, and telomerase reverse transcriptase (). The established cell line maintained its original morphology and had an enhanced proliferation rate. Furthermore, the cells showed a significant, dose-dependent decrease in viability in an irritation test using glycolic acid and Benzalkonium chloride. These cells can now be shared with toxicology scientists and should contribute to increasing the reproducibility of chemical testing .
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http://dx.doi.org/10.3389/fgene.2019.00587DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6646426PMC
July 2019

The Adjuvant Effect of Squalene, an Active Ingredient of Functional Foods, on Doxorubicin-Treated Allograft Mice.

Nutr Cancer 2019 10;71(7):1153-1164. Epub 2019 Jun 10.

Faculty of Applied Life Sciences, Niigata University of Pharmacy and Applied Life Sciences , Niigata , Japan.

Many functional foods or physiologically active ingredients derived from plants and animals are actively being investigated for their role in chronic disease prevention. Squalene (SQ) is found as active ingredient in the functional foods predominantly present in olive oil and shark liver oil. It is known that during chemotherapy anticancer drugs induce inflammation. SQ has been thought to prevent and suppress inflammation; however, there is little direct evidence available. We examined the adjuvant effect of SQ on tumor-transplanted mice along with anticancer drug doxorubicin (DOX). SQ significantly suppressed the DOX-induced increase in prostaglandin E (PGE) concentration ( < 0.05) in plasma of tumor-bearing mice. SQ inhibited the numbers of writhing response ( < 0.05), formalin-induced pain and decreased COX-2 and substance P expression in the tumor tissue compared to control mice and also enhanced the antitumor efficacy of DOX in allograft mice. Thus, SQ reduces inflammation through modulation of PGE production indicating its potential as an adjuvant during chemotherapy in tumor-bearing mice.
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http://dx.doi.org/10.1080/01635581.2019.1597900DOI Listing
June 2020

Comparison of Blood Profiles of γ-Oryzanol and Ferulic Acid in Rats after Oral Intake of γ-Oryzanol.

Nutrients 2019 May 25;11(5). Epub 2019 May 25.

Food and Biodynamic Chemistry Laboratory, Graduate School of Agricultural Science, Tohoku University, Sendai, Miyagi 980-8572, Japan.

γ-Oryzanol (OZ), a bioactive phytochemical abundant in cereals such as rice, has been reported to be mainly hydrolyzed to ferulic acid (FA) in the body. Meanwhile, in our previous study, we revealed that a part of OZ is absorbed into the body and exists in its intact form. However, the comprehensive absorption profile of OZ and its metabolites (e.g., FA) after OZ intake has not been fully elucidated yet. Therefore, in this study, we measured the concentrations of OZ, FA, and FA conjugates (i.e., FA sulfate and glucuronide) in the blood of rats with the use of HPLC-MS/MS after a single oral administration of 300 µmol/kg body weight of rice bran OZ (RBOZ). As a result, intact OZ along with FA and FA conjugates existed in the blood, which implied that these constituents may all contribute to the physiological effects under OZ intake. Additionally, when an equimolar amount of FA (300 µmol/kg body weight) was administered, it was found that the absorption profile of FA was significantly different from that when RBOZ was administered.
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http://dx.doi.org/10.3390/nu11051174DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6567117PMC
May 2019

Physiological Effects and Organ Distribution of Bacillus amyloliquefaciens AS385 Culture Broth Powder Containing 1-Deoxynojirimycin in C57BL/6J Mice.

J Nutr Sci Vitaminol (Tokyo) 2019 ;65(2):157-163

Food and Biodynamic Chemistry Laboratory, Graduate School of Agricultural Science, Tohoku University.

1-Deoxynojirimycin (DNJ) has been known as a potent α-glucosidase inhibitor from mulberry leaves and considered beneficial in prevention of type 2 diabetes. Due to limited amount of DNJ in mulberry leaves, recent studies have focused in finding alternative source that can produce higher amount of DNJ. Previously, we produced a high DNJ-containing culture medium from Bacillus amyloliquefaciens AS385 and constructed a concentration method of bacterial culture medium using cation exchange column. However, less complicated concentration procedure is necessary to save time and cost during the large-scale production. Therefore, we developed a simpler concentration method using anion exchange resin to yield B. amyloliquefaciens AS385 culture broth powder (CBP; 1% DNJ) and evaluated the physiological effects of 5-wk dietary CBP intake in C57BL/6J mice. CBP intake tended to suppress the elevation of blood glucose level during oral glucose tolerance test. Moreover, CBP intake significantly lowered the fasting plasma glucose level and white adipose tissue mass. Next, we evaluated the absorption and distribution of DNJ in mice organs after daily CBP intake. We found detectable amount of DNJ in organs with intestine and kidney as the major targeted organs. We concluded that the DNJ content in CBP is absorbed from digestive tract, distributed and accumulated in organs, which most likely to contribute to the alteration of blood glucose regulation and adiposity in C57BL/6J mice. Our study was the first to report the physiological effects of CBP produced from B. amyloliquefaciens AS385 and the organ distribution of DNJ from CBP.
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http://dx.doi.org/10.3177/jnsv.65.157DOI Listing
December 2019

The differential cellular uptake of curcuminoids in vitro depends dominantly on albumin interaction.

Phytomedicine 2019 Jun 23;59:152902. Epub 2019 Mar 23.

Food and Biodynamic Chemistry Laboratory, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan. Electronic address:

Background: Curcuminoids, mainly present in the plant rhizomes of turmeric (Curcuma longa), consist of mainly three forms (curcumin (CUR), bisdemethoxycurcumin (BDMC) and demethoxycurcumin (DMC)). It has been reported that different forms of curcuminoids possess different biological activities. However, the mechanisms associated with these differences are not well-understood. Recently, our laboratory found differences in the cellular uptake of these curcuminoids. Therefore, it has been inferred that these differences contribute to the different biological activities.

Purpose: In this study, we investigated the mechanisms of differential cellular uptake of these curcuminoids.

Method: Based on our previous study, we hypothesized the differential cellular uptake is caused by (I) polarity, (II) transporters, (III) metabolism rate of curcuminoids and (IV) medium components. These four hypotheses were each investigated by (I) neutralizing the polarities of curcuminoids by encapsulation into poly(lactic-co-glycolic) acid nanoparticles (PLGA-NPs), (II) inhibition of polyphenol-related absorption transporters, (III) analysis of the cellular curcuminoids and their metabolites by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and (IV) use of different mediums in cell study.

Results: The differential cellular uptake was not affected by (I-III). However, when investigating (IV), not only CUR but also BDMC and DMC were incorporated into cells when serum free media was used. Furthermore, when we used the serum free medium containing bovine serum albumin (BSA), only CUR was taken up but BDMC and DMC were not. Therefore, we identified that the differential cellular uptake of curcuminoids is caused by the medium components, especially BSA. Also, the fluorescence quenching study suggested that differential cellular uptake is due to the different interaction between BSA and each curcuminoid.

Conclusion: The differential cellular uptake of curcuminoids was caused by the different interaction between curcuminoids and BSA. The results from this study might give clues on the mechanisms by which curcuminoids exhibit different physiological activities.
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http://dx.doi.org/10.1016/j.phymed.2019.152902DOI Listing
June 2019

Evaluation of γ-oryzanol Accumulation and Lipid Metabolism in the Body of Mice Following Long-Term Administration of γ-oryzanol.

Nutrients 2019 Jan 6;11(1). Epub 2019 Jan 6.

Food and Biodynamic Chemistry Laboratory, Graduate School of Agricultural Science, Tohoku University, Sendai, Miyagi 980-8572, Japan.

γ-Oryzanol (OZ), abundant in rice bran oil, has gained attention due to its physiological activities (e.g., lipid-lowering effects). However, the absorption and metabolism of orally ingested OZ have not yet been fully elucidated. In this study, diets containing normal or high contents of OZ were fed to obesity model mice for 8 weeks, and OZ concentrations in plasma and organs were analyzed by HPLC-MS/MS. To evaluate the relationship between OZ accumulation and lipid metabolism in vivo, lipid concentrations in the mice plasma and liver were also measured. As a result, the accumulation of intact OZ in plasma and organs was seen in mice fed diets containing OZ, where mice fed diets containing higher OZ contents demonstrated higher levels of OZ accumulation and lower amounts of plasma lipids. These results, in combination with our additional data from a single oral administration test, suggest the possibility that intact OZ, along with its metabolites (e.g., ferulic acid), is biologically-active.
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http://dx.doi.org/10.3390/nu11010104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6356301PMC
January 2019

Extended proliferation of chicken- and Okinawa rail-derived fibroblasts by expression of cell cycle regulators.

J Cell Physiol 2019 05 11;234(5):6709-6720. Epub 2018 Nov 11.

Wildlife Genome Collaborative Research Group, National Institute for Environmental Studies, Tsukuba, Japan.

Although immortalized cultured cells are useful for various functional assays or transcriptome analysis, highly efficient and reproducible immortalization methods have not been developed in avian-derived cells. We introduced the simian virus 40 T antigen (SV40T) and human papillomavirus (HPV)-E6E7 to chick and Okinawa rail (endangered species)derived fibroblast. As a result, neither the SV40T nor E6E7 genes could induce avian cell immortality. Accordingly, we attempted to use a recently developed immortalization method, which involved the coexpression of mutant cyclin-dependent kinase 4 (CDK4), Cyclin D, and TERT (K4DT method) in these avian cells. Although the K4DT method could not efficiently induce the efficient immortalization in mass cell population, cellular division until the senescence was significantly extended by K4DT, we succeeded to obtain the immortalized avian cells (chick K4DT: one clone, Okinawa rail K4DT: three clones, Okinawa rail K4DT + telomerase RNA component: one clone) with K4DT expression. We conclude that K4DT expression is used to extend the cell division and immortalization of avian-derived cells.
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http://dx.doi.org/10.1002/jcp.27417DOI Listing
May 2019

Absorption and Metabolism of Luteolin in Rats and Humans in Relation to in Vitro Anti-inflammatory Effects.

J Agric Food Chem 2018 Oct 19;66(43):11320-11329. Epub 2018 Oct 19.

Food and Biodynamic Chemistry Laboratory, Graduate School of Agricultural Science , Tohoku University , Sendai , Miyagi 980-0845 , Japan.

Luteolin is a flavonoid present in plants in the form of aglycone or glucosides. In this study, luteolin glucosides (i.e., luteolin-7- O-β-d-glucoside, luteolin-7- O-[2-(β-d-apiosyl)-β-d-glucoside], and luteolin-7- O-[2-(β-d-apiosyl)-6-malonyl-β-d-glucoside]) prepared from green pepper leaves as well as luteolin aglycone were orally administered to rats. Regardless of the administered luteolin form, luteolin glucuronides were mainly detected from plasma and organs. Subsequently, luteolin aglycone, the most absorbed form of luteolin in rats, was orally administered to humans. As a result, luteolin-3'- O-sulfate was mainly identified from plasma, suggesting that not only luteolin form but also animal species affect the absorption and metabolism of luteolin. When LPS-treated RAW264.7 cells were treated with luteolin glucuronides and luteolin sulfate (the characteristic metabolites identified from rats and humans, respectively), the different luteolin conjugates were metabolized in different ways, suggesting that such difference in metabolism results in their difference in anti-inflammatory effects.
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http://dx.doi.org/10.1021/acs.jafc.8b03273DOI Listing
October 2018

Efficient immortalization of cells derived from critically endangered Tsushima leopard cat (Prionailurus bengalensis euptilurus) with expression of mutant CDK4, Cyclin D1, and telomerase reverse transcriptase.

Cytotechnology 2018 Dec 17;70(6):1619-1630. Epub 2018 Sep 17.

Graduate School of Science and Engineering, Iwate University, Morioka, Japan.

Tsushima leopard cat is the subspecies of Amur cats, and it is classified as the most highest class of critically endangered animals. Although the protection activity is highly recognized, the number of animals is decreasing due to the human activity and invasion of domestic cats and infectious disease. In this study, we succeeded primary culture of normal fibroblasts derived from the Tsushima leopard cat (Prionailurus bengalensis euptilurus). Furthermore, we introduced the human derived mutant Cyclin Dependent Kinase 4, Cyclin D1, and telomere reverse transcriptase. We showed that the expression of these three genes efficiently immortalized cells derived from Tsushima leopard cat. Furthermore, we showed that the chromosome pattern of the established cells is identical with the original one. These data indicate that our method of immortalization is useful to establish cell lines from critically endangered cats, which potentially contributes to the re-generation of critically endangered animals from cultured cell with reproductive technique, such as somatic cloning.
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http://dx.doi.org/10.1007/s10616-018-0254-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6269356PMC
December 2018

Expression of human mutant cyclin dependent kinase 4, Cyclin D and telomerase extends the life span but does not immortalize fibroblasts derived from loggerhead sea turtle (Caretta caretta).

Sci Rep 2018 06 20;8(1):9229. Epub 2018 Jun 20.

Division of Carcinogenesis and Cancer Prevention, National Cancer Center Research Institute, Tsukiji, Chuo-ku, Tokyo, 104-0045, Japan.

Conservation of the genetic resources of endangered animals is crucial for future generations. The loggerhead sea turtle (Caretta caretta) is a critically endangered species, because of human hunting, hybridisation with other sea turtle species, and infectious diseases. In the present study, we established primary fibroblast cell lines from the loggerhead sea turtle, and showed its species specific chromosome number is 2n = 56, which is identical to that of the hawksbill and olive ridley sea turtles. We first showed that intensive hybridization among multiple sea turtle species caused due to the identical chromosome number, which allows existence of stable hybridization among the multiple sea turtle species. Expressions of human-derived mutant Cyclin-dependent kinase 4 (CDK4) and Cyclin D dramatically extended the cell culture period, when it was compared with the cell culture period of wild type cells. The recombinant fibroblast cell lines maintained the normal chromosome condition and morphology, indicating that, at the G1/S phase, the machinery to control the cellular proliferation is evolutionally conserved among various vertebrates. To our knowledge, this study is the first to demonstrate the functional conservation to overcome the negative feedback system to limit the turn over of the cell cycle between mammalian and reptiles. Our cell culture method will enable the sharing of cells from critically endangered animals as research materials.
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http://dx.doi.org/10.1038/s41598-018-27271-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6010431PMC
June 2018

Oxidation of squalene by singlet oxygen and free radicals results in different compositions of squalene monohydroperoxide isomers.

Sci Rep 2018 06 14;8(1):9116. Epub 2018 Jun 14.

Food and Biodynamic Chemistry Laboratory, Graduate School of Agricultural Science, Tohoku University, Sendai, Miyagi, 980-0845, Japan.

Oxidation of squalene (SQ) causes a decline in the nutritional value of SQ in foods, as well as an accumulation of SQ oxidation products in skin lipids which lead to adverse skin conditions. However, mechanistic insights as to how SQ is oxidized by different oxidation mechanisms have been limited, and thus effective measures towards the prevention of SQ oxidation have not been identified. In this study, we oxidized SQ by either singlet oxygen oxidation or free radical oxidation, and monitored the formation of the six SQ monohydroperoxide (SQOOH) isomers, the primary oxidation products of SQ, at the isomeric level. While singlet oxygen oxidation of SQ resulted in the formation of similar amounts of the six SQOOH isomers, free radical oxidation of SQ mainly formed two types of isomers, 2-OOH-SQ and 3-OOH-SQ. The addition of β-carotene during singlet oxygen oxidation, and the addition of α-tocopherol during free radical oxidation lead to a dose-dependent decrease in the formation of SQOOH isomers. Such results suggest that the analysis of SQOOH at the isomeric level allows for the determination of the cause of SQ oxidation in various samples, and provides a foothold for future studies concerning the prevention of SQ oxidation.
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http://dx.doi.org/10.1038/s41598-018-27455-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6002538PMC
June 2018

Modulation of Telomerase Activity in Cancer Cells by Dietary Compounds: A Review.

Int J Mol Sci 2018 Feb 6;19(2). Epub 2018 Feb 6.

Food and Biotechnology Innovation Project, New Industry Creation Hatchery Center (NICHe), Tohoku University, Sendai 980-8579, Japan.

Telomerase is expressed in ~90% of human cancer cell lines and tumor specimens, whereas its enzymatic activity is not detectable in most human somatic cells, suggesting that telomerase represents a highly attractive target for selective cancer treatment. Accordingly, various classes of telomerase inhibitors have been screened and developed in recent years. We and other researchers have successfully found that some dietary compounds can modulate telomerase activity in cancer cells. Telomerase inhibitors derived from food are subdivided into two groups: one group directly blocks the enzymatic activity of telomerase (e.g., catechin and sulfoquinovosyldiacylglycerol), and the other downregulates the expression of human telomerase reverse transcriptase (hTERT), the catalytic subunit of human telomerase, via signal transduction pathways (e.g., retinoic acid and tocotrienol). In contrast, a few dietary components, including genistein and glycated lipid, induce cellular telomerase activity in several types of cancer cells, suggesting that they may be involved in tumor progression. This review summarizes the current knowledge about the effects of dietary factors on telomerase regulation in cancer cells and discusses their molecular mechanisms of action.
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http://dx.doi.org/10.3390/ijms19020478DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5855700PMC
February 2018

Determination of triacylglycerol oxidation mechanisms in canola oil using liquid chromatography-tandem mass spectrometry.

NPJ Sci Food 2018 12;2. Epub 2018 Jan 12.

1Food and Biodynamic Chemistry Laboratory, Graduate School of Agricultural Science, Tohoku University, Sendai, Miyagi 980-0845 Japan.

Triacylglycerol (TG), the main component of edible oil, is oxidized by thermal- or photo- oxidation to form TG hydroperoxide (TGOOH) as the primary oxidation product. Since TGOOH and its subsequent oxidation products cause not only the deterioration of oil quality but also various toxicities, preventing the oxidation of edible oils is essential. Therefore understanding oxidation mechanisms that cause the formation of TGOOH is necessary. Since isomeric information of lipid hydroperoxide provides insights about oil oxidation mechanisms, we focused on dioleoyl-(hydroperoxy octadecadienoyl)-TG (OO-HpODE-TG) isomers, which are the primary oxidation products of the most abundant TG molecular species (dioleoyl-linoleoyl-TG) in canola oil. To secure highly selective and sensitive analysis, authentic OO-HpODE-TG isomer references (i.e., hydroperoxide positional/geometrical isomers) were synthesized and analyzed with HPLC-MS/MS. With the use of the method, photo- or thermal- oxidized edible oils were analyzed. While dioleoyl-(10-hydroperoxy-8,12-octadecadienoyl)-TG (OO-(10-HpODE)-TG) and dioleoyl-(12-hydroperoxy-9,13-octadecadienoyl)-TG (OO-(12-HpODE)-TG) were characteristically detected in photo-oxidized oils, dioleoyl-(9-hydroperoxy-10,12-octadecadienoyl)-TG and dioleoyl-(13-hydroperoxy-9,11-octadecadienoyl)-TG were found to increase depending on temperature in thermal-oxidized oils. These results prove that our methods not only evaluate oil oxidation in levels that are unquantifiable with peroxide value, but also allows for the determination of oil oxidation mechanisms. From the analysis of marketed canola oils, photo-oxidized products (i.e., OO-(10-HpODE)-TG and OO-(12-HpODE)-TG) were characteristically accumulated compared to the oil analyzed immediately after production. The method described in this paper is valuable in the understanding of oil and food oxidation mechanisms, and may be applied to the development of preventive methods against food deterioration.
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http://dx.doi.org/10.1038/s41538-017-0009-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6550225PMC
January 2018

Analysis of Lutein in Mugwort (Artemisia princeps Pamp.) Paste and Evaluation of Manufacturing Processes.

J Oleo Sci 2017 Nov 11;66(11):1257-1262. Epub 2017 Oct 11.

Food and Biodynamic Chemistry Laboratory, Graduate School of Agricultural Science, Tohoku University.

Lutein, a type of xanthophyll, possesses antioxidative properties that contribute to the prevention of various diseases. Preliminary screening has shown that Japanese mugwort (Artemisia princeps Pamp.) contains high amounts of lutein. In this study, we evaluated the lutein concentration in a processed mugwort product (mugwort paste). By using high-performance liquid chromatography coupled with visible light detection or mass spectrometry, the lutein concentration in mugwort paste was determined as 38 mg/100 g dry weight, which indicates that mugwort is a potentially valuable natural food source of lutein. We also investigated the effects of the manufacturing process and found that the lutein content was significantly increased by the boiling and dehydrating processes during the production of mugwort paste. Mugwort paste that is rich in lutein may therefore serve as an effective nutraceutical.
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http://dx.doi.org/10.5650/jos.ess17117DOI Listing
November 2017

Pork Loin Treated with High Hydrostatic Pressure as a Food Processing Technology: Subacute Toxicity of the Freeze-Dried Powder and Cytotoxicity of the Methanol Extracts.

Food Saf (Tokyo) 2017 Sep 29;5(3):98-109. Epub 2017 Sep 29.

Faculty of Applied Life Sciences, Niigata University of Pharmacy and Applied Life Sciences, Niigata, 956-8603, Japan.

High hydrostatic pressure (HP) treatment is used in food processing owing to its sterilization effect. Meat or meat products are sterilized and become tender by HP processing. Therefore, the variety of HP-processed meat products has increased worldwide. However, little is known about the safety of HP-processed meat products. The aim of this study was to determine the effects of HP processing and HP combined with 0.4 M sodium carbonate treatment (HP-Na) on pork loins and to evaluate the subacute toxicity and cytotoxicity of these processing methods. In an study, we performed 90- and 180-day feeding tests in mice and did not detect any adverse effects in HP-processed and HP-Na-processed pork loins. In addition, we evaluated the cytotoxicity of HP-processed meats, and did not observe any obvious toxicity associated with pork loin extracts . These results suggest that HP is not associated with risk factors during processing.
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http://dx.doi.org/10.14252/foodsafetyfscj.2016030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6989177PMC
September 2017

High purity tocotrienols attenuate atherosclerotic lesion formation in apoE-KO mice.

J Nutr Biochem 2017 10 24;48:44-50. Epub 2017 Jun 24.

Food and Biodynamic Chemistry Laboratory, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan. Electronic address:

Previous studies have demonstrated that tocotrienol (T3) has antiatherogenic effects. However, the T3 preparations used in those studies contained considerable amounts of tocopherol (Toc), which might affect the biological activity of T3. There is little information on the effect of highly purified T3 on atherosclerosis formation. This study investigated the effect of high-purity T3 on atherosclerotic lesion formation and the underlying mechanisms. Male apolipoprotein E knockout (apoE-KO) mice were fed a cholesterol-containing diet either alone or supplemented with T3 concentrate (Toc-free T3) or with α-Toc for 12 weeks. ApoE-KO mice fed the 0.2% T3-supplemented diet showed reduced atherosclerotic lesion formation in the aortic root. The 0.2% T3 diet induced Slc27a1 and Ldlr gene expression levels in the liver, whereas the α-Toc-supplemented diet did not affect those expression levels. T3 was predominantly deposited in fat tissue in the T3 diet-fed mice, whereas α-Toc was preferentially accumulated in liver in the α-Toc diet-fed mice. Considered together, these data demonstrate that dietary T3 exerts anti-atherosclerotic effect in apoE-KO mice. The characteristic tissue distribution and biological effects of T3, that are substantially different from those of Toc, may contribute to the antiatherogenic properties of T3.
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http://dx.doi.org/10.1016/j.jnutbio.2017.06.009DOI Listing
October 2017