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1883−1516

東海大学紀要 農学部
第 36 巻
2 0 1 7耐熱性カルボニル還元酵素の結晶構造解析と基質認識機構の解明…………………………………………………………………米田一成・福田雄大・荒木朋洋………１
Lactobacillus plantarum LA 318はヒト大腸ムチンへの Candida albicans ATCC 26555の付着を阻害する………………………………………木下英樹・渡辺真通・井越敬司・石田光晴・川井 泰
北澤春樹・三浦 康・堀井 明・齋藤忠夫………７
Exploring Academic English for the New Curriculum at Tokai University………………………………………………………………Miki Rodriguez-Yagi・Daniel Minor………15
5HFRPPHQGHG0RGL¿FDWLRQVWRWKH.DPEDUD/RFXVRI&RQWURO6FDOH…………………………………………………………………………………… Michael J. Rupp………23
ヤーコン茶葉熱水抽出物のリポキシゲナーゼに対する阻害作用およびRAW264.7マウス由来マクロファージ様細胞を用いた NO 産生抑制作用………………………上田裕人・菅原進太郎・松田 靖・村田達郎・黒田泰弘・星 良和
椛田聖孝・小野政輝・井越敬司・安田 伸………37

東海大農紀要 36：１−６（2017）

耐熱性カルボニル還元酵素の結晶構造解析

耐熱性カルボニル還元酵素の結晶構造解析と基質認識機構の解明
米田一成 *・福田雄大 **・荒木朋洋 *Crystal structure analysis of extremely thermostable carbonyl reductase andstructural basis for substrate recognition
Kazunari YONEDA, Yudai FUKUDA and Tomohiro ARAKI（Received 13 July 2016; accepted 28 July 2016）
ABSTRACTThe highly thermostable NAD（P）H-dependent carbonyl reductase（CR; EC 1.1.1.184）from the hyperthermophilicarchaeon Aeropyrum pernix K1 was crystallized using the sitting-drop vapor diffusion method with L（+）
-tartaric acid（tartrate）
as the precipitant. The crystals were found to belong to hexagonal space group P6122, with unit-cell parameters a = b = 109.8
Å, c = 243.7 Å, α=β= 90Å, and γ= 120ÛDQGGLIIUDFWHGWR1.80 Å resolution on beamline AR-NW12A at the Photon Factory.
The crystal structure of A. pernix CR was determined in the presence of NADPH and the substrate analog, tartrate. The
ternary complex structure of A. pernix&5ZDVGHWHUPLQHGXVLQJWKHPROHFXODUUHSODFHPHQWPHWKRGDQGZDVUH¿QHGDWD
resolution of 1.80 Å with a crystallographic R-factor of 22.5%（R free 23.4%）. The asymmetric unit consisted of one
homodimer. The final structure showed good geometry with no Ramachandran outliers, and consisted of 494 amino acidresidues, two NADPH molecules, two tartrate molecules, and 490 water molecules. In the present model, a portion of thesurface loop in the C-terminal domain（M192-L197 in subunit A）was disordered and not visible on the electron-density map.
Based on the orientation of the tartrate, we modeled the ethyl（S）-4-chloro-3-hydroxybutanoate （S）[ -CHBE] as a product into
the active site cavity of the A. pernix CR. Within this structure, the hydroxyl group of（S）-CHBE forms a hydrogen bond withthe side chain of Ser141 and Tyr155. Therefore, in this paper, we describe the structural determination and structure of CR
based on the（S）-CHBE recognition mechanism and SVSHFL¿FUHGXFWLRQUHDFWLRQPHFKDQLVP

緒

脱水素酵素 / 還元酵素ファミリー（SDR ファミリー）に

言

属す．この SDR ファミリーに属す酵素のアミノ酸配列

カルボニル還元酵素（CR, EC: 1.1.1.184）はアルデヒには，触媒において重要な役割を担うチロシン残基が保ド，ケトンあるいはキノンなどの様々なカルボニル化合

存されているアミノ酸モチーフ（S-YXXXK）および酸化物の酸化還元反応を触媒する NAD（P）H 依存性の酸化還元酵素活性に必須な補酵素 NAD（P）H の結合アミノ還元酵素である．CR はあまりに基質特異性が広い酵素

酸モチーフ（GXXXGXG），の２つの共通する配列が厳密にであるため，生体内において毒物などの生体異物を分解保存されていることが既に知られている（5）．

あるいは排泄するための代謝反応に広く関与する酵素で我々はこれまでにニワトリの脂肪肝特異的に発現するあることが推測されているが，本来の生理的意義はほと機能未知タンパク質が CR 活性を有する酵素であること

んど明らかにされていない．これまでに分子量や酵素化

や，基質特異性の解析から ethyl 4-chloro-3-oxobutanoate

学的諸性質が異なる多様な CR が様々な生物種から発見（COBE）を良好な基質として，NAD（P）H 依存的に還されているにも関わらず（1−4），全ての CR は短鎖型

元 し，ethyl（S）-4-chloro-3-hydroxybutanoate （S）[ -CHBE]を生産する酵素であるという知見を見出している（6，＊東海大学農学部バイオサイエンス学科

7）． こ の 反 応 は 産 業 応 用 上 重 要 な 知 見 で あ り， 基 質＊＊東海大学大学院生物科学研究科

COBE を不斉還元することで，高コレステロール血症治

連絡先 : 米田一成 e-mail: kyoneda@agri.u-tokai.ac.jp

療薬である HMG-CoA 還元酵素阻害剤の原料として有─1─

米田 一成・福田 雄大・荒木 朋洋
用な（S）-CHBE を合成することが可能である（8，9）．

耐熱性 CR の発現，精製および結晶化

また，本酵素の結晶構造解析にも成功しており，他の生超好熱菌 Aeropyrum pernix K1由来 CR の大腸菌を用い物種の CR では見られないユニークな基質結合に関わる

た発現，精製は Fukuda らの方法に従って行った（10）．

アミノ酸残基の特定にも成功している（7）．しかしなが

MICROCON YM-30（Millipore，米国）を使用し精製酵

ら，ニワトリ脂肪肝由来 CR は3日で酵素活性が半減し素を5.1 mg/ml まで濃縮後，還元型補酵素である NADPHてしまうほど非常に不安定な酵素であり，酵素の応用展および酵素反応のプロダクトである（S）-CHBE をそれ

開には大きな問題があることも判明していた．

ぞれ終濃度0.2 mM になるように加え，シッティングド

そこで，我々はゲノムデータベースを利用し，ニワト

ロップ蒸気拡散法を用いて結晶化スクリーニングを行っ

リ脂肪肝由来 CR と相同性を有する好熱菌由来の CR ホ

た．すなわち，96穴結晶化プレートを使用し，精製酵素

モ ロ グ 遺 伝 子 を 検 索 し た 結 果， 超 好 熱 菌 Aeropyrum

１μl とリザーバー溶液１μl を混合した後，リザーバー

pernix K1（最適生育温度95℃）に28% とわずかではあ溶液100 μl と共に密閉し，暗所（20℃）で約１ヶ月静置るものの，CR をコードしていると予測される遺伝子した .

（ORF ID: APE_2503.1）を見出すことに成功した．本酵
素はもともと脂肪酸生合成経路に関わる酵素として知られている3- オキソアシル - アシルキャリアプロテイン還X 線回折データ測定

元酵素とアノテーションされており，CR 活性を有する得られた結晶を用い，筑波の高エネルギー加速器研究のか，また，（S）-CHBE を合成することが可能な酵素で機構（KEK AR-NW12A ビームライン）において X 線回あるか全く不明であったが，酵素のクローニングおよび

折実験を行った．データ測定には気体窒素を吹きかけ，

機能解析を行った結果，本酵素が高度耐熱性を有す CR

180℃の極低温条件下で測定を行うクライオ測定の技術であり，また COBE を良好な基質とすることを明らかを用いた．クライオプロテクタント（抗凍結剤）にはエにしている（10）．超好熱菌由来の CR の報告例はこれチレングリコールを選択し，結晶化条件に終濃度30%までに無く，安定性の高い CR は有用なキラル化合物合（V/V）になるようにエチレングリコール溶液を加え実成などへの有効利用が期待できると考えられる．本酵素

験に用いた．

はすでに NADPH との２者複合体の X 線結晶構造解析

X 線の波長は1.00 Å，振動角度は1イメージにつき１Û，を行い，NADPH の認識機構や安定化のメカニズムを明結晶から X 線二次元検出器（ADSC Quantum 270）まで

らかにしているが，基質複合体結晶の作成や立体構造解の距離は213.91 mm に設定にした．データの処理には析には至っておらず，基質認識に関わる詳細な分子メカ

HKL2000（11）を用いた．

ニズムの情報は不明のままである（10）．そこで，本研
究ではこれまでとは異なった晶系の結晶を作成し，立体

位相決定および構築した構造の精密化

構造解析を行うことで A. pernix CR-NADPH- 酒石酸（基得られた X 線回折データを元に分子置換法を用いて，質アナログ）の3者複合体の構造決定を行うとともに，

A. pernix 由来 CR の NADPH- 基質アナログ３者複合体

基質結合部位に結合していた酒石酸の構造に基づいての 位 相 決 定 を 行 っ た． 分 子 置 換 の プ ロ グ ラ ム に はCR の反応産物である（S）-CHBE をモデリングすること

MOLREP（12）を用いた．サーチモデルには A. pernix

により CR の基質認識機構および反応機構を考察した．

由来 CR-NADPH の２者複合体の立体構造データ（PDB
code = 5B1Y）を用いた．
プログラム COOT（13）を用いて1-250アミノ酸残基

材料および方法

をアミノ酸配列通りに電子密度にフィッティングを行った．その後，プログラム REFMAC5（14）および CNS

試薬
タンパク質結晶化キットである Crystal Screen および（15）により構造の精密化を行った．A. pernix CR の立体

Crystal Screen 2は Hampton Research（米国）から購入し構造の妥当性（ペプチド結合の2面角の妥当性； ϕ，ψ

た．96穴結晶化プレートは Rigaku（東京）から購入した．

角）の評価は MOLPROBITY（16）を用い，ラマチャン- 酒石酸ナトリウムカリウム四水和物，及び [4L（+）

ドランプロットで解析を行った．（S）-CHBE の座標ファ（2-hydroxyethyl）-1-piperazineethanesulfonic acid）] HEPES

イルは ChemDraw（HULINKS Inc.）および PRODRG サー

はナカライテスク（京都）より入手した .

バー（http://davapc1.bioch.dundee.ac.uk/cgi-bin/prodrg/）

その他の試薬は市販の特級または生化学用試薬を用いた．

を用いて作成した．水素結合の検出にはプログラム
─2─

耐熱性カルボニル還元酵素の結晶構造解析
CCP4mg（17）を用い，分子構造の描画にはプログラム

mm のバイピラミッド型の単結晶を作成することに成功

PyMOL（'H/DQR6FLHQWL¿F//&）を用いた．

した（Fig. 1A）．本結晶を用いて X 線回折実験を行った
ところ，最高分解能1.80 Å であり，空間群は六方晶系である P6122であった（Fig. 1B）．すでに報告している A.

結果および考察

pernix 由 来 CR-NADPH の ２ 者 複 合 体（PDB code =5B1Y）の解析に用いた結晶は2.0 M NaCl を沈殿剤とし結晶化およびデータ測定
結晶化条件のスクリーニングを行ったところ，Crystalて得られた結晶であり，最高分解能が2.09 Å，空間群

Screen No. 29（0.1 M HEPES バッファー pH 7.5，0.8 M L-

P43212であったことから，本研究ではこれまでとは異（+）- 酒石酸ナトリウムカリウム四水和物）の条件でのなった晶系の結晶作成および，より高分解能なデータ収単結晶の析出が確認できた．更に，沈殿剤である酒石酸集を行うことができた．

の濃度を0.4-1.0 M の範囲で条件展開することで約0.3
立体構造解析および全体構造

A

解析の結果，活性中心には水以外の分子と予測される明瞭な電子密度が観察されたため，クライオの測定溶媒に用いたエチレングリコール分子と予測して，電子密度にアサインしたものの，エチレングリコールが２分子分以上入る大きさの電子密度であり，カルボキシル基と予測される形状の電子密度も観察された．そこで，結晶化試薬として高濃度使用した酒石酸の存在が予測されたた
Table 1. Data-collection, processing, phase determination, andUH¿QHPHQWVWDWLVWLFVIRUA. pernix CR.
Data collection
Wavelength（Å）
Temperature（K）
Space group
Unit cell parameters（cÛ）

B

aResolution range（Å）
1RPHDVXUHGUHÀHFWLRQV
1RXQLTXHUHÀHFWLRQVaRedundancyaRmergea
Rp.i.m.
aCompleteness（%）
〈I/σ
（I）〉
5H¿QHPHQW
Resolution range（Å）
b
R/RfreeNo. of protein atoms
No. of water molecules
No. of ligands
RMSD
Bond lengths（Å）
Bond angles（Û）
2
Average B-factors（Å ）Protein atoms
Water molecules
NADPH molecules
Tartaric acid moleculescRamachandran quality
Favored regions（%）
Outliers（%）

Fig. 1. Hexagonal crystals and X-ray diffraction pattern of A.
pernix CR. （A）A. pernix CR/NADPH/tartrate ternary complex
at 1 month in the crystallization step using the sitting-drop
vapor-diffusion method at 20℃ . The maximum dimensions of
the crystal were 0.3 ×0.3 ×0.1 mm.（B）The X-ray diffraction
pattern of a native A. pernix CR crystal. The resolution isapproximately 1.80 Å at the edge. The high-resolution area isenlarged（inset）
.

a

1.00
100
P6122
a = b = 109.8, c = 243.7
α = β = 90, γ = 120
50.00 1.80（1.83 1.80）
230761680994
28.5（29.6）
0.080（0.307）
0.016（0.061）
99.9（100）13.1
35.5 1.80
0.225/0.234
3735
490
NADPH, 2
Tartaric acid, 2
0.025
2.3
21.0
36.9
44.1
41.3
96.5
0.0

Values in parentheses are for the last resolution shell.
（5%）.
RfreeZDVFDOFXODWHGZLWKUDQGRPO\VHOHFWHGUHÀHFWLRQVc
MOLPROBITY（16）was used to monitor and validate the structural model.
b

─3─米田 一成・福田 雄大・荒木 朋洋
め，電子密度にアサインしたところ，ぴったり当てはま

酒石酸複合体構造および（S）-CHBE のモデリング

り，活性中心に観測された電子密度を酒石酸として解釈

活性中心に結合している酒石酸の C1カルボキシル基することができた．また，NADPH は電子密度へ容易には触媒残基である Ser141，Tyr155の側鎖と水素結合してフィッティングすることが可能であった．最終的に A.

おり，酒石酸の C4カルボキシル基は Arg248の側鎖およpernix 由 来 CR-NADPH- 酒 石 酸 の ３ 者 複 合 体 を R 値 =び，Val150，Ala151の 主 鎖 の NH と 水 素 結 合 し て い た（Fig. 3A）
．また，酒石酸のカルボニル基は Arg248の側A

Fig. 2. The dimeric structure of A. pernix CR. The subunit A
and B are shown in green and pink, respectively. NADPH（magenta）and tartrate（yellow）molecules are shown as a stickmodel. Oxygen and nitrogen atoms are shown in red and blue,
respectively.
B

0.225，free R 値 = 0.234で構造決定することに成功した（Table 1）．非対称単位中にはホモダイマーが1分子存在しており，494アミノ酸残基，2 分子の NADPH，2分子の酒石酸，490の水分子で構成されていた（Fig. 2）．ま3

−1

た，マシューズ係数は3.49 Å Da ，結晶中の溶媒含有量は64.5% であった．MOLPROBITY を用いたラマチャ
ンドランプロットの解析の結果，全てのアミノ酸残基が許容範囲内であった．モノマー構造はロスマンフォール
ドドメイン（N 末ドメイン）と触媒ドメイン（C 末ドメイン）の２つで構成されていた．
B サブユニットは Met1-Thr250の250アミノ酸残基全てモデル構築することができたが，A サブユニットの分子表面のループ領域である Met192-Leu197の６アミノ酸残基は明瞭な電子密度が観察できず，アミノ酸をアサインすることができなかった．また，本酵素は pET-15b（Novagen，米国）をタンパク質発現用プラスミドとして使用しているため，His-tag 配列（20アミノ酸）が CRの N 末端に付加されているが，構造解析の結果，Histag 配列の電子密度は観察されなかった．結晶化の際にCR の酵素反応プロダクトである（S）-CHBE と共結晶化したものの，結晶化の沈殿剤として使用した高濃度の酒石酸（0.8 M）が基質結合部位を占有し，（S）-CHBE は基質結合部位に結合しなかったと考えられる．
─4─

Fig. 3. The tartrate coordinated within the active site and the
proposed binding model for the（S）-CHBE molecule. （A）
Schematic representation depicting the interactions between
tartrate and A. pernix CR. The networks of hydrogen bonds areshown by dotted lines. The tartrate molecule is shown as a stick
PRGHOLQ\HOORZ7KH¿QDO σA-weighted Fo-Fc omit electron
density map for tartrate is shown at the 1σ level.（B）Comparison of the A. pernix CR active site pocket with that ofchicken fatty liver CR and the proposed binding model of the（S）-CHBE. The C4 atom of the pyridine ring（a hydride
acceptor site）, and si- and re-faces are labeled. NADPH（magenta）and（S）-CHBE（cyan）molecules are shown as a
stick model. The structures of A. pernix and chicken fatty liver
CR are shown in pink and blue, respectively. Oxygen, nitrogenand chloride atoms are shown in red, blue and green, respectively.

耐熱性カルボニル還元酵素の結晶構造解析
鎖と相互作用することを明らかにした．触媒残基である

保存性が全くないユニークな配列である．特に，Y172

Lys159は NADPH のニコチンアミドリボースのヒドロキに関してはアラニン置換変異体の解析により，基質であシル基との水素結合していた（Fig. 3A）．得られた酒石

る9,10-phenanthrenequinone に 対 す る Km 値 が12.5倍 上 昇酸複合体の構造から C1カルボキシル基の部分が CR のすることをすでに明らかにしている（7）．モデリングの

基質のカルボニル基の位置に相当するのではないかと予結果では Y172が活性中心で（S）-CHBE を抑え込むよう測されたため，活性中心に結合した酒石酸（基質アナロ

な形でインタラクションすることが予測されることから，

グ）の配向から CR の酵素反応産物である（S）-CHBE

モデリングした構造と酵素化学的データの結果が良く一の結合モデルを構築し，これまで不明であった基質結合致していると言える．これらの結果より，ニワトリ脂肪に関与するアミノ酸残基を同定した．モデリングの結果，

肝由来 CR では A. pernix CR と比較して（S）-CHBE との（S）-CHBE のヒドロキシル基は触媒残基である Ser141，

インタラクションがより強固になっていることを立体構

Tyr155の側鎖と水素結合しており，（S）-CHBE のカルボ造に基づき説明することが可能となった．これらの結果

ニル基は Arg248の側鎖と水素結合していた（Fig. 3B）．

は，高脂血症治療薬の合成中間体として有用なキラル化

また Val150が（S）-CHBE の酸素原子および疎水性のエ合物である（S）-CHBE に対するハイアフィニティーなチル基と相互作用することが予測され，S 体選択的に反酵素の創製などへの応用利用に役立つ情報となりうる．

応が可能な配向で（S）-CHBE が A. pernix 由来 CR の触媒部位に結合することが示唆された（Fig. 3B）．補酵素

謝NADPH のニコチンアミド−リボース間の結合が syn 型辞の構造であることから NADPH のピリジン環の C4位か本研究を実施するにあたり，貴重なご意見や様々な支らの水素転移は si 面（pro-S; B-type）特異的に起こると援を頂きました大阪工業大学工学部生命工学科 大島敏

考えられることも S 体選択的に COBE の還元反応が可久教授，香川大学農学部応用生物科学科 櫻庭春彦教授

能な理由と言える．

に感謝いたします．また，X 線回折実験を行った機関である高エネルギー加速器研究機構のスタッフの皆様に感

A. pernix CR とニワトリ脂肪肝 CR の構造比較謝します．本研究課題に関する放射光実験は，放射光共

これまでの我々の研究により A. pernix 由来 CR およびニワトリ脂肪肝由来 CR の両酵素において COBE が良好

同利用実験採択課題番号2014G502で実施されたものです．

な基質となることをすでに明らかにしている（7，10）．
COBE に対する親和性はニワトリ脂肪肝由来 CR の方が要

はるかに高い事をすでに報告している（A. pernix CR;

約

Km= 0.7μM，ニワトリ脂肪肝由来 CR; Km= 0.008μM）が，カルボニル還元酵素（CR，EC: 1.1.1.184）は NAD（P）

その分子認識機構は未だに不明であった．そこで，（S）

H を補酵素として様々なカルボニル化合物の還元反応を-CHBE のモデリングを行った A. pernix CR の構造とニワ触媒する酵素である．安定性の高い CR は有用なキラル

トリ脂肪肝由来 CR（PDB code = 3WXB）の重ね合わせ化合物合成などへの有効利用が期待できる．本研究ではを行い，活性中心に結合した（S）-CHBE の周辺構造を超好熱菌である Aeropyrum pernix K1由来の耐熱性 CR の比較することで，基質に対する親和性の違いについて考NADPH- 酒石酸（基質アナログ）との3者複合体の結晶察した．A. pernix CR では（S）-CHBE のエチル基とクロ構造解析を行うとともに，CR の酵素反応産物である

ル 基 の 周 辺 に 存 在 す る ア ミ ノ 酸 は Val150，Phe187，（S）-4- クロロ -3- ヒドロキシ酪酸エチル （S）[ -CHBE] のArg248で あ る が， ニ ワ ト リ 脂 肪 肝 由 来 CR で は（S）

構造を活性中心にモデリングした．0.8 M L（+）
- 酒石酸

-CHBE の疎水性のエチル基を疎水性アミノ酸であるを沈殿剤として NADPH- 酒石酸との複合体結晶を取得

Tyr168，Tyr172が認識するとともに，マイナスにチャー

し，X 線回折実験を行った結果，最高分解能1.80 Å のジしているクロル基と Trp210の NH がインタラクショ

データを取得し，最終的に分子置換法により A. pernix

ンし，（S）-CHBE の酸素原子は構造上，Arg179がインタ

CR-NADPH- 酒石酸の３者複合体の構造を決定することラクトすることが推定された（Fig. 3B）．ニワトリ脂肪に成功した．触媒部位では Ser141，Tyr155が酒石酸の肝 CR の Tyr168，Tyr172は基質結合部位にフタをする役

C1カ ル ボ キ シ ル 基 と の 水 素 結 合 に 関 与 し，Lys159はActive site lid （Ile164-Gln174）内に存在するアミ

NADPH のニコチンアミドリボースのヒドロキシル基とノ酸残基であり，他の生物種の CR ではアミノ酸残基のの水素結合に関与していた．酒石酸の構造に基づいて

割の

─5─

米田 一成・福田 雄大・荒木 朋洋（S）-CHBE のモデリングを行ったこところ，S 体選択的８）Patel, R.N., McNamee, C.G., Banerjee, A., Howell, J.M.,に反応可能な配向で（S）-CHBE が基質結合部位に結合

Robinson, R.S. and Szarka, L.J. 1992, Enzyme Microb.

することが示唆された．これら構造情報は，高脂血症治

Technol. 14, 731 738.

療薬の合成中間体として有用なキラル化合物である

９）清水昌，山田秀明，1991，有機合成化学協会誌，49，（S）-CHBE 生産のための人工 CR 創製への応用利用に役

52 62.
10）Fukuda, Y., Sakuraba, H., Araki, T., Ohshima, T. and立つ情報となりうる．

Yoneda, K., 2016, Enzyme Microb. Technol. 91, 17 25.
11）Otwinowski, Z. and Minor, W. 1997, Methods Enzymol.

引用文献

276, 307 326.

１）Wermuth, B. 1985, Enzymology of Carbonyl Metabolism12）C.C. Project. 1994, Acta Crystallogr. D Biol. Crystallogr.

2（T.G. Flynn, H. Weiner, eds.）Alan R. Liss, Inc., New

50, 760 763.
13）Emsley, P. and Cowtan, K. 2004, Acta Crystallogr. D

York, pp. 209 230.

Biol. Crystallogr. 60, 2126 2132.

２）石倉周平，2005，生物工学会誌，83，181 184.
３）Sgraja, T., Ulschmid, J., Becker, K., Schneuwly, S.,14）Murshudov, G.N., Vagin, A.A. and Dodson, E.J. 1997,
Acta Crystallogr. D Biol. Crystallogr. 53, 240 255.

Klebe, G., Reuter, K. and Heine, A. 2004, J. Mol. Biol.

15）Brünger, A.T., Adams, P.D., Clore, G.M., DeLano, W.L.,

342, 1613 1624.
４）Zaccai, N.R., Carter, L.G., Berrow, N.S., Sainsbury, S.,

Gros, P., Grosse-Kunstleve, R.W., Jiang, J.S., Kuszewski,

Nettleship, J.E., Walter, T.S., Harlos, K., Owens, R.J.,

J., Nilges, M., Pannu, N.S., Read, R.J., Rice, L.M.,

Wilson, K.S., Stuart, D.I. and Esnouf, R.M. 2008,

Simonson, T. and Warren, G.L. 1998, Acta Crystallogr. D

Proteins 70, 562 567.

Biol. Crystallogr. 54, 905 921.
rd16）Lovell, S.C., Davis, I.W., Arendall W.B. 3 , de Bakker,５）田中信忠，2005，生物工学会誌，83，185 187.
６）Yoneda, K., Fukuda, Y., Shibata, T., Araki, T., Nikki, T.,

P.I., Word, J.M., Prisant, M.G., Richardson, J.S. and

Sakuraba, H. and Ohshima, T., 2012, Acta Crystallogr. F
Struct. Biol. Commun. 68, 1568 1570.

Richardson, D.C. 2003, Proteins 50, 437 450.
17）Potterton, E., McNicholas, S., Krissinel, E., Cowtan, K.

７）Fukuda, Y., Sone, T., Sakuraba, H., Araki, T., Ohshima, T.,
Shibata, T. and Yoneda, K., 2015, FEBS J. 282, 3918

and Noble, M. 2002, Acta Crystallogr. D Biol.
Crystallogr. 58, 1955 1957.

3928.

─6─

東海大農紀要 36：７−14（2017）
Lactobacillus plantarum inhibits the adhesion of Candida albicans

Lactobacillus plantarum LA 318 inhibits the adhesion of
Candida albicans ATCC 26555 to human colonic mucin
＊1

2

1

3

4

Hideki KINOSHITA , Masamichi WATANABE , Keiji IGOSHI , Mitsuharu ISHIDA , Yasushi KAWAI ,5

6

6

Haruki KITAZAWA , Koh MIURA , Akira HORII and Tadao SAITO

5（Received 28 October 2016; accepted 7 November 2016）

ABSTRACT
Candida albicans is a fungal organism found in small numbers in the normal human intestinal tract. Normally kept incheck by the bodyʼs own helpful bacteria, C. albicans can increase in numbers when this balance is disturbed to cause
candidiasis of the intestinal tract, or yeast infections in other parts of the body. Here an adhesion inhibition of the yeast andpseudohyphal forms of C. albicans ATCC 26555 to human colonic mucin by Lactobacillus plantarum LA 318 wasinvestigated. SDS-PAGE showed glyceraldehyde-3-phosphate dehydrogenase（GAPDH）at about 30 kDa in Candida albicans
ATCC 26555 using SYPRO Ruby stain while few bands were detected using CBB stain. Forms incubated at 28 °C（induces
yeast）and 37 °C（induces hyphae）showed a dose-dependent adherence of C. albicans ATCC 26555 to human colonic mucin.
Adhesion inhibition tests were performed using three different methods: competition, exclusion, and displacement assays.
Adhesion of the yeast form incubated at 28 ℃ was significantly decreased by about 10 ~ 20 %（p<0.05）; whereas no8

inhibition of fungus adhesion was observed in the competitive assay using 1×10 cells/mL of LAB（equal to fungus
9

numbers）. The highest inhibition was observed using the exclusion assay with 1×10 cells/mL of LAB（10 fold higher thanthe C. albicans ATCC 26555）. Adhesion of the pseudohyphal form incubated at 37 ℃ when using equal numbers of LAB
ZDVVOLJKWO\GHFUHDVHGZKHUHDVWKHSVHXGRK\SKDOIRUPDGKHVLRQZDVVLJQL¿FDQWO\GHFUHDVHGE\21.5 % in the exclusion assay
9

using 1×10 cells/mL of LAB（p<0.05）. This study shows the possible prevention of candidiasis using lactobacilli.

extracellular matrix（6 8）; and glyceraldehyde 3 phosphate

INTRODUCTION

dehydrogenase（GAPDH）is expressed on its cell surface（7）.

Candida species are the most frequent cause of life-

Lactic acid bacteria（LAB）are common microbes usedthreatening invasive fungal infections in immuno-

as probiotics. The genus LactobacillusVKRZVPDQ\EHQH¿FLDO

compromised hosts（1）. Candida colonization often leads toeffects as probiotics, e.g. managing lactose intolerance（9）,opportunistic mucosal or deep organ infection in immuno-

lowering cholesterol（10）, improving immune function（11）,compromised hosts. Invasion of the human gastrointestinal

prevention of colon cancer（12）, and inhibition of adherence

mucosa by C. albicans and its passage across the bowel wallof pathogens（13 15）. Lactobacillus plantarum LA 318 is a

into the bloodstream is an important portal of entry, leading topotential probiotic strain isolated from a normal humansystemic or disseminated candidiasis（2 5）. Adhesion of theintestinal tissue（transverse colon）of a cancer patient and

IXQJXVLVDQLPSRUWDQW¿UVWVWHSLQWKHLQIHFWLRQRIWKHKRVW,W

strongly adheres to human colonic mucin（HCM）and blood

has been reported C. albicans ATCC 26555 adheres to thetype antigens（A type and B type）mediated by a cell surface＊１

Department of Bioscience, School of Agriculture, Tokai University
Research Faculty of Agriculture, Hokkaido University
３
Department of Food Management, School of Food, Agricultural and Environmental Sciences, Miyagi University
４
Graduate School of Bioresource Sciences, Nihon University
５
Graduate School of Agricultural Science, Tohoku University
６
Graduate School of Medicine, Tohoku University
連絡先 木下英樹 e-mail: kinoshita@tokai.ac.jp２

─7─

Hideki Kinoshita, Masamichi Watanabe, Keiji Igoshi, Mitsuharu Ishida, Yasushi Kawai,
Haruki Kitazawa, Koh Miura, Akira Horii and Tadao Saito
GAPDH（16, 17）. The adhesion of C. albicans ATCC 26555

analyzed using SDS-PAGE in a 12.5 % gel. After electrophoresis,

may be inhibited by L. plantarum LA318 through aprotein bands were visualized staining the gels with Comassie

competitive interference, because both the Candida and LAB

brilliant blue（CBB）（Rapid CBB KANTO, Kanto Chemicalstrains possess GAPDH on their cell surface.

Co., Tokyo, Japan）and SYPRO Ruby（BIO-RAD Laboratories,

C. albicans is dimorphic, having a yeast form and a

Hercules, CA, USA）.

pseudohyphal form, according to the temperatureenvironment. Pathogenicity is shown when in pseudohyphal

Carboxyfluorescein diacetate labeling of Candida cells
After the starvation period, C. albicans ATCC 26555 was

growth（18 20）. An adhesion inhibition test to HCM using L.
plantarum LA 318 with the two different forms of C. albicans

cultured at 28℃ or 37℃ for 6 h in YM broth（Difco Laboratories）

ATCC 26555 was investigated.

and centrifuged（5,000 rpm, 10 min, room temperature）. The
pellets were washed with sterilized distilled water three times（8,000 rpm, 5 min, 4 ℃）. The pellets were re-suspended in

MATERIALS AND METHODS

sterilized distilled water and 1 mM carboxyfluoresceindiacetate（CFDA）was added in PBS. After incubation for 30

Microorganisms and growth conditions
L. plantarum LA 318 was directly isolated from human

min at 37 ℃ in the dark, the CFDA labeled microbial cells werecolon tissue using a mass screening. This strain showed highwashed with sterilized distilled water three times（8,000 rpm,

adhesion to HCM and A and B blood type antigens by cell

5 min, 4 ℃）.

surface GAPDH（16, 17）. The strain was cultured twice at
37 ℃ for 24 h in MRS broth（Difco Laboratories, Detroit, MI,

Microtiter plate binding assay
TM

Reacti-BindUSA）using a 2%（v/v）inoculum.

amine-binding maleic anhydride activatedC. albicans ATCC 26555 was purchased from the American

plates（PIERCE, Rockford, IL, USA）that allow attachment

Type Culture Collection（ATCC, Manassas, VA, USA）. Thisof amine-containing protein to the microplate wells were usedstrain was reported to have GAPDH on the cell surface（7）.

for the microtiter plate binding assay. The isolation and

The strain was cultured twice at 28 ℃ for 24 h in YM broth

SXUL¿FDWLRQRI+&0ZDVSHUIRUPHGDVGHVFULEHGSUHYLRXVO\（Difco Laboratories）using a 2%（v/v）inoculum.

（21）
7KHSXUL¿HG+&0（0.3 mg/mL）was dissolved in PBS（pHFor the inhibition assay，C. albicans ATCC 26555 was

7.4）（100 μL）and added to each well and incubated overnight

cultured for 16 h at 28 ℃ . The cells were centrifuged（4,000

at 4 ℃ ; and then the plates were used for the binding assay.

rpm, 10 min）and the supernatant was discarded. The pellets

After immobilization of the HCM, each well was washed threewhere held for 3 or 4 days at 4 ℃（starvation period）. Aftertimes with 250μL PBS. Then the CFDA-labeled C. albicans

starvation, fresh YM broth was added and fungi cells were

ATCC 26555 were added in 100μL distilled water to thecultured for 6 h at either 28 ℃ or 37 ℃ .

wells. Three parallel wells were used in each experiment. The
microbial cells were allowed to adhere for 1 h at 37 ℃ and thewells were washed three times with 250μL distilled water toExtraction and analysis of surface proteins of C. albicans
C. albicans ATCC 26555 were propagated for 16 h at 28 ℃

remove non-adherent cells. The cells bound to the HCM were

and washed three times with PBS（PBS wash solution）. Afterreleased and lysed using 1%（w/v）SDS/0.1 M NaOH

washing, the cells were gently suspended in 2 or 4 M guanidine

solution and incubated for 1 h at 60 ℃ . After incubation, thehydrochloride（GHCl）solution and incubated at 37 ℃ for 2 h.

ÀXRUHVFHQFHLQWHQVLW\（Ex: 485 nm, Em: 538 nm）of the lytic

After centrifugation（8,000 rpm, 20 min, 4 ℃）
, the supernatantsolution was measured using a Fluoroskan Ascent plate reader（extract）was used for the GAPDH activity assay. The PBS（Thermo Fisher Scientific, Inc., Waltham, MA, USA）. PBS

wash solution and GHCl extract were dialyzed against

was used as the control substituting for HCM. The adhesion

distilled water and lyophilized. The lyophilized extracts wereYDOXHZDVGH¿QHGDVWKHYDOXHRI+&0Candida immobilized

used for SDS-PAGE.

on the plate subtracting the control value.

SDS-PAGE

Adhesion inhibition test for C. albicans

The cell surface extracts of C. albicans ATCC 26555 were─8─

The adhesion inhibition tests were performed using threeLactobacillus plantarum inhibits the adhesion of Candida albicans
different methods: competition, exclusion, and displacement
assays. The concentration of CFDA-labeled C. albicans ATCC

RESULTS AND DISCUSSION

826555 was 1×10 cells/mL. The concentrations of non8

9

In this study, inhibition of adherence of C. albicans tolabeled L. plantarum LA 318 were 1×10 cells/mL or 1×10

human intestinal mucus using a high adhesive probiotic LAB,

cells/mL.

L. plantarum LA 318 was considered. It is reported that C.

For the competitive assay, each 50μL of CFDA-labeled

albicans ATCC 26555 possess surface GAPDH and shows

fungus and 50μL of non-labeled LAB strain was mixed and

DGKHVLRQWR¿EURQHFWLQDQGODPLQLQDVWKHIXQJDOSDWKRJHQ
（7）.

added to the HCM-immobilized microtiter wells; andC. albicans ATCC 26555 was propagated at two difference

incubated for 1 h at 37 ℃ . Each well was washed three times

temperatures, 28 and 37 ℃ , after starvation. The fungus morphedwith 250μL distilled water and the fungus cells bound to theinto yeast cells at 28 ℃ or pseudohyphal cells at 37 ℃（Fig. 1）.

HCM were released and lysed using 1%（w/v）SDS/0.1 M

Pathogenicity is increased during pseudohyphal elongation

NaOH solution and incubated for 1 h at 60 ℃ . After incubation,
WKHÀXRUHVFHQFHLQWHQVLW\（Ex: 485 nm, Em: 538 nm）of thelytic solution was measured using a Fluoroskan Ascent plate
reader（7KHUPR)LVKHU6FLHQWL¿F,QF）.
For the exclusion assay, 100μL of non-labeled LAB wasadded to the HCM-immobilized microtiter wells and was
incubated for 1 h at 37 ℃ and washed three times. After washing,
100μL of CFDA-labeled fungus was added and incubated for
1 h at 37 ℃ $IWHUZDVKLQJWKHFHOOVZHUHO\VHGDQGÀXRUHVFHQFH
intensity was measured.
For the displacement assay, the LAB and fungus werereversed in the exclusion assay.
Statistics
All experiments were performed in triplicate and reported
as the mean ± SD. Statistical analyses of all data were performed
using the Scheffeʼs test for the adhesion test and the Tukeyʼs
HSD（KRQHVWO\VLJQL¿FDQWGLIIHUHQFH）test for the inhibition
assays.

Figure 1. The two different forms of C. albicans ATCC 26555 using
incubation at different temperatures（28 ℃ or 37 ℃）. After the
starvation period, C. albicans ATCC 26555 was cultured at 28 ℃（A）or
37 ℃（B）for 6 h in YM broth. Each of cells were stained with Gramʼsstain and observed with microscope.
Arrows indicate a germ tube.

Figure 2. SDS-PAGE of protein extracts from C. albicans ATCC 26555.
LA 318*$3'+SXUL¿HG*$3'+IURPL. plantarum LA 318, PBS: PBS wash fraction,
2M GHCl: 2 M GHCl extracts, 4M GHCl: 4 M GHCl extracts.

─9─

Hideki Kinoshita, Masamichi Watanabe, Keiji Igoshi, Mitsuharu Ishida, Yasushi Kawai,
Haruki Kitazawa, Koh Miura, Akira Horii and Tadao Saito（18 20）. Figure 2 shows the SDS-PAGE of cell surface

surface-bound proteins that play a role in adhesion of L.

extracts of C. albicans. A 30 kDa protein was detected using

plantarum 423 to Caco-2 cells（33）. The strain prevented

SYPRO Ruby stain while few bands were detected using CBBClostridium sporogenes and Enterococcus faecalis fromstain. This suggested the 30 kDa protein was GAPDH because

adhering to Caco-2 cells. Sánchez et al. report GAPDH andthe molecular weight of GAPDH from C. albicans ATCC

phosphoglycerate kinase are expressed on the cell surface of26555 is 33kDa. Further, GAPDH enzymatic activity in theL. rhamnosus GG（34）. Castaldo et al. report L. plantarum

extract was detected. However, the expression level was veryLM3 expresses enolase on the cell surface bound to

low compared to the L. plantarum LA 318（data not shown）.

fibronectin, an extracellular matrix protein（35）. We

Therefore, GAPDH may be not the primary adhesin of C.

previously reported cell surface GAPDH of L. plantarum LA

albicans ATCC 26555 where other adhesins may exist. It was

318 showed high adhesion to HCM and ABO blood type

reported that a 58 kDa fibronectin-binding mannoprotein

antigens, especially A｛GalNAcα1 3（Fucα1 2）Gal ｝（mp58）was expressed on the C. albicans cell surface as an

and B｛Galα1 3（Fucα1 2）Gal ｝antigens where GAPDH

adhesion（6）but it was not detected. Since a lot of protein

may be a GalNAc and Gal recognition lectin-like protein（16ZDVGHWHFWHGDW14 kDa, the possibility of an adhesion factor

17）. Further, cell surface GAPDH activities were detectedat <14 kDa was considered.

from 30 lactobacilli isolated from human intestinal tissues. A
statistically significant positive correlation was shown

Reports show some pathogens possess GAPDH on their
FHOOVXUIDFHDQGWKH\DUHLGHQWL¿HGDVDGKHVLQV6WUHSWRFRFFDO

between GAPDH activity and adhesion using the BIACORE
adhesion assay（0.69, p<0.01）（36）.

surface GAPDH（SDH）from group A Streptococcus shows
multiple binding activities to plasmin（ogen）（22, 23）,

Figure 3 shows the C. albicans ATCC 26555 adhesion

fibronectin, lysozyme, myosin, and actin（24）. SDH

test to HCM using microtiter plates. Both 28 ℃ and 37 ℃

recognizes uPAR/CD87 as its receptor on human pharyngeal

incubated cells adhered to HCM. The amount of C. albicans

cells and mediates bacterial adherence to host cells（25）.

ATCC 26555 adhesion increased in a dose-dependent manner.

Seifert et al. report enzymatically active GAPDH is capable

The detection limit was 1×10 cells/mL for the yeast formof binding to cytoskeletal and extra-cellular matrix proteins（28 ℃）and 1×10 cells/mL for the pseudohyphal form（37and is expressed on the surface of group B Streptococcus（26）.

℃）. C. albicans ATCC 26555 was reported to adhere to

Surface-localized GAPDHs of Staphylococcus aureus and¿EURQHFWLQ（6−7）, laminin（7）, and the 7S（IV）domain of

Staphylococcus epidermidis bind transferrin（27）; whereas

type IV collagen（8）; and other C. albicans strains adhered to

76

the GAPDH of C. albicansELQGV¿EURQHFWLQDQGODPLQLQ（7）.

human buccal epithelial cells, rabbitʼs small intestinal mucin

The fimbriae of Porphyromonas gingivalis, a dominant（37）, and saliva mucin（38, 39）. In this study, C. albicans

periodontal pathogen, mediates co-agglutination using the

ATCC 26555 shows adhesion to HCM.

GAPDH of Streptococcus oralis（28）to bind to human oral
epithelial cells（29）. Tunio et al. report the GAPDH（GapA-1）

The adhesion inhibition tests were performed using threeof Neisseria meningitides, causing life-threatening infections

different methods: competition, exclusion, and displacement

including septicemia and meningitis, adhered to human cells

assays. Adhesion of the yeast form incubated at 28 ℃ were（30）
. Therefore, many pathogenic bacteria possess GAPDH

significantly decreased by about 10 ~ 20 %（p<0.05）;

on their surfaces that show binding to multiple tissues.

whereas no inhibition of fungus adhesion was observed in the
8

In studies of lactobacilli, Hurmalainen et al. reportcompetitive assay when using 1×10 cells/mL of LAB（equal

GAPDH is expressed on cell surfaces with enolase in sevento fungus numbers）（Fig. 4A）. The highest inhibition was

lactobacilli and one lactococci. The proteins boundobserved in the exclusion assay when using 1×10 cells/mL

plasminogen（Plg）and enhanced its activation using a tissue-of LAB（10−fold higher than C. albicans ATCC 26555）.

type Plg activator（tPA）（31）. Antikainen et al. report

Adhesion of the pseudohyphal form incubated at 37 ℃ when

GAPDH bound to lipoteichoic acid on the bacterial cellusing equal numbers of LAB was slightly decreased; whereas

surface using ionic bonds（32）. GAPDH, elongation factor

WKHSVHXGRK\SKDOIRUPDGKHVLRQZDVVLJQL¿FDQWO\GHFUHDVHG

Tu（EF-Tu）and triosephosphate isomerase（TPI）areby 21.5 % in the exclusion assay when using 1×10 cells/mL9

─ 10 ─

9

Lactobacillus plantarum inhibits the adhesion of Candida albicans
may help indigenous bacteriaʼs protective effects.
Reports show the inhibitory effect on candidiasis by
LAB strains. Boris et al. report L. acidophilus inhibited
adhesion of C. albicans and Gardnerella vaginalis to vaginal
epithelial cells in competition and displacement assays;
whereas there was no inhibition using an exclusion assay（40）.
Coudeyras et al. report L. rhamnosus Lcr35 adhered tocervical and vaginal cells and inhibited the growth ofvaginosis-associated pathogens such as C. albicans（41）.
Matsubara et al. report lactobacilli reduced the C. albicans
colonization significantly in the oral mucosa compared tountreated mice（42）. In the group treated with L. rhamnosus,
yeast colonization was significantly reduced compared withthat of the group receiving nystatin. The effect of L. casei
CRL 431 administered as a supplement to a repletion diet wasstudied for resistance of malnourished mice to C. albicans
infection（43）. The addition of L. casei to the repletion dietnormalized the immune response against C. albicans,
DOORZLQJHI¿FLHQWUHFUXLWPHQWDQGDFWLYDWLRQRISKDJRF\WHVDV
well as effective release of pro-inflammatory cytokines.
Martinez et al. reported L. reuteri RC 14 alone or together

Figure 3. Adhesion test for C. albicans ATCC 26555 to HCM
using the microtiter plate assay. A: C. albicans ATCC 26555
incubated at 28 ℃ , B: C. albicans ATCC 26555 incubated at 37℃ 'LIIHUHQWOHWWHUVVKRZVLJQL¿FDQWGLIIHUHQFH
（p<0.05）.

of LAB（p<0.05）（Fig. 4B）
. The difference in the amount of
LAB and Candida cells may be one cause. The cell size of C.
albicans ATCC 26555, a eukaryote, is ten times larger than
LAB, a prokaryote. When the concentration of LAB was 1×
8

10 cells/mL（equal to C. albicans ATCC 26555）, LAB
occupied a smaller volume of the well. When concentration of
9

LAB was 1×10 cells/mL（10 hold higher）, the occupied
LAB volume of the well was equal to C. albicans ATCC
26555. LA 318 showed adhesion to HCM in a dose-dependent
7

10

manner at from 1×10 cells/mL to 1×10 cells/mL in the
microtiter plate binding assay（data not shown）. If LAB cells
are added 100 times or more as much as C. albicans ATCC
26555 cells, there may be further inhibition. It is well known
indigenous bacteria protect a human body from various
pathogens including C. albicans. The ingestion of LA 318
─ 11 ─

Figure 4. Adhesion of C. albicans ATCC 26555 to HCM in theinhibition assay with L. plantarum LA 318. A: using C. albicans
ATCC 26555 incubated at 28 ℃ , B: using C. albicans ATCC
26555 incubated at 37 ℃ .
'LIIHUHQWOHWWHUVVKRZVLJQL¿FDQWGLIIHUHQFH
（p<0.05）.

Hideki Kinoshita, Masamichi Watanabe, Keiji Igoshi, Mitsuharu Ishida, Yasushi Kawai,
Haruki Kitazawa, Koh Miura, Akira Horii and Tadao Saito
1992b, Infect. Immun. 60, 4221 4229.

with L. rhamnosus GR 1 has the potential to inhibit yeast
growth, and their cell-free supernatants may up-regulate IL 8

7）Gozalbo, D., Gil-Navarro, I., Azorin, I., Renau-Piqueras,

and IP 10 secretion by epithelial cells from the normal humanJ., Martinez, J.P. and Gil, M.L., 1998, Infect. Immun. 66,

vagina that may possibly play an important role in clearing

2052 2059.

vulvovaginal candidiasis（44）. Further, LAB produces

8）Alonso, R., Llopis, I., Flores, C., Murgui, A. andantifungal substances such as lactic acid and hydrogen

Timoneda, J., 2001, Microbiology. 147, 1971 1981.

peroxide（H2O2）（45, 46）. Therefore, there are potential9）Savaiano, D.A. and Kotz, C., 1989, ASDC J. Dent. Child.

EHQH¿WVDJDLQVWCandida infection using LAB.

56, 228 233.
10）Danielson, A.D., Peo, E.R., Shahani, K.M., Lewis, A.J.,
Whalen, P.J. and Amer, M.A., 1989, J. Anim. Sci. 67,

CONCLUSION

966 974.

In this study, the adhesion inhibition to HCM by L.

11）Perdigón, G., Maldonado-Galdeano, C., Valdez, J.C. andMedici, M., 2002. Eur. J. Clin. Nutr. 56, S21 26.

plantarum LA318 using yeast and pseudohyphal forms of C.
albicans ATCC 26555 was investigated. SDS-PAGE showed12）Lim, B.K., Mahendran, R., Lee, Y.K. and Bay, B.H.,
2002, Jpn. J. Cancer Res. 93, 36 41.

GAPDH in C. albicans ATCC 26555 while it is suggested thatGAPDH is not a primary adhesin because few bands are13）Mack, D.R., Michail, S., Wei, S., McDougall, L. anddetected using the CBB stain. In the adhesion inhibition tests,

Hollingsworth, M.A., 1999, Am. J. Physiol. 276, G941

the inhibition of C. albicans ATCC 26555 adhesion to HCM

950.

using the probiotic LAB, L. plantarum LA 318, especially

14）Chen, X., Xu, J., Shuai, J., Chen, J., Zhang, Z. and Fang,
W., 2007, Int. J. Food Microbiol. 115, 307 312.

with the exclusion assay was found. The exclusion assay canbe regarded as preventative. Therefore we believe a highly15）Varma, P., Dinesh, K.R., Menon, K.K. and Biswas, R.,
2010, J. Food Sci. 75, 546 551.

adhesive LAB may be used for the prevention of candidiasis.
This study shows the possibility of prevention of candidiasis

16）Kinoshita, H., Uchida, H., Kawai, Y., Kawasaki, T.,
Wakahara, N., Matsuo, H., Watanabe, M., Kitazawa, H.,

using lactobacilli.

Ohnuma, S., Miura, K., Horii, A. and Saito, T., 2008a, J.
Appl. Microbiol. 104, 1667 1674.

Acknowledgements: The study was supported by a Grant-inAid for Scientific Research from the Japan Society for the17）Kinoshita, H., Wakahara, N., Watanabe, M., Kawasaki, T.,

Promotion of Science（JSPS）to H. Kinoshita（No. 19002318）.

Matsuo, H., Kawai, Y., Kitazawa, H., Ohnuma, S.,
Miura, K., Horii, A. and Saito, T., 2008b, Res. Microbiol.
159, 685 691.

REFERENCES

18）Cutler, J.E., 1991, Annu. Rev. Microbiol. 45, 187 218.

1）Beck-Sagué, C. and Jarvis, W.R., 1993, Secular trends in19）Casanova, M., Lopez-Ribot, J.L., Martinez, J.P. andthe epidemiology of nosocomial fungal infections in the

Sentandreu, R., 1992a, Infect. Immun. 60, 4898 4906.

United States, 1980 1990. National Nosocomial Infections20）Sherwood, J., Gow, N.A., Gooday, G.W., Gregory, D.W.
and Marshall, D., 1992, J. Med. Vet. Mycol. 30, 461

Surveillance System. J. Infect. Dis. 167, 1247 1251.
2）Guentzel, M.N. and Herrera, C., 1982, Infect. Immun. 35,

469.
21）Kinoshita, H., Uchida, H., Kawai, Y., Kitazawa, H.,

222 228.

Miura, K., Shiiba, K., Horii, A. and Saito, T., 2007, J.

3）Cole, G.T., Lynn, K.T., Seshan, K.R. and Pope, L.M.,

Appl. Microbiol. 102, 116 123.

1989, J. Med. Vet. Mycol. 27, 363 380.
4）de Repentigny, L., Phaneuf, M. and Mathieu, L.G., 1992,22）Winram, S.B. and Lottenberg, R., 1996, Microbiology.

Infect. Immun. 60, 4907 4914.

142, 2311 2320.

5）Cole, G.T., Halawa, A.A. and Anaissie, E.J., 1996. Clin.

23）DʼCosta, S.S. and Boyle, M.D., 2000, Methods. 21, 165

Infect. Dis. 22, S73 88.

177.

6）Casanova, M., Lopez-Ribot, J.L., Monteagudo, C.,24）Pancholi, V. and Fischetti, V.A., 1992, J. Exp. Med. 176,

Llombart-Bosch, A., Sentandreu, R. and Martinez, J.P.,─ 12 ─

415 426.

Lactobacillus plantarum inhibits the adhesion of Candida albicans25）Jin, H., Song, Y.P., Boel, G., Kochar, J. and Pancholi, V.,36）Kinoshita, H., Imoto, S., Suda, Y., Ishida, M., Watanabe,M., Kawai, Y., Kkitazawa, H., Miura, K., Horii, A. and2005, J. Mol. Biol. 350, 27 41.

Saito, T., 2013, Anim. Sci. J. 84, 150 158.

26）Seifert, K.N., McArthur, W.P., Bleiweis, A.S. and Brady,

37）de Repentigny, L., Aumont, F., Bernard, K. and

L.J., 2003, Can. J. Microbiol. 49, 350 356.

Belhumeur, P., 2000, Infect. Immun. 68, 3172 3179.

27）Modun, B. and Williams, P., 1999, Infect. Immun. 67,

38）Hoffman, M.P. and Haidaris, C.G., 1993, Infect. Immun.

1086 1092.
28）Maeda, K., Nagata, H., Yamamoto, Y., Tanaka, M.,
Tanaka, J., Minamino, N. and Shizukuishi, S., 2004,

61, 1940 1949.
39）Ogasawara, A., Komaki, N., Akai, H., Hori, K.,
Watanabe, H., Watanabe, T., Mikami, T. and Matsumoto,Infect. Immun. 72, 1341 1348.

T., 2007, Biol. Pharm. Bull. 30, 284 286.

29）Sojar, H.T. and Genco, R.J., 2005, FEMS Immunol. Med.

40）Boris, S., Suárez, J.E., Vázquez, F. and Barbés, C., 1998,

Microbiol. 45, 25 30.

Infect. Immun. 66, 1985 1989.

30）
7XQLR6$2OG¿HOG1-$ODʼAldeen, D.A., Wooldridge,41）Coudeyras, S., Jugie, G., Vermerie, M. and Forestier, C.,

K.G. and Turner, D.P., 2010, BMC. Microbiol. 10, 280

2008, Infect. Dis. Obstet. Gynecol. 2008, 549640.

289.
31）Hurmalainen, V., Edelman, S., Antikainen, J., Baumann,42）Matsubara, V., Silva, E., Paula, C., Ishikawa, K. andNakamae, A., 2012, Oral Dis. 18, 260 264.

M., Lähteenmäki, K. and Korhonen, T.K., 2007,43）Villena, J., Salva, S., Agüero, G. and Alvarez, S., 2011,

Microbiology. 153, 1112 1122.

Microbiol. Immunol. 55, 434 445.

32）Antikainen, J., Kuparinen, V., Lähteenmäki, K. and44）Martinez, R.C., Seney, S.L., Summers, K.L., Nomizo, A.,

Korhonen, T.K., 2007, J. Bacteriol. 189, 4539 4543.

dee Martinis, E.C. and Reid, G., 2009, Microbiol.

33）Ramiah, K., van Reenen, C.A. and Dicks, L.M., 2008,

Immunol. 53, 487 495.

Res. Microbiol. 159, 470 475.
34）Sánchez, B., Schmitter, J.M. and Urdaci, M.C., 2009,45）Gil, N.F., Martinez, R.C.R., Gomes, B.C., Nomizo, A.
and de Martinis, E.C.P., 2010, Braz. J. Microbiol. 41, 6

Lett. Appl. Microbiol. 48, 618 622.
35）Castaldo, C., Vastano, V., Siciliano, R.A., Candela, M.,
Vici, M., Muscariello, L., Marasco, R. and Sacco, M.,

14.
46）Wagner, R.D., Johnson, S.J. and Tucker, D.R., 2012,
Open J. Med. Microbiol. 2, 54 64.

2009, Microb. Cell. Fact. 8, 14 23.

─ 13 ─

Hideki Kinoshita, Masamichi Watanabe, Keiji Igoshi, Mitsuharu Ishida, Yasushi Kawai,
Haruki Kitazawa, Koh Miura, Akira Horii and Tadao Saito

Lactobacillus plantarum LA 318はヒト大腸ムチンへのCandida albicans ATCC 26555の付着を阻害する
木下英樹・渡辺真通・井越敬司・石田光晴・川井 泰
北澤春樹・三浦 康・堀井 明・齋藤忠夫
要約Candida albicans はカンジダ症を引き起こす日和見感染菌である．C. albicans は培養条件により酵母型と仮性菌
糸型の二形成を示す．本試験ではヒト大腸ムチン（HCM）に高い付着性を示す Lactobacillus plantarum LA 318を用いて C. albicans ATCC 26555の付着阻害の可能性を検討した．SDS-PAGE により C. albicans ATCC 26555の菌体表層タンパク質中にグリセルアルデヒド 3 リン酸脱水素酵素（GAPDH）の発現を確認したが，その発現量は非常に少なかった．このことから GAPDH は，C. albicans ATCC 26555のメインとなるアドヘシンではない可能性が考えられた．また，C. albicans ATCC 26555は酵母型，仮性菌糸型ともに HCM に対し菌数依存的な付着性を示した．カンジ
ダの付着阻害試験は，競合，排除，置換試験の３つのパターンで行った．28 ℃培養のカンジダ菌（酵母型）では，
8

1×10 cells/mL の乳酸菌の競合阻害以外は10 ~ 20 % の付着阻害が見られた（p<0.05）．最もカンジダ菌の付着阻害9が見られたのは1×10 cells/mL の乳酸菌を用いた排除試験だった．37 ℃培養のカンジダ菌（仮性菌糸型）では，18×10 cells/mL の乳酸菌を用いた試験においては，競合，排除，置換試験のいずれにおいても僅かな阻害しか示さ9なかったが，1×10 cells/mL の乳酸菌では排除試験において21.5% のカンジダ菌の付着の減少が見られた（p<0.05）．
これらの結果より乳酸菌を用いた C. albicans の感染阻害の可能性が示された．

─ 14 ─

東海大農紀要 36：15−21（2017）
Exploring Academic English for the New Curriculum at Tokai University

Exploring Academic English for the New Curriculumat Tokai University
Miki Rodriguez-Yagi・Daniel Minor（Received 29 November 2016; accepted 11 January 2017）

Summary
In this paper the authors outline the steps taken to prepare for the development of a new class to be called “Academic
English” for the upcoming curriculum change at Tokai University. The definition of Academic English for the School of
Agriculture, what exactly is meant by Academic English in the context of the Tokai University School of Agriculture, ispresented and methods are suggested. A review of pertinent literature on related topics and methods was done. Based on the¿QGLQJVLQWKHOLWHUDWXUHUHYLHZDSLORWFODVVZDVFRQGXFWHGLQWKHIDOOVHPHVWHURI2015. The outcome of the pilot class was
positive, but it was also noted that the self-selected participants of the pilot class were more motivated and better in English
than the average student in the School of Agriculture. This made clear the need for more thought on the development ofmaterials for the new class. The inclusion of Academic English in the new curriculum is an opportunity to start a tailor madeEnglish educational program at the School of Agriculture with the cooperation of academic teachers. This change is hoped tolead to better opinions toward studying English, better understanding of why students should study English, and better
performance in their study of English.

was a questionnaire to the teachers in the agriculture

Introduction

department. A review of the relevant literature was conducted,
and subsequently a pilot class was conducted in the fall

BackgroundAt the Tokai University School of Agriculture, a newsemester of 2015. The outcome of the pilot class was positive,

English curriculum will start in 2018. Eight credits of English

but this was tempered by the fact that the self-selectedclasses are required at present and that will stay the same. As

participants of the pilot class were generally higher in bothfor the subjects, there will be two new ones, Academic

motivation and English level than the average student in the

English and Global Skills. These will replace the current

School of Agriculture. These differences in motivation andReading and Writing 2 and Listening and Speaking 2. These

English ability between the pilot class and the general student

changes will produce a new English curriculum which

population made clear the need for further consideration of

consists of four subjects, namely; Listening and Speaking,the choice and adaptation of materials for the new class.

Reading and Writing, Global Skills, and Academic English.
Consistent with the current class offerings, Academic English

Questionnaire to Academic Teachersand Global Skills will both be two credits and class will meet

The Tokai University Educational Council presented atwice a week for 15 weeks. The definition of Academic

QHZVXEMHFW$FDGHPLF(QJOLVKKRZHYHUWKHGH¿QLWLRQZDVEnglish provided by the Tokai University Educational Council

not provided in detail. No New Paragraph The subject was

was lacking in detail, but the council suggested that English

described in broad strokes with instructions to cooperate andlanguage instructors cooperate with instructors of the

discuss the details with the School of Agriculture. As part ofacademic subjects in the development and teaching of thethe development of the new Academic English class, theAcademic English classes. The first step in this cooperation

original plan was to have English teachers interview thefaculty in the School of Agriculture. However, due to various

Aso Liberal Arts Education Center

logistical considerations, information was collected by the─ 15 ─

Miki Rodriguez
Yagi・Daniel Minor
authors by means of a questionnaire given to the agriculture'H¿QLWLRQRI$FDGHPLF(QJOLVK

teachers. There are three departments within the School ofIn order to be able to develop a curriculum with an

Agriculture, Bio Science, Plant Science, and Animal Science.

Academic English class, one should be clear about what

In the present curriculum, each department has two classes

exactly is meant by “Academic English”. A review of relevant

titled “Reading Academic Papers in English.” All agriculturalliterature provided valuable insight into several important

students are expected to take these classes in which the focus

considerations. For example, it is important to consider theis to read academic papers written in English. In therelationship between language classes and content classes（1）.

questionnaire, the academic teachers were asked what types ofIn order to investigate what methods are currently employedmaterials were used in those classes. Among the teachers

by English teachers to teach English using content relevant to

responding to the questionnaire, sixteen said the students read

VWXGHQWVWKH¿HOGRI7HDFKLQJ(QJOLVKWR6SHDNHUVRI2WKHU

academic papers, ten said they read overseas textbooks, six

Languages was researched. It became clear that as Brinton etsaid they read general science books, four said they readal.（2）state, “The claim that language is most effectively

English textbooks for junior and senior high school students

learned in context is hardly a new or revolutionary one.”and university liberal arts level students. With regard to the

Immersion education started in the 1960ʼs in Canada（3）andquestion of what agriculture teachers want the students to

the United States, however, it has been found that learninglearn in Academic English, the responses were grammar,

content in a second language was taking place about 5000

translation, vocabulary, how to use a dictionary and web

years ago in the area which is modern-day Iraq. At that time,translation, and academic writing. When asked about the skills

it was the Sumerians teaching theology, botany and zoologythat are necessary for academic study besides Reading

to the conquering Akkadians using Sumerian（4, 5）. The

Academic Papers in English, the responses were oral

focus on learning the content while learning the target

communication, writing, and listening to lectures. As to thelanguage came naturally. Content-Based Instruction（CBI）,

question “How much can you cooperate with English teachers

Language across the Curriculum, Language for Specificin Academic English classes?”, seven indicated they would

Purposes, Content and Language Integrated Learning（CLIL）,

like to give advice to curriculum development and material

and Task-Based Language Teaching（TBLT）are the same inselection, six said it was difficult to cooperate, three

the sense that all combine language learning and content

responded that they would be able to help students as follow-

learning. However, there can be differences in the focus andup to Academic English, but no one wanted to participate in

the actual methods used（6, 7）.
For example, Immersion focuses on content and by

the teaching of Academic English classes.
From the questionnaire result it became clear that the

studying the content students learn a target language. Moreacademic teachers do not seem to be content with the present

specifically, immersion uses the learnersʼ second language

English education at the School of Agriculture, wondering（L2）as the medium of classroom instruction. In an

what the students learn in the first two years of university

immersion classroom, students study school subjects, such as

English classes. By the time students have completed thealgebra, biology, and history, in their L2. The main purpose of

required eight credits of English, the agriculture teachers

this method is to promote bilingualism. This incorporates the

expect the students to have mastered basic English grammar

development of learners' communicative competence orand vocabulary, and to be able to read basic science passages.

language proficiency in their L2 in addition to their first or

The faculty want to know why, after six years of junior andnative language（L1）. “Intensive exposure to the target

senior high school and then two years at university studying

language through natural communication with a native

English, students still have not mastered these basic English

speaker was considered essential, as was starting at a young

skills. One reason is that the students can graduate junior andage（2）.” Due to this fact, this method has most commonly

senior high school without learning English. The students thenbeen found in programs for primary or secondary school

expect the same to hold true at university. They feel thatstudents.
Content-based instruction（CBI）is a form of communicative

English is not necessary for study or for daily life. They are
QRWLQWHUHVWHGRUFRQ¿GHQWLQOHDUQLQJ(QJOLVKlanguage teaching. In CBI, language instruction is integrated
with academic content instruction. CBI implies the total
─ 16 ─

Exploring Academic English for the New Curriculum at Tokai University
integration of language learning and content learning（8）. TheEnglish can be defined as theme or topic-based language

intention behind CBI is to provide second-language learners

courses which seem most appropriate as the English teachers

instruction in content and language. In this context, content iswill not have much support by content teachers as thetaken to mean the use of subject matter as a vehicle for second

questionnaire results show.

or foreign language teaching/learning. In CBI, it is expectedthat learners acquire language together with content through

7RSLFVDQG0DWHULDOVRI$FDGHPLF(QJOLVK

the awareness-raising activities the teacher makes use of. For

An academic English class is not a class geared toward

this reason, the learner is expected to process language

reading academic papers in English class, therefore English

consciously as well as intuitively. In the case of CBI, teachers

teachers should not utilize the same methods such as reading

are required to familiarize themselves with difficult andacademic papers, American high school biology textbooks, or

unfamiliar content and often need to develop their own

general science books in English. English teachers are notcourses or choose and adapt materials that provide a basis forbiology or agriculture teachers. Therefore, they are not

CBI（9）.

responsible for teaching content. However, as intelligent,

As for CLIL the focus is on both a target language andresponsible global citizens, English teachers are interested incontent（10 12）. “The term ʻContent and Language Integrated

current topics related to agriculture, such as the water crisis,

learningʼ（CLIL）was adopted in 1994（Marsh, Maljers andvirtual water, ecological footprint, food mileage, GMO, andHartiala, 2001）within the European context to describe andorganic food. They have access to such information infurther design good practice as achieved in different types ofEnglish, and that is what they can use in the Academic

school environment where teaching and learning take place inEnglish classes. By studying this information, the students

an additional language（13）.” This methodology focuses onwill have more knowledge of the topic while learning the

supporting language within context. Language teachers arelanguage used to discuss it. English teachers are not requiredteaching through a language rather than in another language.

to teach the content. English teachers should be able to rely on

This serves to assist learners in acquiring both language andacademic teachers to explain in detail any content or concepts

content as they learn a school subject（12）. Both CBI andrelated to the subject matter that they are not familiar with.

CLIL require the learners to actively participate, and share theEnglish teachers can encourage students to consult withgoal of moving toward learner autonomy.

academic teachers or English classes can have guest speakers.

As Academic English will be taught by English teachers,
the focus is more on language. Therefore, CBI would seem to

However, it should be noted that the questionnaire results do
not show much eagerness on the academic teachersʼ side.

be an appropriate model for Academic English. There are

The material used for CBI should be authentic（2）.

three types of CBI. One is Theme-Based Language

However, considering the English level of the students,

Instruction, another is Sheltered Content Instruction, and theEnglish teachers cannot use the material as it is. Brinton et al.

third is Adjunct Language Instruction（2）. Sheltered Courses（2）bring up the need to adapt authentic materials. In

are content courses taught in the second language to a group“Reading Academic Papers in English” classes, academicof learners by a content area specialist. In the adjunct model,teachers are expected to use authentic materials. Therefore, it

students are enrolled concurrently in two courses, a language

is a good stepping stone to use adapted authentic material incourse and a content course. These two courses share theAcademic English to work as a bridge to Reading Academic

content base and complement each other in terms of mutually

Papers in English.

coordinated assignments. The use of theme or topic-based
language courses is one way to increase the use of subject

6NLOOVRI$FDGHPLF(QJOLVK

matter content in language classes. In such courses theChoosing good tasks is the key to the success of CBI.

language class is structured around topics or themes, with the

Stryker（8）introduces sheltering activities, such as lectures,

topics forming the backbone of the course curriculum. The

small- and large-group activities, student projects andlanguage teacher provides the basis for language analysis andpresentations, invited speakers, and the use of a variety ofpractice through the content material presented（2）. In theaudio-visual aids. Academic teachers expressed in thecase of the Tokai University School of Agriculture, Academic

questionnaire that searching the internet in English is a

─ 17 ─

Miki Rodriguez
Yagi・Daniel Minor
necessary skill. Also, not only reading, but also writing,

explicit, deductive manner.” Therefore, although Academic

listening, and speaking are important. Critical thinking is the

English should be a topic-based language course and not abase of all these skills. The sheltering activities Stryker

grammar- or lexical- based language course, that does not

introduced above are similar to the skills academic teachers

mean the elimination of teaching grammar. When it isthink are important. The following are the kind of activities

necessary to focus on form, it should be done.

that can be employed:（1）Listening to mini lectures$3UHOLPLQDU\3LORW6WXG\（2）Reading（3）Internet search&ODVV（4）Oral presentation with power point, discussion

To get some insight on how the Academic English（5）Writing

suggested above would work, in the fall semester of 2015 a
small-scale pilot class was conducted. The class was in thefirst period on Tuesday, when the first-year Class 2 and the/DQJXDJHRI$FDGHPLF(QJOLVK
17 out of 21 academic teachers commented in thesecond-year Class 1GLGQRWKDYH(QJOLVKFODVVHVDVWKH¿UVW

questionnaire that they want students to have basic vocabulary

year Class 1 and the second-year Class 2 had English classes

and grammar. A command of grammar and basic vocabulary

DWWKDWWLPH)LUVWDÀ\HUZDVFUHDWHGWRLQWURGXFHVWXGHQWVWR

are necessary to read academic papers, but there is a concern

a pilot study for the Academic English class. The flyer was

about classes centered around grammar and vocabulary. The

KDQGHGWRDOOWKH¿UVW\HDU&ODVV2 and the second-year Class

students had such classes at junior and senior high school and1 students. There were about 250 students. The pilot class met

repeating the same type of lessons does not seem to be aonce a week for 9 weeks.

preferable method. Mohan（1）says, “Both in research and inclassroom practice it makes little sense to disconnect language

Participantslearning from learning about the world. There is no reason for

Eight students participated in the pilot class and on

research to be restricted to the study of language learning in

DYHUDJH¿YHRIWKHPSDUWLFLSDWHGHYHU\ZHHN7KHLUSODFHPHQW

isolation from content learning and from contexts ofEnglish scores ranged from 38 to 66 out of 100. One of themcommunication. There is no reason for the language

was a Masterʼs degree student. Four of the undergraduate

classroom to be restricted to teaching language for its own

students were in the top class.

sake.” In a class of German students studying English, Brinton
et al.（2）chose a theme-based model stating that, “The0DWHULDO

theme-based model was selected as an effective way of

The material from New Internationalist Easier English

providing a meaningful context for refining the English

Wiki（14）was used. There were such topics as food waste,

language skills of advanced students. Since the German

cowspiracy, seeds of change.

students had been exposed extensively to English grammar
and vocabulary in their high school English classes, aTeaching Method

curriculum designed around a grammatical syllabus would

As this was a pilot class conducted with self-selectedhave been very repetitious. In addition, the program designers

participants from among the students in the School of

hoped that the lagging motivation of advanced students could

Agriculture, reasonably high levels of motivation werebe revitalized in a theme-oriented course which capitalizes on

expected. The volunteers did display good motivation andwhat the students already know and fills in the gaps in ainterest in the topics. Even so, it was felt that there should notcontextualized manner.” This brings us back to “The claim

be too great a demand placed on students to work on the pilotthat language is most effectively learned in context is hardly aclass material outside of the class time. Materials were

new or revolutionary one（2）.” Stryker（8）says, “However,prepared ahead of time and distributed during the class.

many of us who, in our initial enthusiasm for CBI, minimized

Generally, a “packet” of material included various reprints ofor removed the formal study of grammar, found that our adultreading material, vocabulary and grammar exercises,

students wanted − and needed − to deal with grammar in ancomprehension questions, and discussion points relating to the─ 18 ─

Exploring Academic English for the New Curriculum at Tokai University
topic. While the readings were short, being only 2 to 6 pages

Table 3.

in length on average, there were often several related readings

Familiarity with content

for a single topic. Therefore, this material was of sufficient

Very familiar0

volume to allow the same packet to be used over a period of

Fairly familiar

13

several classes. The students were allowed, but not required,

Not so familiar27

to take the material home between class sessions to study at

Not familiar at all

2

Familiarity with content

Number of student responses

their leisure.
Since students were not required to do any outside

The level of the English seemed adequate, however, thepreparation, a portion of class time was devoted to reading

English level of the participants was quite high. Given the fact

and comprehending the material. As the material was gearedthat there will be students with lower English proficiency

toward non-native speakers of English, the grammar

taking Academic English, adaptation of this easier English to

structures were generally familiar to the students. Vocabulary

an even easier level will be necessary. Next, although theprovided a challenge, as many of the words used in theparticipants were agriculture students, they were not overly

material were specific to the topic. Although students werefamiliar with the topics. One of the possible reasons was thatslightly familiar with the topics, they had almost exclusively

the participants were mainly first year. However, there wasbeen exposed to the material in Japanese until the time of the

one graduate student who participated, and he also said thatpilot class.

WKH(QJOLVKZDVGLI¿FXOWDQGWKDWKHZDVQRWIDPLOLDUZLWKWKH

The class was very much teacher centered. Students

topic. It seems that what the students learn in academic

received English language input from both the instructor andclasses is not related to our everyday life or what is happening

the reading materials provided. Language output from thein the present world. The research is more particular,

students was almost entirely verbal as no written assignments

VFLHQWL¿FDQGDGYDQFHG

were given. The only writing was in the form of short answers
to some comprehension and discussion questions. While the&RQFOXVLRQlack of written output might be considered less than ideal foran academic English class, for the purposes of this pilot study
it was felt to be acceptable.

This paper describes the steps taken in the developmentof Academic English for the School of Agriculture. A review
of pertinent literature on related topics and methods was done.
Based on the findings in the literature review, Academic

4XHVWLRQQDLUH5HVXOWVA questionnaire was conducted after each class. There

English was defined as theme or topic-based language

were a total of 42 responses from students who participated incourses, methods and materials were determined, and a pilot

the pilot class. The following are the results.

class was conducted in the fall semester of 2015. The outcome

Table 1.

of the pilot class was positive. However, the need for more

Content of material

Content of material

Number of student responses

Very interesting40

Fairly interesting2

Not so interesting0

Not interesting at all0

thought on the development of materials for the new class wasmade clear.

6XJJHVWLRQVIRU
The students of the Academic English pilot class

Table 2.

'LI¿FXOW\RI(QJOLVK

Difficulty of English

voluntarily participated with a high level of attendance. That
Number of student responses

fact, in addition to the questionnaire results, make it evident

Very easy0that the students generally had positive attitude toward theQuite easy

3

Just right20

A little difficult

18

Very difficult1class. It should be noted, however, that four out of seven
undergraduate students were in the top class, therefore, thestudents who will be required to take this Academic English
FODVVZRXOGOLNHO\KDYHORZHU(QJOLVKSUR¿FLHQF\DQGEHOHVV─ 19 ─

Miki Rodriguez
Yagi・Daniel Minor
motivated. One thing that can be done to overcome this is to

語授業デザイン：学習空間づくりの教授法と実践，

adapt the pilot class English material to make it even easier.

大修館書店，東京

Another possibility is to increase cooperation between the（8）Stryker, S. B. and Leaver, B. L.（eds.）, 1997, Content-

English and academic teachers. It should not be that English

Based Instruction in Foreign Language Education:teachers teach English and then send the students into

Models and Methods, Georgetown University Press,

academic classes. Instead, the academic teachers should comeWashington, D. C.

in at an earlier stage and emphasize the necessity of English.

（9）Jack C. Richards and Theodore S. Rodgers, 1986,

The CBI approach to language learning, the blending ofApproaches and Methods in Language Teaching − Third

language and content, would help to motivate the students.

Edition, Cambridge University Press, Cambridge

The focus is placed on content and not on grammar or general（10）渡部良典，池田真，和泉伸一（共著），2011，ＣＬreading. This shift in focus makes it different from the English

ＩＬ内容言語統合型学習：上智大学外国語教育の新classes they had at junior and senior high school.

たなる挑戦

The idea of Academic English was imposed by Tokai

第１巻

原理と方法，上智大学出版，

東京

University Educational Council. It is, after all, a great（11）和泉伸一，池田真，渡部良典，（共著），2012，Ｃ

opportunity to start a tailor made English educational program

ＬＩＬ内容言語統合型学習：上智大学外国語教育のat the School of Agriculture with cooperation of academic

新たなる挑戦 第２巻 実践と応用，上智大学出版，

WHDFKHUV,WLVKRSHGWKDWWKHVWXGHQWVZLOO¿QDOO\PDVWHUWKH

東京（12）Dale, L. and Tanner, R., 2012, CLIL Activities: A

English skills that they will need in their academic classes.

resource for subject and language teachers, Cambridge
Handbooks for Language Teachers（Thornbury, S. ed.）,

5HIHUHQFHVCambridge University Press, Cambridge（1）Mohan, B. A., 1986, Language and Content, Second（13）Coyle, D., Hood, P., and Marsh, D., 2010, CLIL:

Language Professional Library（Savignon, S. J. ed.）,Content and Language Integrated Learning, Cambridge

Addison-Wesley Publishing Company, Reading

University Press, Cambridge（2）Brinton, D. M., Snow, M. A., and Wesche, M. 2003,（14）http://eewiki.newint.org/index.php/Main_Page

Content-Based Second Language Instruction, Michigan
Classics Edition, The University of Michigan Press,
Michigan, pp. 1 10.
（3）Baker, C.（2011）. Foundations of Bilingual Education
and Bilingualism 5 ed. Clevedon: Multilingual Matters.
th（4）Mehisto, P., Marsh, D., and Frigols, M. J., 2008,
Uncovering CLIL: Content and Language Integrated
Learning in Bilingual and Multilingual Education,
Macmillan Books for Teachers, Macmillan Education,
London, pp.9 23.
（5）笹島茂（編者）Mehisto, P., Marsh, D., Frigols, M. J.,
斎藤早苗，池田真，鈴木誠，佐藤ひな子，Yassin, S.
M., and Hemmi, C.（著）2011，ＣＬＩＬ新しい発想の授業−理科や歴史を外国語で教える !? −，三修社，東京
（6）全国英語教育学会第40回研究大会記念特別誌編集委
員会，2014，英語教育学の今−理論と実践の統合−，
全国英語教育学会（7）山岸信義，高橋貞雄，鈴木政浩（編集）大学英語教育学会（監修）2011，英語教育学大系 第11巻 英─ 20 ─

Exploring Academic English for the New Curriculum at Tokai University

東海大学新カリキュラムのアカデミック英語の開発に向けて
ロドリゲス八木美樹
Daniel Minor
抄録
東海大学農学部では2018年度から新英語カリキュラムが始まり，アカデミック英語が新設される．まず，農学部
教職員に対して行ったアンケート調査の報告として現カリキュラムの外書講読で扱っている教材，アカデミック英語で学生に学習してほしい内容，外書講読以外に専門分野の学習に必要な英語スキルが挙げられた．又，アカデミック英語への協力は限定的になることと現英語カリキュラムには不満をもっていることが明らかになった．次に，
内容と言語の両方に焦点をあてた様々な教授法を紹介する．そして，それを基に農学部でのアカデミック英語の教授法を提案する．具体的には農学分野のトピックの一般的なリーディング，短い英語での講義のリスニング，イン
ターネットを使っての調査，パワーポイントを使った口頭発表，議論，まとめのライティングである．高校までの英語教育の繰り返しにならず，学生の興味・関心を引くように内容中心の授業になるが必要に応じて文法や語彙の指導も行う．2015年度秋学期にはアカデミック英語の試行授業を行った．その参加者，教材，教授法，参加者に対して行ったアンケート調査を報告する．希望者に対するものであったので当然，英語力，意欲はもともと高いが，結果を見るとアカデミック英語に対して殆どが肯定的な感想をもっていた．最後に2018年度開始に向けた提案と改善案として，実際は試行授業の参加者より英語力も意欲も低いので，教材の内容は変えずに英語を更に平易にすることと農学部教員が専門の勉強には英語が必要であることを早い段階で示す必要性を挙げている．英語教員と農学部教員との連携により学生の英語力向上が可能になると考える．

─ 21 ─

東海大農紀要 36：23−36（2017）
5HFRPPHQGHG0RGL¿FDWLRQVWRWKH.DPEDUD/RFXVRI&RQWURO6FDOH

5HFRPPHQGHG0RGL¿FDWLRQVWRWKH.DPEDUD/RFXVof Control Scale
Michael J. Rupp
（Received 29 November 2016; accepted 10 January 2017）

SummaryThis paper reports on the results of a mixed-methods approach towards the evaluation of a psychometric instrument
commonly used in the Japanese research context for measuring the construct known as locus of control（LoC）, namely the
Kambara Locus of Control Scale（K-LoC）. Kambara（1987）created this 43-item scale in order to measure LoC in Japanesehigh school students. With the goal of verifying whether the K-LoC actually measures what it purports to measure, the author
FRQGXFWHGDFRQ¿UPDWRU\IDFWRUDQDO\VLV（CFA）with an a priori set of data collected for this purpose. The dimensionality ofthe scores produced by the instrument were found to not be structurally valid, indicating unsatisfactory model fit for theoriginal conception of the instrument（Rupp, 2016a）. Based on this outcome, the author subsequently conducted a focus
group study in order to provide a qualitative perspective from the Japanese university student population with which to further
inform the results of the CFA（Rupp, 2016b）. The results of the focus group study revealed numerous potential problems withthe scale such as issues with the Likert scale, item content and survey length. In order to further explore the scale, the author
decided to conduct an exploratory factor analysis（EFA）which added further explanatory power to the previous two studies,particularly with regard to item factor loadings（Rupp, in press）. Taking into account the previous research on the K-LoC, thispaper, applying a sequential explanatory mixed-methods approach（Cresswell, 2014）, represents the combined analysis of the
EFA and focus group studies, with the goal of elucidating changes which are recommended be made to the K-LoC in order to
UHPHG\WKHGH¿FLHQFLHVRXWOLQHGLQWKHSUHYLRXVVWXGLHVThe Locus of Control Construct

Language Learning and Locus of Control

This study is situated in a larger research context ofGhonsooly and Moharer（2012）, conducted a Persian

attempts at measuring learner autonomy through notionallyversion of the Duttweiler（1984）LoC（5-point Likert Scale）

related constructs, such as attribution theory（e.g. Heider,

to investigate the correlation between I-LoC and translation

1958; Kelley, 1967; McLeod, 2010）, self-efficacy theory

studentsʼ achievement. Using statistical analysis on data

（Bandura, 1977）, self-determination theory（Deci & Ryan,gathered from university seniors in the English translation

1985）and locus of control theory（Rotter, 1966）. The LoC

department（N=151）, they found a significant positive

construct refers to a unidimensional continuum, ranging from

relationship, which was further backed up by results from ainternal to external, for which people are supposed to fallqualitative interview phase of the study. In another study

upon to the degree to which they believe that outcomes in（Ghonsooly & Elahi, 2010）, using the 28-item（Duttweiler,

their lives are controlled by internal factors（I-LoC）or1984）, five-point Likert scale, a significant positive

external factors（E-LoC）. Oxford（2003, 2008）posits that

relationship was found between I-LoC and general English

having a higher degree of I-LoC corresponds to possessing

achievement among university students（N=240）, as well as

higher degrees of agency and self-efficacy, which are key¿QGLQJGLIIHUHQFHVLQ,/R&OHYHOVDPRQJJURXSVRIVWXGHQWV

components of learner autonomy.

EDVHGRQWKHLUPDMRU¿HOGVRIVWXG\（humanities, sciences, andengineering）. Studying the relationship between LoC and L2

Aso Liberal Arts Education Centerreading and writing achievement with university English
─ 23 ─

Michael J. Rupp
major students in Iran（N=136）, using the Internal Control

studies conducted in Japanese on a Japanese high school

Index（ICI）created by Duttweiler（1984）, Ghonsooly andpopulation, both the K-LoC18 and K-LoC43 were found to

Shirvan（2011）found a positive correlation between I-LoC

SURGXFHVFRUHVZKLFKGLGQRWVXSSRUWPRGHO¿WDFFRUGLQJWR

and higher achievement in reading and writing. They conclude

CFA（Rupp, 2016a）analysis. This result was followed by athat teachers can empower students by applying attribution

focus group study involving Japanese university students totheory through reattribution training to help students view

analyze the K-LoC43（Rupp, 2016b）the outcomes of which

their failures as being due to controllable factors such as

highlighted many potential problems with the K-LoC43.

effort, thus moving them from E-LoC orientations to I-LoC

These outcomes are analyzed below in light of a subsequentorientations, and thus enabling students “to become

EFA（Rupp, in press）conducted by the author. This combined

autonomous L2 readers or L2 writers”（p. 238）.

analysis leads to a summary of changes which are recommended

A study conducted in Taiwan（Chang & Ho, 2009）using
Crandallʼs（Crandall, Crandall, & Katkovsky, 1965）I-E LoC

to be made to the K-LoC for future use in the Japanese
research context.

instrument with university students not majoring in English
found a positive correlation between I-LoC and comprehension

Summary of Focus Group Outcomes

DVZHOODVVHOIHI¿FDF\XVLQJDZHEEDVHGLQWHUDFWLYHLQVWUXFWLRQDO
system with two types of control: learner and program. The

This section will summarize the themes which arose inhighest scores on the test were received by students who had

the focus group discussions（Rupp, 2016b）about theboth a high I-LoC orientation, as well as those who learned

K-LoC43. Appendix A shows a translation of the K-LoC43

from the learner-control version of the learning environment.

along with the tabulation of votes by focus group members on

In recent research exploring the relationship between

item appropriateness for the university level. This tabulation

learner autonomy and the language learning locus of control

was conducted as the K-LoC43 was originally designed forin multilinguals, Peek（2016）used data from an online

KLJKVFKRROVWXGHQWVLQRUGHUWR¿QGLWHPVZKLFKZRXOGQHHG

language learning beliefs survey（N=842）
WRGH¿QHODQJXDJH

to be modified or eliminated for university student

learning experience as it related to the participantsʼ

measurement studies. The general feedback on the K-LoC43

multilingual capacities. Using statistical analyses, they foundfrom the 27 focus group members is found in Appendix B andDVLJQL¿FDQWSRVLWLYHFRUUHODWLRQEHWZHHQPXOWLOLQJXDOLVPDQG

WKHLWHPVSHFL¿FFRPPHQWVDUHIRXQGLQ$SSHQGL[&higher degrees of I-LoC in a domain specific study of

Individual survey general criticisms and comments

language learning of locus of control（LLLOC）
6SHFL¿FDOO\

included criticisms about the length of the survey, occasional

they found that language learners might be more autonomous

XQQDWXUDO-DSDQHVHODQJXDJHXVDJHGLI¿FXOWLWHPFRQWHQWDQ

learners when they subscribe to beliefs that align with a more

unexpected Likert scale ordering, an overly rough scale range,internal LLLOC.

age/life-stage inappropriate items and item similarity. There
were also positive comments including praise of a unique andthought provoking items which surprised some students and

The Kambara Locus of Control Scale

FDXVHGRWKHUVWRHQJDJHLQGHHSVHOIUHÀHFWLRQThe K-LoC（see Appendix A）emerged as a prominent

Regarding the numerous complaints about survey lengthscale used in the Japanese research context in a wide range ofand repetitious nature of some items, it should be noted thatdomains over the last 30 years, including employee

the original Kambara LoC Scale（1982）was only 18 items,

psychology（Fushimi, 2011）, developmental psychology

referred to here as the K-LoC18. Kambara expanded the scale（Kanda, 2006）, education（Kambara, 1987）and English

in order to focus more on items related to high school life.

language achievement（Hosaka, 2007）. The K-LoC was

While the students experienced these kinds of questions as

originally developed in a short（K-LoC18）18-item form

redundant, there is in fact a measurement rationale for（Kambara, Higuchi, & Kiyomizu, 1982）which was latermeasuring a construct from both a positive and negative frame

expanded（K-LoC43）to 43 items（Kambara, 1987）, and it is

as a means of addressing response set bias and issues of item

the K-LoC43 which is the scale which was found to be

skew. Nevertheless, based on the studentsʼ comments in thispredominantly used in Japan. Based on the authorʼs previous

study, it appears that such a shorter scale would be preferable─ 24 ─

5HFRPPHQGHG0RGL¿FDWLRQVWRWKH.DPEDUD/RFXVRI&RQWURO6FDOH
or that more care is needed in constructing the negative andSummary of EFA Results for K-LoC43
and K-LoC18

positive items so that they are not merely inverse statements.
This is supported by the results of the EFA as will be
discussed further below. The reduced implementation time

The EFAs used data collected from 1125 high school

required for an instrument with fewer items scale would be an

students from three local high schools, two private and one

DGGLWLRQDOEHQH¿W

public, from all three grade levels with 57% male and 43%

A common complaint was that it was felt to be impossible

female participants. The data was analyzed using IBM/to form an opinion about certain items, often due to theStatistical package for the Social Sciences（SPSS）software

content being perceived as more appropriate to an older, more

Version 16.0. Table 1 shows the two-component extraction

established working adult. Thus many students were forced to（Rupp, in press）for the K-LoC43.

choose either 2 or 3（on a 4-point Likert scale, corresponding
to a “slightly disagree” or “slightly agree” answer）even
though the real answer would have been a 3RQD¿YHSRLQWTable 1.

Two Component Extraction on K-LoC43

Likert scale（corresponding to a “neither agree nor disagree”

Pattern Matrix for 2 Component Extraction on K-LoC43
Component

answer）. This desire for a more refined Likert scale was1reiterated by the fact that many students expressed an explicit
Item13（I）
Item02（I）

0.685
0.661

Item19（I）

0.627

Item38（I）

0.627

Item21（I）

0.601

important to force a choice, rather than leaving an easy middle

Item24（I）

0.570

answer which can be chosen with less serious consideration.

Item42（I）

0.563

In this case more nuanced results might be possible with a

Item39（I）

0.559

6-point scale.

Item10（I）

0.558

There were a few students who indicated that in their

Item34（I）

0.506

experience Likert scales generally start from 1 being a

Item11（I）

0.499

positive answer to 4（or 5）being a negative answer, although

Item17（I）

0.456

Item03（I）

0.414

desire for a middle choice, such as would be available on a
5-point Likert scale, due to the fact that they felt they could
not give accurate answers when there was truly no preference.
This can be a tricky issue as some researchers feel it is

this scale used the original Kambara（1987）scale going fromnegative to positive. In other words, many students felt that it
was more common for the positive response to begin on the2

Item23（I）
Item35（I）
Item04（I）

left, and the negative part of the scale to be on the right.

Item12（I）

Though not a universal sentiment, students who pointed this

Item14（I）

out appeared to feel quite strongly about it.

Item30（I）

Another common complaint was that the intentions

Item29（I）

and/or assumptions underlying some of the questions wereItem43（E）

unclear, causing frustration and test fatigue. Regarding the

Item06（E）

0.585

Japanese, although some students indicated there were no

Item15（E）

0.549

problems with the Japanese, other felt that some items should
be reworded, often by making the question sound less extremeby adding an “I think...” before the question. There were also

Item16（E）

0.522

Item27（E）

0.502

Item09（E）

0.500

Item26（E）

0.473

a number of students who expressed frustration at not

Item33（E）

0.452

understanding the goals of the survey and how it could relate

Item01（E）

0.449

to English education. Other students found questions about

Item20（E）

0.434

making friends with the opposite sex to have an unnecessary

Item07（E）

0.430

emphasis on gender.

Item05（E）

0.430

─ 25 ─

Michael J. Rupp
Item18（E）

-0.4

0.427

Item08（E）

0.426

Item36（E）

0.422

Item22（E）

0.421

Analysis of K-LoC43 based on EFA and Focus
Group Outcomes

Item28（E）

In looking at the items of the K-LoC43, it is important to

Item31（E）

note the large number of items which failed to load in a two-

Item37（E）

factor EFA extraction. This was not the case with the

Item41（E）

K-LoC18 where all items loaded at the .40 threshold, with the

Item32（E）

exception of Item 18（Rupp, in press）. Thus these non-

Item25（E）
Item40（I）

loading items should be foremost in consideration for

Extraction Method: Principal Component Analysis.
Rotation Method: Oblimin with Kaiser Normalization.
Rotation converged in 5 iterations.
（E）denotes E-LoC items and（I）denotes I-LoC items
according to original conception for the instrument.

elimination from future revisions of the K-LoC43. The 13
items which failed to load in the two-factor EFA extraction
include the following: I-LoC Items 04, 12, 14, 23, 29, 30 and
35; and E-LoC Items 28, 31, 32, 37, 41, and 43.
Beginning with the I-LoC items which failed to load,

For comparison, the two-component EFA on the K-LoC18

Item 04（I decide my own life）received no comments fromis show in Table 2. It can be seen that nearly all of the items

the students, one of only six items to be thusly ignored by theload according to their respective constructs, whereas in thestudents. This may be due to the fact that it is too obvious and

K-LoC43, numerous items failed to load.

for students to comment upon; perhaps even laboring under acertain level of cliché or platitude. Also, as an I-LoC item, it

Table 2. Two Component Pattern Matrix K-LoC18

did not engender the level of indignation, or perhaps protest,

Pattern Matrix on 2 Component Extraction K-LoC18

that the E-LoC items tended to. Item 12（Usually, things turn

Component
1
Item13（I）
Item11（I）

0.689
0.684

out better if I make my own decisions）received only one

2comment, “[t]his is a mediocre item, because it is important tolisten to othersʼ opinions.”7KLVFRXOGEHVHHQDVDUHÀHFWLRQRI

Item02（I）

0.681

the traditional group-oriented nature of Japanese society

Item03（I）

0.571（Benedict, 1967; Doi, 1973）wherein conventional members

Item14（I）

0.525

of society tend to look askance at individuals who overtly

Item10（I）

0.513

express their individuality. Item 14（I will be able to live my

Item17（I）

0.502

entire life as I plan to）received more comments, with “Iʼm

Item12（I）

0.465

Item04（I）

0.416

still only a university student” being the most sobering one
among them. It is true that while many young people have

Item06（E）

0.659

Item15（E）

0.658

Item05（E）

0.557

bubble economy and the troubling and competitive job market

Item09（E）

0.549

which no longer assumes a guarantee of lifetime employment,

Item16（E）

0.515

they also have anxiety about the future, and cannot necessarily

Item01（E）

0.513

buy into the idea that life is going to be easy for them.

Item07（E）

0.453

Anecdotally, many young people now complain that the future

Item08（E）
Item18（E）

0.411

of the pension system and higher taxes due to the aging

high hopes for the future, especially in light of the end of thesociety, combined with a lack of clear support for young

Extraction Method: Principal Component Analysis.
Rotation Method: Oblimin with Kaiser Normalization.
Rotation converged in 5 iterations.
（E）denotes E-LoC items and（I）denotes I-LoC items
according to original conception for the instrument.

families, leaves them feeling with unease about their future
lives in Japanese society. Item 23（It is easy to get a goodscore on tests if I prepare for lessons and review afterwards）
received dismissive remarks such as “[n]o kidding” and “[p]reparation, review and understanding are all different … [i]f

─ 26 ─

5HFRPPHQGHG0RGL¿FDWLRQVWRWKH.DPEDUD/RFXVRI&RQWURO6FDOH
you donʼt understand, all of it would be meaningless.” These

comment, “[t]his item is mediocre because we should not bekinds of comments underscore the importance of not having

so insipid.” This comment gives more support to the idea that

items in the instrument which are expressed in a facilephrases which could be interpreted as invoking a negative

manner. The wording and concept should be subtler to avoid

sense of person or self should be reconsidered. Item 41（Mythese kinds of negative reactions from the subjects. Item 29（Ifgrades depend on the teacher）received four comments froma class in school is boring, it is because you are not interested

the students, “[i]t may be so, but I donʼt think we can ask suchin that subject）received a comment stating “[n]ot a good item

a question,” “[t]his is common sense,” “[i]f there needs to bebecause, havenʼt university students come to study what theysome improvement, I think it is necessary,” and “[e]ven if you

wanted to study?” This makes the case for this item being

say this, nothing can be done about it.” These comments again

targeted at high school students, although it could be argued

show that the students tend to dislike items which are obviousthat even university students have to take classes as a part ofor have no apparent purpose, or perhaps in this case, items

their major, which they might not be interested in. Item 30（Iwhich carry a taboo connotation, for example, here being thealways decide what Iʼm going to do）was criticized in its only

idea that grades are more less objective than society expects

comment, “[t]his is rather obvious, isnʼt it?” It would appear

WKH\VKRXOGEH7KH¿QDOLWHPZKLFKIDLOHGWRORDGZDV,WHP

that comments which are too obvious do not fare well in the

43（,RIWHQ¿QGP\VHOIGRLQJWKLQJVWKDW,GRQʼt like to do）,

EFA factor loadings. This item could be considered a truism,

which was discussed at the beginning of this section as it wasas it is perhaps impossible to do something without deciding

the item with the highest number of NA votes.

to do it, regardless of the underlying factors for that decision.

With regards to the tabulated votes from the studentsʼ

7KH¿QDOQRQORDGLQJ,/R&LWHPZDV,WHP35（If necessary, I

individual surveys in Appendix A, upon comparing the top

can suppress my desires at any time）, which had the negative

items voted as appropriate（AP）, and the top items voted

comment, “[t]his is just common sense,” and which could be

most inappropriate（NA）, with the EFA factor loadings in the

categorized as another of the overly obvious items.

K-LoC43 two-factor extraction from Table 1, it became

Continuing with the E-LoC items which failed to load inevident that the items which received the highest numbers of

the K-LoC43 two-factor EFA, Item 28（When you takeAP votes tended to be items measuring I-LoC, and conversely,

actions, it is more often the case that others have suggested

the items with the highest numbers of NA votes were thosethem to you rather than you acting upon your own desires）

measuring E-LoC. This was regardless of the factor loadings,

received just one student comment, “I always act based on myand many unpopular items loaded strongly, while someown ideas.” This comment could also be seen as categorizing

popular items failed to load. This may be due to the fact thatthe item as being too obvious or overly simplistic in its

the I-LoC items express positive sounding sentiments, suchconception. This is another item which might have been betteras: “If I work hard;” “If I try hard;” “I decide my own life;” “If

if it had been rephrased in a subtler manner. Most people, it

I am kind;” “make my own decisions;” “try my best;” et cetera.

would seem, would tend not to think of themselves as puppets

In the case of E-LoC items, the sentiments expressed arein the hands of others, but rather acting from a sense of free

typically those of a more negative world view, including

will when they take actions. The next E-LoC item whichphrases such as: “determined by fate;” “depends on thefailed to load was Item 31（In your case, when it comes to test

situation;” “others have suggested;” “we canʼt change;”

UHVXOWVWKH\DUHRIWHQLQÀXHQFHGE\\RXUSK\VLFDOFRQGLWLRQ“depends on luck;” “it is hard for me;” “doing things I donʼtor other random events）.

like to do;” “life is like gambling;” “I often do impulsive

This item was already discussed

earlier in the section in conjunction with its appearing in thethings;” et cetera. The students tended to dislike answering

top 12 items voted NA. The next non-loading item was Item

such questions, even on a Likert scale where they could

32（It is hard for me to do things as I have planned）, whichstrongly disagree with them, perhaps because such questions

was another one of the rare items to receive no comments. It

were experienced as attacking their character. Numerous

is hard to account for this, but it could be speculated that thisstudents also registered complaints about the premise of aitem taps into a personal discipline construct rather than anumber of E-LoC items, such as “I donʼt like luck” about Item

LoC construct. Item 37（My actions tend to end up going

36, “[w]e should not be so insipid” about Item 37, “[n]ot aalong with the flow of circumstances）received one student

good item because we should know how to make efforts”

─ 27 ─

Michael J. Rupp
about Item 33, “I donʼt like chance” about Item 31, “I hate the

It is the authorʼs opinion, based on the evidence, that at a

idea of fate deciding things” about Item 15, et cetera.

very minimum, given 1）the multitude of criticisms of the

Nevertheless, according to the factor loadings, the number oflength of the K-LoC43 2）it having failed the CFA test forvotes tabulated, both for AP and NA, is not correlated with themodel plausibility 3）the shorter K-LoC18 two-factor EFA

loading strength, and therefore, when considering a revision

showed much better factor loadings for the underlying LoC

of the instrument, it appears that a priority should be placed

constructs on an item per item basis, that the K-LoC18 beon the factor loadings, rather than the appropriateness voteused in place of the K-LoC43. This should be until such time

tabulations.

as a revised Kambara LoC scale which is even shorter, lessredundant, and more refined in terms of Likert scale
discrimination points can be tested and confirmed with a

5HFRPPHQGHG0RGL¿FDWLRQVIRU./R&

priori data from the Japanese population. The K-LoC18 also

Based on the feedback from the students, the changes

KDVWKHEHQH¿WRIEHLQJPRUHRIDJHQHUDO/R&LQVWUXPHQWDQG
thus useable in the university context with no explicit itemthat should be made to the instrument are as follows:

alterations required.
1

In terms of a future revision of the K-LoC43, a useful

7KHLQVWUXPHQWQHHGVWREHVKRUWHQHGSUHIHUDEO\WR¿YH
or six items per construct.

starting point would be to make a ten to twelve item scale

2

Repetitive items should be eliminated.

using the 5 or 6 items（taking into consideration redundancy）

3

Unclear and age inappropriate items should be avoided

from both I-LoC and E-LoC, which had the highest factor

The Likert scale should be more refined by adding

loadings（Table 1）. Table 3 shows the top ten highest factor

further points of discrimination to it, and the ordering ofloading items for both constructs.

4

Based on the principles of avoiding redundancy, athe scale should be reversed to conform with the what5

the students perceive to be the standard ordering.

modified K-LoC43 could be created using five items fromThe focus group suggestions for Japanese language

each construct, I-LoC and E-LoC. For example, Item 13 andchanges should be adopted where appropriate or

Item 6, while both being the highest loading items in the EFA,

necessary

should not be included together as they are saying the samething in opposite wording which, on the basis of responses

Table 3.

Top Ten Items based on EFA Loadings

Top Ten Items based on EFA Loadings
Item

I-LoC Items

Item

13 I

My happiness or sadness is determined by my
6E
own efforts.

My happiness and sadness are determined bychance.

2I

I can be a great success if I work hard.

My future is determined by fate or chance.

19 I

If I am careful about my actions, people will
16 E
trust me.

What happens does not depend on my efforts.

38 I

The results are far better when I prepare for
27 E
exams in advance.

Being able to maintain long friendships depends
on the external situation.

21 I

If I try hard, I will be able to get the job I want.

15 E

E-LoC Items

9E

Life is a gamble.

26 E

Getting into my first choice university depends
more on luck than ability.

24 I

I can make friends with the opposite sex if I try.

42 I

If I am kind to my friends, someday they will help
33 E
me.

We can't change how smart or stupid we are.

39 I

It is important to think about my actions in order
1E
to have good relationships with my friends.

It is best to just go with the flow.

10 I

It is useful（meaningful）to think about what I
20 E
want to be in the future.

My ability to make good friends depends on theclass or club's atmosphere.

34 I

Maintaining friendships depends on your effort.

What happens depends on the situation.

7E

─ 28 ─

5HFRPPHQGHG0RGL¿FDWLRQVWRWKH.DPEDUD/RFXVRI&RQWURO6FDOH
from the qualitative data, respondents do take positively. Also,

Research and Treatment, 2011. doi:10.1155/2011/731307.

such oppositely-worded items serve also to make the point of

Ghonsooly, B., & Elahi, M.（2010）. Validating locus of

the survey too obvious. Thus, for example, a short 10-Item

control questionnaire and examining its relation tosurvey could be creating using for I-LoC, Items 13, 19, 38, 21

general English（ge）achievement. The Journal ofand 42; and for E-LoC, Items 15, 16, 27, 33, and 1. The cost

, 2（1）, 117 143.
Teaching Language Skills（JTLS）

in terms of time for such a survey is minimal and the low

Ghonsooly, B., & Elahi, M.（2012）. Validating locus of

QXPEHURILWHPVPDNHVWKHSXUSRVHXQFOHDU\HWLWLVVXI¿FLHQW

control questionnaire and examining its relation toto measure the constructs in question. Furthermore,

General English（GE）achievement. Journal of Teaching
Language Skills, 2（1）, 117 143.

respondents are more likely to answer authentically with ashorter survey as they will not suffer from fatigue, or even theGhonsooly, B., & Moharer, A.（2012）. Examining the

expectation of fatigue which might develop as they are

relationship between translation studentsʼ locus of control

presented with 43 items to respond to. This survey should also

and their translation achievement. International Journal

use a 5 to 7SRLQW/LNHUWVFDOHIRUPRUHUH¿QHPHQWDQGPD\of Linguistics, 4（2）, 244 259.

require the scale directionality to be reconsidered from

Ghonsooly, B., & Shirvan, M. E.（2011）. On the relation of

positive（I agree/I think so）to negative（I disagree/I donʼtlocus of control and L2 reading and writing achievement.

think so）, though this is a matter of judgement, opinion andEnglish Language Teaching, 4（4）, 234.

convention, and should not matter if the scaling is clearly

Heider, F.（1958）. The psychology of interpersonal relations.
New York, NY: Wiley.

labeled.

Hosaka, Y.（2007）. An empirical study on streaming in
English class: With a focus on the relationship between

References

LoC and achievement. Ritsumeikan Studies in Language

Benedict, R.（1967）. The chrysanthemum and the sword:
Patterns of Japanese culture.%RVWRQ+RXJKWRQ0LIÀLQand Culture, 18（3）: 193 200.
Kambara, M., Higuchi, K., Kiyomizu, N.（1982）Locus of

Harcourt.

control no shakudo no sakusei to shinraisei, datousei no

Chang, M. M., & Ho, C. M.（2009）. Effects of locus of

kentou [Creating and Researching the Reliability andcontrol and learner-control on web-based language

Validity for a Locus of Control Scale]. Kyouiku

learning. Computer Assisted Language Learning, 22（3）,Shinrigaku Kenkyuu, 30（4）, 302 307.
Kambara, M.（1987）. Koukousei no locus of control ni kan

189 206.
Crandall, V. C., Crandall, V. J., & Katkovsky, W.（1965）. A

suru kenkyuu: kitai oyobi gakushuu douki to no kanren

children's social desirability questionnaire. Journal of[Locus of Control belief in high school students: On theConsulting Psychology, 29（1）, 27.

relation to expectancy and motives for learning]. Toukyou

Creswell, J. W.（2013）. Research design: Qualitative,
quantitative, and mixed methods approaches. London,

Daigaku Kyouiku Gakubu Kiyou. 26, 107 117.
Kanda, N.（2006）. Development change of perceived control
in children. Life Science Studies. Bunkyou University, 28,England: Sage.
Deci, E. L., & Ryan, R. M.（1985）. Intrinsic motivation andself-determination in human behaviour. New York, NY:

35 42, 2006 03.
Kelley, H. H.（1967）. Attribution theory in social psychology.
In D. Levine（Ed.）, Nebraska Symposium on Motivation

Plenum.

（Volume 15, pp. 192-238）. Lincoln, NE: University ofDoi, T.（1973）. The anatomy of dependence. Tokyo:

Nebraska Press.

Kodansha International.
Duttweiler, P. C.（1984）. The internal control index: A newly

McLeod, S. A.（2010）. Attribution Theory. Retrieved fromwww.simplypsychology.org/attribution-theory.html

developed measure of locus of control. Educational andOxford, R. L.（2003）. Toward a more systematic model of

Psychological Measurement, 44（2）, 209 221.
Fushimi, M.（2011）. The relationship between individual

L2 learner autonomy. In D. Palfreyman & R. C. Smith

personality traits（internality-externality）and（Eds.）, Learner autonomy across cultures: Language

psychological distress in employees in Japan. Depression

education perspectives（pp. 75 91）. Basingstoke,─ 29 ─

Michael J. Rupp
Rupp, M. J.（2016a）. Examining the Kambara Locus of

England: Palgrave Macmillan.

Control Scale. International Journal of Social andOxford, R. L.（2008）. Hero with a thousand faces: Learnerautonomy, learning strategies and learning tactics inCultural Studies, 9, 47 69.

independent language learning. In S. Hurd & T. Lewis

Rupp, M. J.（2016b）. Using focus groups for qualitative（Eds.）, Language learning strategies in independent

feedback on psychometric instruments. In G. Brooks, M.

. Bristol: Multilingual Matters.
settings（pp. 41 63）

Porter, D. Fujimoto, & D. Tatsuki（Eds.）, The 2015
PanSIG Journal（pp. 162 179）. Kobe, Japan: JALT.

Peek, R.（2016）. Exploring learner autonomy: language
learning locus of control in multilinguals. International

Rupp, M. J.（in press）. An exploratory factor analysis ofscores generated by the Kambara Locus of Control Scale

Journal of Multilingualism, 13（2）, 230 248.
Rotter, J. B.（1966）. Generalized expectancies for internalversus external control of reinforcement. Psychologicalin the Japanese high school population. Kumamoto
University Studies in Social and Cultural Science, 15.

Monographs: General and Applied, 80（1）, 1.

Appendices
Appendix A: Author's Translation of K-LoC43 with Focus Group Results
Kambara 43-Item LoC Scale（Translated by author; including the Item Appropriateness Focus Group Voting Results for appropriateness to the university context− AP= Appropriate NS= Not Sure, NA = Not Appropriate）
Note: I refers to items measuring internal LoC and E for those measuring external LoC. Students taking the surveys do not see
such information.
Item

I/E

AP

NS

NA

1

E

2

I

It is best to just go with the flow.

10

4

20

I can be a great success if I work hard.

5

1

20

3
4

I

Anyone will be able to understand me if I try my best to communicate with them.

5

1

19

I

I decide my own life.

6

1

10

5

E

My life is decided by fate.

7

9

14

6

E

My good fortune and misfortune are determined by chance.

6

5

16

7

E

What happens depends on the situation.

8

1

13

8

E

My friends can't understand me no matter how hard I try.

9

2

15

9

E

Life is like gambling.

4

8

21

10

I

It is useful（meaningful）to think about what I want to be in the future.

3

0

17

11

I

If I try hard, I can do anything on my own.

3

4

17

12

I

Usually, things turn out better if I make my own decisions.

8

0

20

13

I

My happiness or sadness will be determined by my own efforts.

4

0

14

14

I

I will be able to life my entire life as I plan to.

7

3

15

15

E

My future is determined by fate or chance.

4

6

12

16

E

What happens does not depend on my efforts.

5

7

18

17

I

I can be a friend with anyone if I try.

6

1

15

18

E

Your efforts and success are not really related to each other.

4

5

16

19

I

If I am careful about my actions, people will trust me.

6

2

16

20

E

My ability to make good friends depends on the class or club's atmosphere.

5

4

18

21

I

If I try hard, I will be able to get the job I want.

6

1

14

22

E

Marrying an ideal partner depends on fate or luck.

7

4

18

23

I

It is easy to get a good score on tests if I prepare for lessons and review afterwards.

7

1

16

─ 30 ─

5HFRPPHQGHG0RGL¿FDWLRQVWRWKH.DPEDUD/RFXVRI&RQWURO6FDOH
24

I

I can make friends with the opposite sex if I try.

6

2

14

25

E

I often do impulsive things without being aware of them.

9

2

11

26

E

Getting into my first choice university depends more on luck than ability.

7

7

17

27

E

Being able to maintain long friendships depends on the external situation（environment?）.

6

1

19

28

E

When you take actions, it is more often the case that others have suggested themrather than you acting upon your own desires.

5

1

17

29

I

If a class in school is boring, it is because you are not interested in that subject.

5

2

20

30

I

I always decide what I'm going to do.

4

0

13

31

E

In your case, when it comes to test results, they are often influenced by yourphysical condition or other random events.

6

6

18

32

E

It is hard for me to do things as I have planned.

5

1

15

33

E

We can't change how smart or stupid we are.

5

5

19

34

I

Maintaining friendships depends on your effort.

5

0

16

35

I

If necessary, I can suppress my desires at any time.

7

1

10

36

E

There is no use in thinking about how to make friends with members of the
opposite sex as such things are determined by fate/depend upon luck.

7

7

17

37

E

My actions tend to end up going along with the flow of circumstances.

6

1

21

38

I

The results are far better when I prepare for exams in advance.

3

0

20

I

It is important to think about my actions in order to have good relationships with
my friends.

4

1

17

40

I

Even if my friends have different ideas, I place a priority on my own actions.

7

1

18

41

E

My grades depend on the teacher.

3

3

18

42

I

If I am kind to my friends, someday they will help me.

6

2

0

43

E

I often find myself doing things that I don't like to do.

2

17

6

39

Appendix B: General Focus Group Feedback on K-LoC43
Student

Author’s Translation of Written Student Comments

1

Some of the Japanese seems a bit unnatural. It was difficult as these are questions that we usually never think
about.

2

The contents of many of these questions are a bit hard for people around 20 years old. For number 1, maybe
adding "I think.. " to it would be better.

3

Overall, there are many questions that are hard to answer. The Japanese used in the questions is sometimes
unnatural, so I would fix those areas.

4

Overall the content is rather deep. There were many questions that I didn't understand the intentions of.

5

There were many questions which didn't take into account various degrees or levels or gradations of answer.
Also, there were a few questions for which it was hard to understand what was really being asked.

6

I felt like these questions were trying to find out what kind of a human being I am!

7

It seems like a lot of the questions are similar, merely asking the same thing in the opposite fashion.

8

There were a lot of hard items. Numbers 24 and 36 seemed the same. There were many questions that seemed
the same.

9

I can't figure out what these questions have to do with English class. There were many questions that seemedsimilar.

10（no comment）

11

This was difficult.

12This was hard. It seemed like philosophy. There is no answer even after thinking for a long time. Many
questions were similar. For example, you could just have Item 4 or Item 5, and that would be sufficient.

13

The questions about "life" were really hard to answer. Some of the questions were good for self-reflection.

14

These made me think about life and my actions.

─ 31 ─

Michael J. Rupp

15

I think you could see a person's viewpoints on life through this questionnaire. Basically it seems to be asking"effort" or "luck"?

16

There were some hard questions. I couldn't figure out what the aim of this questionnaire was.

17

There were a number of questions that we don't see on typical questionnaires, so it was kind of interesting. I
would love to see the results of this questionnaire. It was unusual to see questions using the words "luck" and"fate".

18

People act on their own volition, so I think the answers are going to depend on the person.

19（no comment）

20

I was surprised by this questionnaire because I was expecting something related to our classwork. However, I
thought it was meaningful.

21

At the end of the day, I think I decide my own outcomes.

22

I felt like the contents weren't fully fleshed out, the questions were too abrupt and this could cause
misunderstandings. It seems like there were a lot of questions that were more like "digs" at us. LOL

23

I don't get this questionnaire.

24

I don't understand the point of this questionnaire.

25

I was made to rethink how I live my life. Many questions were very similar, gradually it became like leaving it
up to luck.

26

There is no need to make friends with members of the opposite sex. I think answering all these questions from
1 to 4 is not as good as expressing your opinions about these items.

27

There are a lot of similar questions and negative questions. Some of the questions were helpful, though.

Appendix C: Author’s Translation of Student Comments on each item of K-LoC43,WHP

,WLVEHVWWRMXVWJRZLWKWKHÀRZ*This resembles question 9.

*

This is not good because I am at an age where my choices are respected.

*

I don't think everything would necessarily go well.

*I think you could get the wrong idea with this one and end up not making any effort.

,WHP
*,WHP
*,FDQEHDJUHDWVXFFHVVLI,ZRUNKDUG

I think the word "great success" is a bit abstract.
$Q\RQHZLOOEHDEOHWRXQGHUVWDQGPHLI,WU\P\EHVWWRFRPPXQLFDWHZLWKWKHPThis is too idealistic.

,WHP

,GHFLGHP\RZQOLIH(No Comments),WHP

0\OLIHLVGHFLGHGE\IDWH

*I have only lived for 20 years.

*

University student life depends upon hard work*

I have only live for 20 years.

*

This is not a good question. It seems like it is asking the same thing as number 4.

*

People can change their lives.

*

This is similar to item 4.

*

I don't want this to be true.

*

It is not only fate but also your destiny. Both of these decide your life.

,WHP

0\KDSSLQHVVDQGVDGQHVVDUHGHWHUPLQHGE\FKDQFH

*This is a heavy topic for university students.

*

I still don't know.

*

I don't want this to be true.

*

I think this depends on my efforts.

*

I feel that the word "chance" lacks a sense of responsibility.

─ 32 ─

5HFRPPHQGHG0RGL¿FDWLRQVWRWKH.DPEDUD/RFXVRI&RQWURO6FDOH,WHP

:KDWKDSSHQVGHSHQGVRQWKHVLWXDWLRQ*This is a heavy topic for university students.

*

A questionable item as it ends up making me look like I am disregarding my own person.

*

What happens around you depends on your attitude.

,WHP
*0\IULHQGVFDQ WXQGHUVWDQGPHQRPDWWHUKRZKDUG,WU\

Not a good item because university students should be able to make themselves understood, at least to some extent.

,WHP/LIHLVDJDPEOH

*This resembles question 1*

This really depends on the person.

*

This is the same as no. 5. Also, I'm pretty sure there aren't any university students who would say something this outrageous.

*I don't like the idea of things being decided by fate.

*

I don't understand this question.

,WHP
*,WLVXVHIXO PHDQLQJIXO WRWKLQNDERXWZKDW,ZDQWWREHLQWKHIXWXUH

Depending on the person it might turn out bad.

,WHP

,I,WU\KDUG,FDQGRDQ\WKLQJRQP\RZQ

*

If you try your best things may be only easier to do.

*

The people around you are definitely going to be involved.

,WHP
*,WHP
*8VXDOO\WKLQJVWXUQRXWEHWWHULI,PDNHP\RZQGHFLVLRQV

This is a mediocre item, because it is important to listen to others’ opinions.
0\KDSSLQHVVRUVDGQHVVLVGHWHUPLQHGE\P\RZQHIIRUWVHappiness depends on how you view things.

,WHP

,ZLOOEHDEOHWROLYHP\HQWLUHOLIHDV,SODQWR*

I'm still only a university student.

*

I plan my own life.

*

Does this mean will I be able to overcome obstacles?

*

I hate the idea of fate deciding things.

,WHP

0\IXWXUHLVGHWHUPLQHGE\IDWHRUFKDQFH

*I don't think you can say everything is up to chance or fate.

*

I'm still only a university student.

*

I hate the idea of fate deciding things.

*

It is not only chance but also effort combined with it, as a minimum requirement.

,WHP

:KDWKDSSHQVGRHVQRWGHSHQGRQP\HIIRUWV*This might be better asked of adults.

*

I don't think this is how things work.

*

Not a good item, because we have to make great efforts in order to find a job.

*

This question seems like "giving up on it all".

*

You can do something on your own. This question is not appropriate for university students.

,WHP

,FDQEHIULHQGVZLWKDQ\RQHLI,WU\(No Comments),WHP

<RXUHIIRUWVDQGVXFFHVVDUHQRWUHDOO\UHODWHGWRHDFKRWKHU*I don't think this is how things work.

*

This depends on the person.

,WHP

,I,DPFDUHIXODERXWP\DFWLRQVSHRSOHZLOOWUXVWPH

*This item is mediocre because we should be careful about our own actions in any event.

*If you are careful about your words and actions and take consideration to your situation, at some point people are going*

There can sometimes be problems with personality/character.

to have to trust you.
,WHP
*0\DELOLW\WRPDNHJRRGIULHQGVGHSHQGVRQWKHFODVVRUFOXE VDWPRVSKHUH

Not a good item because we should be able to make friends regardless of the atmosphere.

─ 33 ─

Michael J. Rupp*

In university, unlike up until high school, it depends on the person.

,WHP
*,I,WU\KDUG,ZLOOEHDEOHWRJHWWKHMRE,ZDQW

Sometimes there are job "ice ages".

,WHP

0DUU\LQJDQLGHDOSDUWQHUGHSHQGVRQIDWHRUOXFN*I can’t imagine.

*

I really don't know, as I am still a college student.

*

I think you should ask someone around 30 years old.

*

University students are rarely married, so this is hard to think about.

*

Ideals and reality can be different, if you try hard, you might get lucky.

,WHP

,WLVHDV\WRJHWDJRRGVFRUHRQWHVWVLI,SUHSDUHIRUOHVVRQVDQGUHYLHZDIWHUZDUGV*Preparation, review and understanding are all different. If you don't understand, all of it would be meaningless.

*

No kidding!

,WHP
*,FDQPDNHIULHQGVZLWKWKHRSSRVLWHVH[LI,WU\

We don't need this question about "opposite sex".

,WHP,RIWHQGRLPSXOVLYHWKLQJVZLWKRXWEHLQJDZDUHRILW*

I don't really get what this question is asking.

*I'm not sure what the aim of this question is.

,WHP

*HWWLQJLQWRP\¿UVWFKRLFHXQLYHUVLW\GHSHQGVPRUHRQOXFNWKDQDELOLW\*Maybe you should change this to getting into "a company".

*

This is a question for high school students.

*

I hate things like "chance".

*

You get into university based on your ability.

*

For those students who are attending a university that wasn't their first choice, this is a tough question.

*

Getting into the first choice university requires both effort and luck.

*

We have already entered university, so this is unnecessary.

,WHP

%HLQJDEOHWRPDLQWDLQORQJIULHQGVKLSVGHSHQGVRQWKHH[WHUQDOVLWXDWLRQ HQYLURQPHQW *

Not a good item because university students should be able to maintain friendships regardless of the situation.

*

Personal relationships are getting weaker these days, so it depends on the person.

Item 28

When you take actions, it is more often the case that others have suggested them rather than you acting upon yourRZQGHVLUHV*I always act based on my own ideas.

,WHP
*

Not a good item because, haven't university students come to study what they wanted to study?

,WHP
*,IDFODVVLQVFKRROLVERULQJLWLVEHFDXVH\RXDUHQRWLQWHUHVWHGLQWKDWVXEMHFW,DOZD\VGHFLGHZKDW, PJRLQJWRGRThis is rather obvious, isn't it?

,WHP

,Q\RXUFDVHZKHQLWFRPHVWRWHVWUHVXOWVWKH\DUHRIWHQLQÀXHQFHGE\\RXUSK\VLFDOFRQGLWLRQRURWKHUUDQGRPHYHQWV*This is just an excuse, so I don't think there is any need for this question.

*

Not good, I'm pretty sure we can't use our physical condition as an excuse (LOL).

*

I don't like "chance".

*

This is just an excuse.

*

Are we talking about children here?

,WHP

,WLVKDUGIRUPHWRGRWKLQJVDV,KDYHSODQQHG(No Comments),WHP

:HFDQ WFKDQJHKRZVPDUWRUVWXSLGZHDUH

*

Not a good item because we should know how to make efforts!

*

One's intelligence or lack thereof is based on effort.

*

Someone's efforts can have an effect.

*

I think you get smarter and smarter by working hard.

,WHP

0DLQWDLQLQJIULHQGVKLSVGHSHQGVRQ\RXUHIIRUW─ 34 ─

5HFRPPHQGHG0RGL¿FDWLRQVWRWKH.DPEDUD/RFXVRI&RQWURO6FDOH(No Comments),WHP
*,IQHFHVVDU\,FDQVXSSUHVVP\GHVLUHVDWDQ\WLPH

This is just common sense.

Item 36

There is no use in thinking about how to make friends with members of the opposite sex as such things areGHWHUPLQHGE\IDWHGHSHQGXSRQOXFN*

Not good because we should be positive about any aspects of our lives.

*

I don't like "luck".

,WHP
*0\DFWLRQVWHQGWRHQGXSJRLQJDORQJZLWKWKHÀRZRIFLUFXPVWDQFHVThis item is mediocre because we should not be so insipid.

,WHP

7KHUHVXOWVDUHIDUEHWWHUZKHQ,SUHSDUHIRUH[DPVLQDGYDQFH

(No Comments),WHP
*,WLVLPSRUWDQWWRWKLQNDERXWP\DFWLRQVLQRUGHUWRKDYHJRRGUHODWLRQVKLSVZLWKP\IULHQGVThis is common sense.

,WHP

(YHQLIP\IULHQGVKDYHGLIIHUHQWLGHDV,SODFHDSULRULW\RQP\RZQDFWLRQV

*This is common sense.

*

This is a mediocre item because university students should not be so self-centered. LOL.

,WHP

0\JUDGHVGHSHQGRQWKHWHDFKHU

*It may be so, but I don't think we can ask such a question.

*

This is common sense.

*

If there needs to be some improvement, I think it is necessary.

*

Even if you say this, nothing can be done about it.

,WHP

,I,DPNLQGWRP\IULHQGVVRPHGD\WKH\ZLOOKHOSPH

*I have a problem with the phrasing "If I am kind".

*This is common sense.

*

This is a mediocre item because I don't think I would try to make friends with the idea of someday being helped bythem.

,WHP,RIWHQ¿QGP\VHOIGRLQJWKLQJVWKDW,GRQ WOLNHWRGR(No Comments)

─ 35 ─

Michael J. Rupp
要旨
本論文は英語教育などにおける学習者の自律（learner autonomy: LA）と密接にかかわるデータ抽出手法である，鎌原（1982, 1987）が開発したアンケート「統制の所在 : LoC」（The Kambara Locus of Control Scale（K-LoC）（18項
目版および43項目（拡大）版）によって得られたデータを，心理測定学的（psychometric）観点から実証的に解析・検証したものである．研究にあたって，1125名の高校生からデータを収集し，得られたデータを確認的因子分析（CFA）および探索的因子分析（EFA）により解析している．併せて，定性的研究手法である「フォーカス・グルー
プ」を用いて，27名の大学生からグループディスカッション形式の発話データなどを収集し，計量的解析に質的解析を加えることで検証の妥当性を高めることを試みている．量的のデータ分析に加えて質的のデータ分析でK-LoC への改善や変更を目指している．K-LoCS への修正提案や「フォーカス・グループ」法によって収集したデータから明らかになったことを念頭に，本論文のまとめが記述されている．

─ 36 ─

東海大農紀要 36：37−43（2017）
ヤーコン茶葉のリポキシゲナーゼ阻害と細胞を用いた NO 産生抑制

ヤーコン茶葉熱水抽出物のリポキシゲナーゼに対する阻害作用およびRAW264.7マウス由来マクロファージ様細胞を用いた NO 産生抑制作用
上田裕人 *・菅原進太郎 *・松田,,

靖 * **・村田達郎 * **・黒田泰弘 ***・星

,

,

,

椛田聖孝 * **・小野政輝 * **・井越敬司 * **・安田,良和 * **,

伸 * **

Effects of Hot-water Extract of Herbal Tea Leaves from Yacon (Smallanthus sonchifolius): Lipoxygenase
Inhibition and Suppression on Nitric Oxide Generation in RAW264.7 Mouse Macrophage-like Cells
Yuto UEDA, Shintaro SUGAHARA, Yasushi MATSUDA, Tatsuro MURATA, Yasuhiro KURODA, Yoshikazu HOSHI,
Kiyotaka KABATA, Masateru ONO, Keiji IGOSHI and Shin YASUDA（Received 5 December 2016; accepted 17 January 2017）
ABSTRACT
Yacon (Smallanthus sonchifolius), has been believed as a potent alternative food source for patients who require a dietary
cure in regional area. It has been used as a folk medicine for the people suffering from diabetes and digestive/renal disorders.
The leaf part of this plant is locally consumed as herbal tea, and recognized as a health food material due to the rich inpolyphenol. However, the mechanisms underlying the functional properties of this plant and even its processed foods remain to
be completely established. We have previously evaluated antioxidant capacity of herbal tea leaves from yacon. Therefore, it is
LPSRUWDQWWRHYDOXDWHWKHPXOWLIXQFWLRQDOLW\RIWKLVKHUEDOWHDOHDYHV7KLVVWXG\DLPVWRH[DPLQHDQWLLQÀDPPDWRU\HIIHFWVRI
hot-water extract of herbal tea leaves from yacon. In lipoxygenase (LOX) inhibition assay, the hot-water extract resulted indemonstrating a concentration-dependent inhibition, with IC50 value of 39.5 μg/ml. Subsequently, macrophages cultured in thepresence of hot-water extract showed suppressing effect on nitric oxide (NO) production in a lipopolysaccharide-activated cell
model, with IC50 value of 35.4 μg/ml under the non-toxic concentration. In addition, the hot-water extract showed weak NO
scavenging activity. These results may indicate the potential of the herbal tea leaves from yacon to manage LOX and NOPHGLDWHGLQÀDPPDWLRQ緒て利用されている（1,2）．最近では，ヤーコンは地域に言

よっては食事療法を必要とする患者のための潜在的な代ヤーコン（Smallanthus sonchifolius）はキク科スマラ

替食として考えられている（2）．また，糖尿病や消化／ンサス属の一種であり，南米アンデス山脈地方原産の多腎障害を患う人々のための民間薬としても使用されてい年生草木である．過去数十年に渡り，ヤーコンはアジア

る．これは豊富な難消化性フラクトオリゴ糖と有益なポ（日本，韓国，台湾，海南およびフィリピン），オセアニ

リフェノールに起因する可能性が示唆されている（3）．

ア（ニュージーランド），そしてヨーロッパ（チェコ共さらにヤーコン塊根は，抗酸化作用（4），ラットにおけ和国）などの様々な場所に導入されてきた（1）．この植る抗糖尿病作用（5），ヒトにおける抗肥満（6）および物の塊根は，サツマイモのような形状であり，歴史的に糖尿病ラットに対する脂質低下作用（7）が研究されて果物／野菜として摂取されており，現在では，南米にている．ヤーコン葉部の一部はハーブティーとして地域限

シロップ，ジュース，マーマレードなどの加工食品とし定ながらも市販化ならびに消費されているものの，そのほとんどは食材として利用されておらず，有効利用の開＊東海大学大学院農学研究科

発が必要である．また葉部には，豊富なフェノール性化＊＊東海大学農学部

合物が含有されている．その主成分としてクロロゲン酸，＊＊＊東海大学工学部

カフェ酸，フェルラ酸等が挙げられ（8），さらにはヤー

連絡先：安田 伸 e-mail: shin.yasuda@agri.u-tokai.ac.jp

コン葉特有のカフェオイル誘導体が含有されていること
─ 37 ─

上田 裕人・菅原 進太郎・松田 靖・村田 達郎・黒田 泰弘・星 良和
椛田 聖孝・小野 政輝・井越 敬司・安田 伸（9）が報告されている．またヤーコン葉部は，抗真菌作材料および方法

用（10），抗酸化作用（11），正常および糖尿病ラットモ
デルにおける血糖降下作用（12）を有することが報告さ

原材料および試薬類れている．これまでに我々は，ヤーコン茶葉の機能性研

サラダオトメ（旧系統名：SY201） は，日本で品種究に着手しており，これまでに複数のフリーラジカル種

登録されているヤーコンの一つである（26）．本研究でに対する抗酸化作用，特に活性酸素種の１つであるスー用いた乾燥ヤーコン茶葉は，本品種を菊池市（熊本県）

パーオキシドアニオン（O2 ）ラジカルに対する抑制作

で栽培，茶葉への加工およびハーブティーとして販売さ用について酸化酵素やヒト顆粒球様好中球細胞モデルをれているものを2011年３月に入手したものである．ノル

用いて明らかにしてきている（13）．我々は，本植物な

ジヒドログアイアレチン酸（NDGA）は MP Biomedicals

らびにその加工食品の新たな機能性として，抗炎症作用

LLC（米国）より，メチレンブルー，リン酸二水素ナトに着目している．

リウム，リン酸水素二ナトリウム，リノール酸，ジメチ

炎症は，病原体，化学または物理的損傷の存在に対す

ルスルホキシド（DMSO），ペニシリン G，ストレプトるヒトおよび他の哺乳動物の応答反応である．炎症反応

マイシン，N-1- ナフチルエチレンジアミン二塩酸塩，には，いくつかの起炎酵素が炎症性メディエーターの産

水酸化ナトリウム，塩化ナトリウム，塩化カリウムはナ

生や組織損傷の亢進に寄与することが知られている．リ

カライテスク（京都）より入手した．DMEM 培地，ポポキシゲナーゼ（LOX）は，喘息，乾癬，癌転移およリ オ キ シ エ チ レ ン（20） ソ ル ビ タ ン モ ノ ラ ウ レ ー トびアテローム性動脈硬化症のような多くの病態生理学的（Tween20），リン酸は和光純薬工業株式会社（大阪）よプロセスにおいて，炎症性ロイコトリエン，リポキシン

り，ペンタシアノニトロシル鉄（Ⅲ）酸ナトリウム二水およびヒドロキシエイコサテトラエン酸を生成する重要和物（SNP），（±）-6- ヒドロキシ -2,5,7,8- テトラ - メ

な酵素である（15,16）．また，LOX が急性皮膚炎（17）チルクロマン -2- カルボン酸（trolox），ダイズ（Glycine

や in vivo でのヒスタミン誘発性浸潤（18）に関与してMax） 由 来 リ ポ キ シ ゲ ナ ー ゼ， 大 腸 菌（Escherichia

いることが報告されている．このように様々な炎症関連coli）由来リポ多糖（LPS），スルファニルアミドはシグプロセスに本酵素が寄与していることから，アラキドン

マアルドリッチ社（米国）より購入した．Cell Counting

酸カスケードにおける LOX 経路を天然物が阻害しうる

Kit-8（WST-8）は同仁化学研究所（熊本）より，クルク

かどうかを調べることに関心が寄せられている（19,20）．

ミンはキシダ化学株式会社（大阪）より，RAW264.7マ

血液中の単球に由来するマクロファージ細胞は生体防御

ウス由来マクロファージ様細胞（ECACC91062702）はの上で免疫反応に重要な役割を担う．サイトカインや微DS ファーマバイオメディカル株式会社（大阪）より購

生物性の物質はマクロファージを活性化し，そこでは過入した．ウシ胎児血清（FBS）はギブコ，インビトロジ

剰量の一酸化窒素（NO）が産生されうるため，NO レエン（米国）より購入した．Griess 溶液は，１% スル

ベルは in vitro および in vivo でのマクロファージの炎症

ファニルアミド，0.1% N-1- ナフチルエチレンジアミン

状態を反映する（21,22）．また，過度の NO 産生によっ

二塩酸塩および2.5% リン酸を含む溶液から成る．10て敗血症，関節リウマチの他に出血性ショック，全身性mM リノール酸溶液は，リノール酸56 mg，Tween20 112

エリテマトーデス，シェーグレン症候群，血管炎，変形

mg，MilliQ 35.2 μl を転倒混和し，１M 水酸化ナトリウ性関節炎，乾癬，接触性皮膚炎のような炎症性疾患の進

ム24 μl，MilliQ 776 μl を含む溶液から成る．

行が生じることが指摘されている（23）．我々はこれま
で に，薬 用 植 物 で あ る ツ ル ボ（Scilla scilloides DRUCE

ヤーコン茶葉熱水抽出物の調製（Liliaceae））や地域の特色ある食材としての食用イグサ

ヤーコン茶葉熱水抽出物は，以前の報告で調製された（Juncus effusus L.（Juncaceae））の有する抗炎症作用につものを使用した（11）．簡潔に述べると，粉砕した茶葉

いて研究を行い，両者が起炎酵素である LOX を阻害す

５g をコニカルビーカーに入れ，90〜100℃の MilliQ 水

ること，マクロファージ様の細胞モデルにおいて NO 産

250 ml 中で45分間浸漬した．茶葉の懸濁液を遠心分離し，生抑制作用を示すことを報告してきた（24,25）．

濾過した．その後，濾液を凍結乾燥させ，水で再溶解さ

本研究では，ヤーコン茶葉熱水抽出物の潜在的な抗炎せたものを以降の実験に使用した．

症作用について調べるため，in vitro で LOX に対する阻害作用およびマクロファージ様細胞モデルにおける NO

リポキシゲナーゼ（LOX）阻害活性測定
LOX 阻害活性は，酵素反応中にリノール酸の酸化に産生抑制作用について調べることとした．
─ 38 ─ヤーコン茶葉のリポキシゲナーゼ阻害と細胞を用いた NO 産生抑制伴うメチレンブルーの退色を指標に測定した（24）．こ加し，さらに24時間培養した．培養後上清中に放出されれは既報の異なる LOX 活性測定技術（27,28）を我々た NO 量の測定は Marcocci ら（29）の Griess 法によりが改良したものである．96ウェルプレートに試料溶液を測定した．即ち，培養後に得られた培養上清100 μl と17.5 μl，0.2 M リン酸緩衝液（pH 6.0）を140 μl を混合

Griess 溶液100 μl を混合させ，マイクロプレートリー

させ，さらに MilliQ を102.9 μl，５mM リノール酸溶液

ダーを用いて550 nm における吸光度を測定した．

を5.6 μl，250 μM メ チ レ ン ブ ル ー を14 μl，212 kU/ml

このとき100 ng/ml LPS で処理した際に認められた NO

LOX を70 μl それぞれ混合した．マイクロプレートリー

産生量を100% として，サンプル添加時の NO 産生率

ダ ー（SH-1000Lab． コ ロ ナ 電 気， 茨 城 ） を 用 い て660（%）を以下の式により算出した．

nm における吸光度を測定した．その後，遮光下で30分

NO Production(%) = (ASample - ABlank) / (ALPS – ABlank) ×100

間反応後同様に測定を行なった．ポジティブコントロー

ASample：LPS 刺激時の試料処理後の吸光度

ルには NDGA を使用した．

ALPS：LPS 刺激のみの吸光度
ABlank：ブランクの吸光度阻害活性（%）は，次式により算出した．
Inhibition Activity(%)=[１- (ASample) / AControl] ×100
ASample：試料の30分反応後の吸光度より0分の吸光度をNO 消去活性の測定
NO 消去活性は，SNP を用いて PBS(-) 中にて自発的差し引いた値
AControl：試料を含まないコントロールの30分反応後のに生じる NO 量を Griess 試薬を使用し測定する Marcocci

吸光度より０分の吸光度を差し引いた値

ら（29）の方法に基づいて行なった．1.5 ml マイクロチューブに試料溶液を５μl，PBS を45 μl，10 mM SNP
溶液を50 μl とを混合させ，室温で150分間静置した．そRAW264.7マウス由来マクロファージ様細胞の培養法
RAW264.7マウス由来マクロファージ様細胞は，10%の後，Griess 試薬100 μl により発色させ，マイクロプ

FBS および１% ペニシリン ストレプトマイシンを含む

レートリーダーを用いて550 nm における吸光度を測定

DMEM 培地を用いて，37℃，５% CO2の条件下で培養した．ポジティブコントロールには trolox およびクルク

した．

ミンを使用した．
NO 消去活性は次式で求め，消去率（%）で表した．
NO Scavenging Activity(%) =［１-（ASample - ABlank)] /WST-8法による細胞生存率の測定(AControl - ABlank) ×100

細胞生存率の測定は，WST-8法を用いて行った．即ち，
4

RAW264.7細胞を50×10 cells/ml となるように調整し，ASample：試料の吸光度

96ウェルプレートに100 μl ずつ播種した．３時間前培養

AControl：試料を含まないコントロールの吸光度した後，試料溶液を25 μl ずつ添加し，さらに24時間培

ABlank：ブランクの吸光度

養 し た． 培 養 後， リ ン 酸 緩 衝 生 理 食 塩 水（PBS(-)
2+

2+（Phosphate buffered saline,Mg ,Ca -free））で希釈された

統計解析

WST-8溶液を20 μl 添加し，さらに1．5時間培養を行なっ

実験結果は４つの異なるデータより得た平均値±標準

た．その後，マイクロプレートリーダーを用いて450 nm

偏差（mean±S.D.）で表記した．また統計的な差はス

における吸光度を測定した．

チューデントの t 検定を用いて P < 0.001で有意であると細胞生存率（%）は，次式により算出した．

みなした．

Relative Cell Number =（ASample - ABlank) / (AControl - ABlank)
ASample：試料処理後の吸光度結果および考察

AContol：試料を含まないコントロールの吸光度
ABlank：ブランクの吸光度

ヤーコンは主に塊根部分が食用とされる植物であるが，茎葉部はハーブティーとして利用されている．近年では，
ヤーコンは地域によっては食事療法を必要とする患者の活性型 RAW264.7細胞の NO 産生抑制効果の測定
4

RAW264.7細胞を50×10 cells/ml となるように調整し，ための潜在的な代替食として考えられている（2）．我々

96ウェルプレートにそれぞれ100 μl ずつ播種した．３時は，本植物ならびにその加工食品の新たな機能性として，間前培養を行なった後，試料溶液または終濃度100 ng/

抗炎症作用に着目している．これまでに我々は，ヤーコ

ml になるように調製した LPS を含む培地を合計25 μl 添

ン茶葉が複数のフリーラジカル種に対して抗酸化作用を─ 39 ─

上田 裕人・菅原 進太郎・松田 靖・村田 達郎・黒田 泰弘・星 良和
椛田 聖孝・小野 政輝・井越 敬司・安田 伸
示すこと，特に活性酸素種の１つである O2 ラジカルにほどの阻害作用を有することが認められた．

対する抑制作用について明らかにしてきた（13）．近年，
健康志向食品素材として注目されつつあるヤーコンの健
康維持および増進に有益な情報を提供するべく，本研究では，ヤーコン茶葉の熱水抽出物を用いて，起炎酵素である LOX に対する阻害活性およびマクロファージ様細胞モデルにおける NO 産生抑制作用を調べることとし，潜在的な抗炎症作用について検証を行うこととした．
ヤーコン茶葉の LOX 阻害活性
最初に，ヤーコン茶葉熱水抽出物が LOX 酵素活性を阻害しうるか検討した．その結果，ヤーコン茶葉熱水抽Fig. 1. Effect of concentration of hot-water extract from yacon
tea leaves in lipoxygenase inhibition assay. Data shown
represent mean±S.D. from four different experiments. NDGA
was used as the standard sample. NDGA; nordihydroguaiareticacid.

出物は濃度依存的な阻害活性の上昇を示した（Fig. 1）．
ヤーコン茶葉熱水抽出物の半数阻害濃度（IC50）値は39.5 μg/ml であった．ポジティブコントロールとして用いた NDGA の IC50値は3.67 μg/ml であった．我々はこれまでに，薬用植物であるツルボ，食用イグサおよび抹茶

ヤーコン茶葉のマクロファージ細胞モデルにおける NOの LOX 阻害活性について調べてきた．そこでは，ツル

産生抑制

ボ酢酸エチル抽出物の IC50値には31.5 μg/ml（24），食用

次に，ヤーコン茶葉熱水抽出物が LPS で刺激した活

イグサの熱水抽出物およびエタノール抽出物にはそれぞ性型マクロファージ様細胞の過剰な NO 産生を抑制しうれ123 μg/ml および143 μg/ml（25），さらに抹茶の熱水

るかについて調べた．まず初めに細胞毒性試験を行うべ

抽出物およびエタノール抽出物にはそれぞれ138 μg/ml

く，細胞をサンプルとともに24時間培養し，WST-8法をおよび145 μg/ml の値を得ており（25），それぞれに異な用いて比色定量により生細胞数を求め，これを細胞生存るレベルで LOX 阻害作用を認めている．本研究で用い率の指標とした．その結果，未処理の生細胞数を100%た抽出物は，我々が以前報告した際に調製したものであとしたとき，10〜200 μg/ml の濃度範囲でヤーコン茶葉

り，熱水抽出物１mg 当たり0.279 mg のポリフェノール

熱水抽出物は，有意な生細胞数の低下を示さず，細胞生が含まれていること，収率より乾燥茶葉１g 当たり0.0776

存率に影響を及ぼさなかった（Fig. 2A）．これにより，

g のポリフェノールが含まれていることが明らかとなっ200 μg/ml までの非毒性濃度範囲でマクロファージ細胞ている（13）．また，これまでに植物体に含有されるモデルにおける NO 産生抑制作用を調べることとした．

フェノール性化合物によっては，LOX 酵素阻害を示す

RAW264.7細胞を100 ng/ml の LPS により刺激し，これものがあることが報告されており（30-32），緑茶由来カを炎症時の活性型マクロファージのモデルとした．ヤー

テキン類の摂取は，LOX 阻害を介して in vivo での急性

コン茶葉熱水抽出物を LPS とともに24時間処理し，上炎症を抑制することが報告されている（33）．ヤーコン

清中の NO 産生量に及ぼす影響を調べた．その結果，

葉のフェノール性の主成分にはクロロゲン酸，カフェ酸，

LPS 誘導の NO 産生量を100% としたとき，ヤーコン茶

フェルラ酸等が挙げられ（8），これらが成分レベルで

葉熱水抽出物は10〜200 μg/ml の濃度範囲において濃度

LOX 阻害作用を示すことが報告されている（34,35）．

依存的に有意な NO 産生抑制作用を示した（P < 0.001）．

現時点ではヤーコン茶葉熱水抽出物中に含まれる LOX算出された IC50値は35.4 μg/ml であった（Fig. 2B）．これ阻害に関する成分レベルでの知見は未だ得られていない．

までに異なる８種類のハーブティー（デイジーティー，

ヤーコン葉特有のカフェオイル誘導体（9）が特に強い

ジャスミンティー，ラベンダーティー，ローズティー，

LOX 阻害作用を示すことが報告されており（36），これ

モクセイティー，レモングラスティー，ローズマリー

らに類するポリフェノール成分が LOX 阻害に対して大ティー，緑茶）のマクロファージ様細胞における NO 産

きく貢献している可能性がある．従って，ヤーコン茶葉

生抑制作用について報告があり，その中では113 μg/ml

熱水抽出物は，食用イグサや抹茶の熱水抽出物あるいはの IC50値とともに緑茶が最も強い抑制作用を有している

エタノール抽出物よりも LOX に対して強い阻害作用を（37）．また我々は，食用イグサおよび抹茶の RAW264.7

有し，薬用植物のツルボの酢酸エチル抽出物に匹敵する細胞を用いた NO 産生抑制作用についても調べており，

─ 40 ─ヤーコン茶葉のリポキシゲナーゼ阻害と細胞を用いた NO 産生抑制
食用イグサの熱水抽出物およびエタノール抽出物の IC50

ヤーコン茶葉熱水抽出物が RAW264.7細胞の NO 産生

値はそれぞれ120 μg/ml および35.2 μg/ml，ならびに抹茶を抑制することが確認されたものの，これらの結果は，の熱水抽出物およびエタノール抽出物の IC50値はそれぞ

細胞中の NO 合成経路を直接的または間接的に抑制する

れ > 250 μg/ml および > 100 μg/ml であることを報告してことによるのか，または細胞より放出された NO 自体をいる（25）．ヤーコン茶葉熱水抽出物で得られた本結果特異的に消去したためか疑問が残る．そこで PBS 中には，他の報告例と比べてもより強力なものであった．

NO ドナーである SNP を入れることにより，疑似的にヤーコン葉は TNF-α，PGE2といったエイコサノイド類

溶液中に NO を生成させ，その NO をヤーコン茶葉熱水の産生に対しても阻害作用を有しており，セスキテルペ

抽出物が消去しうるかについて調べた．その結果，濃度

ンラクトン類が局所抗炎症作用に寄与している（38）．

依存的な消去活性の上昇が認められ，738 μg/ml の EC50

現時点ではヤーコン茶葉中に含まれるマクロファージモ値が得られた（Fig. 3）．しかしながら，前述の細胞実験

デル細胞の NO 産生抑制成分に関する知見は得られていに使用した10-200 μg/ml の濃度範囲では約8-45% の消去

ないものの，本研究で用いたヤーコン茶葉熱水抽出物は，率を示すのみであったことより，ヤーコン茶葉熱水抽出

細胞レベルで強い NO 産生抑制作用を示したことより，

物は弱いながらも高濃度では NO を直接的に消去しうるNO が引き起こす炎症性疾患のリスクを軽減させる可能こと，200 μg/ml 以下の低濃度領域では直接的または間性が考えられた．

接的に細胞に作用し，NO 合成経路に干渉して細胞のNO 産生を抑制する可能性が考えられた．この時のポジ
ティブコントロールとして用いたクルクミンおよび
trolox の EC50値はそれぞれ18.0 μg/ml および733 μg/ml でFig. 3. Effect of concentration of hot-water extract from yacon
tea leaves in NO scavenging assay. Data shown represent mean
±S.D. from four different experiments. Curcumin and trolox
were used as the standard sample. NO; nitric oxide.

あった．
ヤーコン茶葉の機能特性に関する科学的エビデンスのFig. 2. Effect of hot-water extract from yacon tea leaves on cell
viability (A), and LPS-stimulated NO production in RAW264.7
mouse macrophage-like cells (B). In (A), cells were incubated in
the presence of varying concentrations of the extract for 24 h.
Cell viability was measured using Cell-counting kit-8. In (B),cells were incubated in the presence of the extract plus 100 ng/
ml LPS for 24 h. The culture media were collected and subjected
to determination of NO levels by the Griess method. Data shown#
represent mean±S.D. of four different experiments. 6LJQL¿FDQW
difference from untreated cells (control) was considered at P <***0.001, and VLJQL¿FDQWGLIIHUHQFHIURP/36WUHDWHGFHOOVP <0.001. N.S.LQGLFDWHVQRVLJQL¿FDQFHIURPFRQWUROLQ $ 12nitric oxide, LPS; lipopolysaccharide.

付 与 を 目 的 に， ヤ ー コ ン 茶 葉 熱 水 抽 出 物 を 用 い て
in vitro での抗炎症作用を評価した．LOX に対して濃度依存的な阻害作用を有し，既報の薬用植物と同等の阻害能を持つことを見出した．また，LPS 刺激した活性型
RAW264.7マウス由来マクロファージ様細胞より過剰産生される NO に対して濃度依存的かつ有意な阻害能を持
つこと，作用メカニズムは NO に対して直接消去するよりも，マクロファージ様細胞内の NO 合成経路に直接的
または間接的に干渉して，抑制的に作用する可能性が考えられた．本研究で用いたヤーコン茶葉熱水抽出物は，
─ 41 ─

上田 裕人・菅原 進太郎・松田 靖・村田 達郎・黒田 泰弘・星 良和
椛田 聖孝・小野 政輝・井越 敬司・安田 伸
O2 ラジカルを中心に多彩な抗酸化能を有することを

引用文献

我々は報告しており（13），多機能性を有する高機能性食品として，本加工食品の有効活用が期待される．しか

１）Ojansivu, I., Ferreira, C.L. and Salminen, S., 2011,
Trends Food Sci. Tech. 22, 40-46.

しながら，ヤーコン茶葉が有しうる抗炎症作用の解明には，ヤーコン茶葉の NO 産生抑制メカニズムの解明，

２）Delgado, G.T., Tamashiro, W.M., Marostica, J.M.R. andヤーコン茶葉由来の有効成分の分離同定や定量，分子レ

Pastore, G.M., 2013, Plant Foods Hum. Nutr. 68, 222-

ベルでの評価，生体レベルでの評価など，更なる検証が228.
３）Campos, D., Betalleluz, P.I., Chirinos, R., Aguilar, G.A.,

必要である．

Noratto, G. and Pedreschi, R., 2012, Food Chem. 135,

謝1592-1599.

辞４）Yan, X., Suzuki, M., Ohnishi, K.M., Sada, Y., Nakanishi,
T. and Nagata, T., 1999, J. Agric. Food Chem. 47, 4711-

本研究の一部は，東海大学総合研究機構プロジェクト

4713.

研究，総合農学研究所プロジェクト研究，および先進生
命科学研究所プロジェクトの資金援助により実施された

５）Oliveira, G.O., Braga, C.P. and Fernandes, A.A.H., 2013,
Food Chem. Toxicol. 59, 256-260.

ものです．

６）Genta, S., Cabrera, W., Habib, N., Pons, J., Carillo, I.M.,
Grau, A. and Sanchez, S., 2009, Clin. Nutr. 28, 182-187.

要７）Habib, N.C., Honore, S.M., Genta, S.B. and Sanchez,

約

S.S., 2011, Chem. Biol. Interact. 194, 31-39.

ヤーコン（Smallanthus sonchifolius）は，地域によっ

８）Valentova, K., Lebeda, A., Dolezalova, I., Jirovsky, D.,ては食事療法を必要とする患者のための潜在的な代替食

Simonovska, B., Vovk, I., Kosina, P., Gasmanova, N.,として考えられ，糖尿病や消化／腎障害を患う人々のた

Dziechciarkova, M. and Ulrichova, J., 2006, J. Agric.

めの民間薬としても使用されている．一方，ヤーコン葉

Food Chem. 54, 1347-1352.

部の一部はハーブティー等として地域限定ながら流通し

９）Chagas, P.D.A., Oliveira, T.B., Faleiro, D.P.V., Oliveira,ており，豊富なポリフェノールを含む健康食品素材としR.B. and Costa, F.B.D., 2015, Planta Med. 81, 1296-て注目される．我々は，本植物ならびにその加工食品の1307.

新たな機能性として，抗炎症作用に着目している．これ10）Lin, F., Hasegawa, M. and Kodama, O., 2003, Biosci.
Biotechnol. Biochem. 67, 2154-2159.

までに我々は，ヤーコン茶葉の抗酸化作用について評価を行ってきた．ヤーコン茶葉の持つ多角的な機能性を評

11）Valentova, K., Sersen, F. and Ulrichova, J., 2005, J. Agric.
Food Chem. 53, 5577-5582.

価することは重要であり，抗炎症作用の観点よりヤーコ
ン茶葉の機能性について調査を開始することとした．

12）Aybar, M.J., Riera, A.N.S., Grau, A. and Sanchez, S.S.,
2001, J. Ethnopharmacol. 74, 125-132.

ヤーコン茶葉熱水抽出物が，起炎酵素であるリポキシゲ
ナーゼに対して阻害作用を有するかを調べた結果，

13）Sugahara, S., Ueda, Y., Fukuhara, K., Kamamuta, Y.,

39.5 μg/ml の IC50値とともに濃度依存的な阻害作用が認

Matsuda, Y., Murata, T., Kuroda, Y., Kabata, K., Ono, M.,められた．次に RAW264.7マウスマクロファージ様細胞

Igoshi, K. and Yasuda, S., 2015, J. Food Sci. 80,の NO 産生に及ぼす影響を調べた結果，毒性を示さない

C2420-C2429.

濃度で35.4 μg/ml の IC50値とともに濃度依存的な抑制作

14）Samuelsson, B., 1983, Science 220, 568-575.

用が認められた．一方 , 本熱水抽出物は，それほど強い

15）Lewis, R.A., Austen, K.F. and Soberman, R.J., 1990, N.
Engl. J. Med. 323, 645-655.

NO 消去作用を示さなかった．以上より，ヤーコン茶葉
熱水抽出物にリポキシゲナーゼ阻害や NO 産生抑制が見16）Henderson, W.R.Jr., 1994, Ann. Intern. Med. 121, 684697.

出されたことから，同抑制作用を機序とする抗炎症作用に注目した応用利用に期待がもたれる．

17）Yamazaki, R., Aiyama, R., Matsuzaki, T., Hashimoto, S.
and Yokokura, T., 1998,QÀDPP5HV47, 182-186.
18）Giannaras, A., Selig, W., Ellis, J. and Hullinger, T., 2005,
Eur. J. Pharmacol. 506, 265-271.
─ 42 ─ヤーコン茶葉のリポキシゲナーゼ阻害と細胞を用いた NO 産生抑制19）Gleason, J.G., Perchonock, C.D. and Torphy T.J., 1986,

29）Marcocci, L., Maquire, J.J., Droy, L.M.T. and Packer, L.
1994, Biochem. Biophys. Res. Commun. 201, 748-755.

Annu. Rep. Med. Chem. 21, 73-83.
20）Komoda, T., Kishi, M., Abe, N., Sugiyama, Y. and Hirota,30）Puerta, R., Gutierrez, V.R. Robin, J. and Hoult, S., 1999,
Biochem. Pharm. 57, 445-449.

A., 2004, Biosci. Biotechnol. Biochem. 68, 903-908.

31）Schewe, T., Kuhn, H. and Sies, H., 2002, J. Nutr. 132,

21）Nathan, C., 1992, FASEB J. 6, 3051-3064.
22）Bogdan, C., 2001, Nat. Immunol. 2, 907-916.

1825-1829.

23）大塚真理，磯前和男，石井二三夫，2003，炎症・再生，

32）Maqsood, S. and Benjakul, S., 2010, Food Chem. 119,

23，93-98.

123-132.

24) Nishida, Y., Sugahara, S., Wada, K., Toyohisa, D.,33）Choi, J.H., Chang, H.W. and Rhee, S.J., 2002, Asia Pac. J.
Clin. Nutr. 11, 292-297.

Tanaka, T., Ono, M. and Yasuda, S., 2014, Pharm. Biol.

34）Oszmianski, J. and Lee, C.Y., 1990, J. Agric. Food Chem.

52, 1351-1357.
25）Nishimura, T., Kabata, K., Koike, A., Ono, M., Igoshi, K.
and Yasuda, S., 2016, Food Sci. Technol. Res. 22, 395-

38, 688-690.
35）Huang, M.T., Lysz, T., Ferraro. T., Abidi, T.F., Laskin,
J.D. and Conney, A.H., 1991, Cancer Res. 51, 813-819.

402.
26）Sugiura, M., Nakanishi, T., Kameno, T., Doi, Y. and36）Kimura, Y., Okuda, H., Okuda, T., Hatano, T. and Arichi,
S., 1987, J. Nat. Prod. 50, 392-399.

Fujino, M., 2007, Bull. NARO. West. Reg. Agric. Res.

37）Tsai, P.J., Tsai, T.H., Yu, C.H. and Ho, S.C., 2007, Food

Cent. 6, 1-13.

Chem. 103, 181-187.

27）Suda, I., Hajika, M., Nishiba, Y., Furuta. S. and Igita, K.,

38）Oliveira, R.B., Chagas, P.D.A., Secatto, A., Gasparoto,

1995, J. Agric. Food Chem. 43, 742-747.
28）Anthon, G.E. and Barrett, D.M., 2003, Food Chem. 81,

T.H., Faccioli, L.H., Campanelli, A.P. and Costa, D.F.B.,
2013, Braz. J. Pharmacogn. 23, 497-505.

275-279.

─ 43 ─

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PROCEEDINGSOFSCHOOL OF AGRICULTURE
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