JP4150050B2 - ほそ麦花粉アレルゲン - Google Patents
ほそ麦花粉アレルゲン Download PDFInfo
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- JP4150050B2 JP4150050B2 JP2006242106A JP2006242106A JP4150050B2 JP 4150050 B2 JP4150050 B2 JP 4150050B2 JP 2006242106 A JP2006242106 A JP 2006242106A JP 2006242106 A JP2006242106 A JP 2006242106A JP 4150050 B2 JP4150050 B2 JP 4150050B2
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Description
関連出願との関連性
本出願は1991年8月16日提出の米国出願第 746,702号の一部係属出願であり、換言すれば1990年3月24日提出の米国出願第 585,086の一部係属出願であり、その両者の開示内容は本明細書に組込まれる。
a)組換 DNA分子であってその上に載っているほそ麦プロモーター配列又はその同族体もしくは縮重体及びイネ科に由来する細胞の中で有害な機能を有するポリペプチドをコードするヌクレオチド配列を含んで成る分子を保有する植物を発育させ(前記ヌクレオチド配列は前記プロモーターより転写可能であり、そして前記組換 DNA分子は花粉生産細胞の中に安定的に含まれている)、次いで
b)前記植物を、前記プロモーターから前記ヌクレオチド配列の発現を引き起こさせるよう、その発育段階にとって十分な条件及び時間にわたって成長させ、これにより花粉形成が阻害されるか又は前記花粉が不活性されるように前記花粉生産細胞の中で有害な機能を有するポリペプチドを生産させること;
を含んで成る。
ベクターラムダ−gt11中のcDNA発現ライブラリーを、成熟ほそ麦花粉のポリアデニル化mRNAより調製した(Beall & Mitchell(1986) J.Immunol. Methods 86: 217−223)。このライブラリーをまずモノクローナル抗体(MAb)FMC−A1(40.1)でスクリーンした(図1a)。
実施例2−クローン化アレルゲン12Rの同定
この研究において用いた4種の MAbは全てクローン化アレルゲン12Rを認識した(図1a)。
実施例3−アレルゲンの花粉特異性発現
ポリA+ RNAを種々の植物組織、即ち、種子、葉、根及び花粉から単離した。これらの種々の組織に由来する20μgの全 RNAをホルムアミド及びホルムアルデヒドの存在下において1.2 %w/wのアガロースゲル上で電気泳動させ(Sambrook ら、前掲) 、ハイボンド−Cエキスラ(Amersham,Arlington Heights,1L) に移し、そして80℃で2hフィルターベークに付した。その1.2 kbの12R cDNA を32Pでラジオラベルし、そして50%v/vのホルムアルデヒドの存在下で65℃でNCフィルターとインキュベートした。この膜を0.1 %w/vの SDSを含む2Xの SSC(0.3MのNaCl、0.3 Mのクエン酸ナトリウム、pH7.0 )で65℃で洗った。タンパク質は種々の組織(花、葉、根及び花粉)を1mMのPMSFを含む10mMのPBS の中で粉砕し、次いで標識抗体でイムノブロット(レーン当り10μgのタンパク質)に付することにより単離した。結合性は MAbに対する125I−ヤギ抗マウス IgG(Amersham,Arlington Heights,1L) 及びポリクローナル125I−ヤギ抗−ヒト IgE(Kallestad,Chaska,MN.)を用い、次いでオートラジオグラフィーを行うことにより識別化させた。
実施例4−一次構造分析
cDNAクローン12Rを単離し、そしてpGEM−3zベクター(Promega,Madison,Wl) の中にサブクローンし、次に制限地図化に付した。様々なサイズの制限フラグメントをpGEMベクターの中にサブクローンした。
実施例5− IgE−及び Mab−反応性エピトープの描写
MAb及び IgE決定基の位置決めをするため、E.コリ組換発現系を利用した(Smith and Johnson(1988)Gene 67:31−40) 。この系を利用し、いくつかの制限フラグメントを発現プラスミドpGEX1−3の中にサブクローンした。pGEXの中への全長cDNAの「フレーム内」サブクローニングは、 IgE並びにMAb 40.1及び12.3の両者により認識される61kDの融合タンパク質を発現せしめた。
実施例6−ほそ麦花粉中の Lol p Ib の細胞内標的化
ほそ麦の成熟花粉を樹立された方法(Staffら(1990) HistochemJ.22: 276−290)に従って走査電子顕微鏡のために調製した。免疫細胞化学のため、成熟葯を無水条件のもとで2,2−ジメトキシプロパン中の0.1 %のグルタルアルデヒド、1%のパラホルムアルデヒドの中で4℃で2h固定し、次いで透過型電子顕微鏡のために処理した(Staffら、前掲)。この方法は水性媒質の中でのアレルゲンのその細胞部位からの拡散を抑えるために開発されている。ブロックを1%のベンジルを有するLP金樹脂の中で、−25℃でUVイルミネーションのもとで重合し、そして80nmの薄切片を金グリット上で拾い上げた。イムノラベリングはまず第一抗体MAb 12.3(Lol p Ibに特異的) 、次いで金−ヤギ−抗−マウス IgGプローブ(15nmの粒径)による。このラベルを40nmの粒径にまで銀増強させた(Danscher &Norgaard(1983)J.Histochem.Cytochem. 31:1394−1398の改良法) 。第2ラベリングは同じ切片上で、3種の MAb、3.2 、21.3及び40.1の混合物、それに続く15nmの粒径の金−ヤギ−抗マウス IgGプローブにより行った。前述した通りに実施した抗体特異性及び方法のコントロール(Staffら、前掲) はこれらの部位での全粒子を示さなかった。
実施例7−免疫系に対する Lol p Ib の提供
ほそ麦花が開花するとき、その葯は広がり、そしてこの花粉は各葯の基底において開いている孔を通じて大気中に放出される。ほそ麦はあらゆる草類のうちで最大の花粉生産を示し、ヘクタール当り約960 kgの抗原を刈り入れ又は放牧されていない牧草地における大気中へと放出させる。この花粉の99%がその起源の1km内に蓄積(及び再蓄積)する。草類の花粉は寿命が短いが、しかしそれは大気中で数日間残っていることがある、花粉は放出後数時間しか生存していないことが実験で示されている。
実施例8− Lol p Ib.1 に対するモノクローナル抗体
実施例5由来の融合タンパク質 GST−1Hに対するモノクローナル抗体(MAb) を、当業者に公知の技術(例えばKohler and Milstein 、前掲及びKohler and Milstein 、前掲を参照のこと) を利用して調製した。フラグメント1H(図4)によりコードされ、 IgE結合性ドメインに相当する融合タンパク質は抗原性である。
実施例9− Lol p Ib.2 をコードするcDNAクローン19Rの単離免疫学スクリーニング
実施例1におけるcDNA発現ライブラリーのデュープリケートフィルターをプールしたヒトアレルギー血清由来の特異的 IgEでスクリーンした。結合 IgEは125I−ラベル化抗−ヒト IgE(Kallestad Laboratories,Chaska,MN)を用いて検定した。両デュプリケートフィルター上の抗体陽性プラークを拾い、精製し、次いで再プレートに付し、そして MAbへの結合性について試験した。
DNAのサブクローニング及びシーケンシング
Messe,E.ら(1990)Nucleic Acids Res., 第18巻:1923に記載の液体リゼート法を利用して DNAをプラーク精製相から調製した。EcoRI消化より回収したインサートをpGEM 4-Z(Promega,Madison,W1)へとリゲートし、そしてpGEMベクター(pGEM4-Z) の中に様々なサイズの制限フラグメントとしてサブクローンした。全てのシーケンシングは二本鎖プラスミド鋳型を用いて行った。これらの鋳型はQuiagen,Inc., Chatsworth,CA,USA に記載の通りに調製した。ジデオキシシーケンシング(Sanger ら(1977)Proc.Natl. Acad.Sci.USA.74:5460−5463) を実施例4に記載の通りに実施した。クーデアザdITPを強いGCバンド圧縮を解くために用いた。シーケンシングはExo III 及びS1ヌクレアーゼによってインサートの両端から入れ子欠損を作り上げることにより助長した。必要に応じて内部シーケンシングプライマーを合成した。
配列分析
配列分析はメルボルンデーターベースシステム(MELBDBSYS) 、即ち、オーストラリア、メルボルン大学のWalterとEliza Hall,LuduigとHoward Florey Institutes及びPC Gene Clnttelligenetics,Mount-ain View,CA)で開発された分析プログラムコレクションを用いて実施した。このシステムは下記の起源由来のデーターベースを含んでいる:GenBank,EMBL、及びBPRF核酸ライブラリー;NBRF PRIタンパク質、 PSD−キョート(オオイ)、GBトランス、スイスプロット及びDoolittle タンパク質ライブラリー。探索中、EMBL及びGenBankデーターベースはそれぞれ28.0及び68.0リリースした。
i)提唱の翻訳開始部位とそのフランキング配列(ヌクレオチド21−29)とが単子葉植物の共通配列と89%の相同性を共有した。最も重要なヌクレオチド、即ちATG 出発コドン(図10aにおけるヌクレオチド21)に対して−3の位置のプリンが保存されていた(Cavener,D.R.,and Ray,S.C.(1991)Nucleic Acids Research, 19:3185−3192);
ii)cDNAは完全な3′−未翻訳領域である標準AATTAAポリアデニル化シグナル(Birnsteilら(1985)Cell,41: 349−359)、それに続くポリ(A)テールを有する;及び
iii )3′未翻訳領域はmRNA安定性にかかわるATTTA も含む。
花粉タンパク質の単離及びイムノブロッティング
可溶性タンパク質をほそ麦花粉から、PBS 及び1mMのフェニルメチルスルホニルフルオリドの中で氷上で3時間強く撹拌することにより抽出した。 SDS−PAGEのための条件は本質的にOng ら(1990)Int.Arch.Allergy Appl.lmmunol., 93: 338−343 に記載の通りである。電気泳動の直後、分離させたタンパク質を銀染色するか(Angorge,W.(1982) 「Electrophoresis'82:Advanced Methods,Bioc-hemical and Clinical Applications,Proceedings of the Interna-tional Conference on Electrophoresis,Athens,Greece, 1982年4月21−24日、編集者:D.Stathakos,Walter de Gruyeer,BerlinとNewYork, 1983年、頁 235−242)、又はニトロセルロースに4℃で移した(Towbin ら(1979)Proc.Natl.Acad.Sci.USA,76 :4350−4354) 。
IgE抗体のアフィニティー精製
アレルゲンをコードするラムダgt11ファージ中のcDNAクローンをエッシェリヒアコリの中で融合タンパク質として発現させた。組換融合タンパク質(rfp) を含むプラークリフトを次にプール血清の中でインキュベートした。結合 IgE抗体を 0.2MのグリシンHCl,pH2.6 /0.5 %のBSA /0.1 %のアジ化ナトリウムで溶離させ、そしてウェスタンブロットをプローブするために用いた。 IgE結合は125I−ラベル化抗−ヒト IgE(Kallestad, Chaska,MN)、次いでオートラジオグラフィーを用いて識別させた(Ongら、Int.Arch.Allergy Appl.Immunol.,93 :338 −343)。
RNAブロットハイブリダイゼーション
RNAゲルブロット分析のため、全 RNAを20mMの3−(N−モルホリノ)−プロパンスルホン酸、50%の脱イオンホルムアミド及び2.2 Mのホルムアミドの中で65℃で5分間変性させ、2.2 Mのホルムアルデヒドを含む1.2 %のアガロースゲルで電気泳動させ、そしてニトロセルロースにエレクトロブロットさせた。 RNAスロットブロット分析は、全 RNAを20mMの3−(N−モルホリノ)プロパンスルホン酸、5mMの酢酸ナトリウム及び1mMのEDTAの中で65℃で10分間変性させ、そのサンプルを20XのSSC (SSC−3Mの塩化ナトリウム、1.0 Mのクエン酸ナトリウムで飽和せしめたミニホールド11濾過用マニホールド(Schleicher & Schuell,Dassel,Germany) の中に納めたニトロセルロース上に適用することで実施した。両フィルターは50%の脱イオン化ホルムアミド、2XのSSPE (SSPE−3Mの塩化ナトリウム、0.2 Mのリン酸ナトリウム、0.02MのEDTA) 、1%のドデシル硫酸ナトリウム(SDS) 、0.5 %のBlotto(リン酸バッファー食塩水中の10%の脱脂乳)、10%の硫酸デキストラン、及びオリゴラベリングキット(Bresatec,Adelaide,Australia) を用いてランダムオリゴヌクレオチドプライミングにより調製した。32P−ラベル化cDNAプローブを含む溶液の中で42℃で2〜6時間にわたり予備ハイブリダイズさせておいた。これらのフィルターを2XのSSC 、0.1%のSDS で42℃で2時間で4回洗い、次いでX線フイルムに暴露させた。
実施例10〜Lol p Iアレルゲンの特性化
材料及び方法
花粉をGreer Laboratories,Lenoir,NCより入手した。可溶液タンパク質をGriffithら(1991)FEBS Letters, 279: 210−215 に記載の通りに抽出した。粗花粉抽出物を獲得し、そしてそのタンパク質濃度をOng ら(1990)Int.Arch.Allergy Appl. Immunol.,93: 338−343に記載の通りに決定した。MAb LpIX3A及びLpIX4Aを実施例8に記載の通りに、クローン12Rの IgE結合部分によりコードされる組換タンパク質に対して発生せしめた。MAb 7Eは実施例8に記載の通り Lol p Ia 及び実施例1に記載の通りFMC-A1に特異的であった。
二次元ゲル電気泳動及びイムノブロット分析
2次元(2D)−PAGEをミニ−プロテアンII2−Dセル(Biorad,Richmond,CA)において、その製造者の仕様書に従って実施した。タンパク質を9%のCHAPS の中で1:1に希釈した。ゲル当り13mgのタンパク質のアリコートを適用し、そしてサンプルの上に一次方向サンプルオーバーレイバッファーを載せた。一次元ゲルを3.5 時間泳動させた。二次元ゲルは45分泳動させた。2D−PAGEゲル上のタンパク質を銀染色してタンパク質プロフィールを表示させた。
花粉抽出物中のアレルギーアイソフォームの同定
アレルギー個体の血清による、 SDS−PAGEにより分けた花粉タンパク質のウェスタンブロットのプロービングは IgEに結合する28〜35kDのMW範囲にある4種の分子量でのタンパク質バンドを示した。2D−ゲルのウェスタンブロットの類似の処理はこれら4本のバンドを、図15に示す通り12のアレルゲンスポットへと分解した。多数の MAb及び IgE調製品を利用することにより、これらのアレルゲン間の抗原性の関係を研究した。
新鮮な頭状花をオーストラリアのほそ麦草から集め、凍結し、そして米国に輸送した。500 mgの頭状花をドライアイス上の乳鉢及び乳棒によりつぶし、そして0.1 %のDEPCでFrankis and Mascarhenas(1980)Ann.45:595-599に記載の通りに一夜処理した0.2MのNaCl、1mMのEDTA、0.1 %の SDSを有する50mMのトリスpH9.0 5mlの中に懸濁した。フェノール/クロロホルム/イソアミルアルコール(25:24:1で混合)で1回の抽出の後、この材料をフェノール/クロロホルム/イソアミルアルコール中で60秒間音波処理し、そして再抽出した。音波処理を3回目の抽出において30秒間繰り返した。最後の2回の抽出は音波処理抜きで行った。RNA が 0.1容量の2Mの酢酸ナトリウム及び2容量のエタノールを有する水性相から沈殿した。そのペレットを遠心により回収し、dH2Oの中に再懸濁し、そして65℃で5分間熱した。2mlの4Mの塩化リチウムを RNA調製品に加え、そして0℃で一夜沈殿させた。この RNAペレットを遠心により回収し、1mlのdH2Oの中に再懸濁し、そして再び3Mの酢酸及びエタノールにより氷上で3時間かけて沈殿させた。最終ペレットを70%のエタノールで洗い、風乾し、そして 100μlのDEPC処理H2O の中に再懸濁し、次いで−80℃で保存した。
鋳型の変性 DNA、94℃、1分;オリゴヌクレオチドのアニール、65℃、1分30秒;伸長、72℃、2分;24サイクル反復;4℃に保持。
Claims (11)
- 前記核酸配列が、
(a)請求項1に示している、ほそ麦アレルゲン(Lol p Ib.2)のアミノ酸−25〜314、または
(b)請求項1に示している、ほそ麦アレルゲン(Lol p Ib.2)のアミノ酸1〜314、をコードする請求項1に記載の核酸。 - 請求項1または2に記載の核酸配列を含んで成る発現ベクター。
- 請求項1または2に記載の核酸によりコードされるタンパク質またはペプチドを発現するように形質転換された宿主細胞。
- ほそ麦タンパク質アレルゲンLol p Ib.2に特異的なT細胞を刺激することができる単離ほそ麦タンパク質アレルゲン(Lol p Ib.2)の製造方法であって、請求項4に記載の宿主細胞を適宜培地中で培養し、細胞と、前記ほそ麦タンパク質アレルゲン(Lol p Ib.2)を含む培地との混合物を作り;任意に前記混合物を精製して精製ほそ麦タンパク質アレルゲン(Lol p Ib.2)を製造することを含んで成る方法。
- 請求項1または2に記載の核酸によってコードされ、且つほそ麦タンパク質アレルゲンLol p Ib.2に特異的なT細胞を刺激することができる、ほそ麦タンパク質アレルゲン(Lol p Ib.2)。
- 請求項1に示しているアミノ酸配列から成る請求項6に記載のほそ麦タンパク質アレルゲン(Lol p Ib.2)。
- 化学的に合成される、請求項6または7に記載のアレルゲン。
- 天然源から単離される請求項6または7に記載のアレルゲン。
- 請求項1または2に記載の核酸で形質転換した宿主細胞中で製造される、請求項6または7に記載のアレルゲン。
- ほそ麦花粉アレルゲンに対する哺乳動物の感受性を検出するためのin vitro 方法であって、血液成分とタンパク質アレルゲンとの結合のために適した条件下で、前記哺乳動物から得られた血液サンプルを、請求項6〜10のいずれか1項に記載の単離タンパク質アレルゲンと組合せて、そしてかかる結合が生じた度合いを決定し;任意にT細胞機能、T細胞増殖、B細胞機能、血中に存在する抗体に対するタンパク質アレルゲンの結合を評価することにより、結合が生じた度合いを決定すること、を含んで成る方法。
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US74670291A | 1991-08-16 | 1991-08-16 |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP5503961A Division JPH06509941A (ja) | 1991-08-16 | 1992-08-14 | ほそ麦花粉アレルゲン |
Publications (2)
Publication Number | Publication Date |
---|---|
JP2007054071A JP2007054071A (ja) | 2007-03-08 |
JP4150050B2 true JP4150050B2 (ja) | 2008-09-17 |
Family
ID=25001978
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP5503961A Withdrawn JPH06509941A (ja) | 1991-08-16 | 1992-08-14 | ほそ麦花粉アレルゲン |
JP2006242106A Expired - Lifetime JP4150050B2 (ja) | 1991-08-16 | 2006-09-06 | ほそ麦花粉アレルゲン |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP5503961A Withdrawn JPH06509941A (ja) | 1991-08-16 | 1992-08-14 | ほそ麦花粉アレルゲン |
Country Status (9)
Country | Link |
---|---|
EP (1) | EP0665888B2 (ja) |
JP (2) | JPH06509941A (ja) |
KR (1) | KR100263393B1 (ja) |
AT (1) | ATE260342T1 (ja) |
AU (1) | AU651728B2 (ja) |
CA (1) | CA2115579C (ja) |
DE (1) | DE69233311T3 (ja) |
NZ (2) | NZ270897A (ja) |
WO (1) | WO1993004174A1 (ja) |
Families Citing this family (16)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5736362A (en) | 1990-10-26 | 1998-04-07 | The University Of Melbourne | Ryegrass pollen allergen |
NZ254688A (en) * | 1992-08-14 | 1997-11-24 | Univ Melbourne | Lol p v grass pollen allergens, peptides containing t-cell binding sites, detection of sensitivity, antibodies and recombinant production of the allergens |
US5480972A (en) * | 1992-10-30 | 1996-01-02 | The University Of Melbourne | Allergenic proteins from Johnson grass pollen |
US5710126A (en) * | 1993-03-12 | 1998-01-20 | Immulogic Pharmaceutical Corporation | T cell epitopes of ryegrass pollen allergen |
NZ263913A (en) * | 1993-03-12 | 1997-10-24 | Immulogic Pharma Corp | Lol pi protein allergen from ryegrass and related peptides coding sequences, vectors, protein production and use |
AU7559794A (en) * | 1993-08-13 | 1995-03-22 | Immulogic Pharmaceutical Corporation | T cell epitopes of ryegrass pollen allergen |
US5882851A (en) * | 1993-12-08 | 1999-03-16 | Novartis Finance Corporation | Cytochrome P-450 monooxygenases |
US7112333B1 (en) | 1994-08-05 | 2006-09-26 | Heska Corporation | T cell epitopes of ryegrass pollen allergen |
AUPN056395A0 (en) * | 1995-01-16 | 1995-02-09 | University Of Melbourne, The | Production of genetically transformed forage grasses |
FR2809416B1 (fr) * | 2000-05-29 | 2004-12-10 | Tabacs & Allumettes Ind | Clonage et sequencage de l'allergene dac g5 du pollen de dactilys glomerata, sa preparation et son utilisation |
AUPR779201A0 (en) | 2001-09-20 | 2001-10-11 | University Of Melbourne, The | Immunotherapeutic and immunoprophylactic reagents |
AU2003903132A0 (en) | 2003-06-20 | 2003-07-03 | Molecular Plant Breeding Nominees Ltd. | Plant promoter |
AU2004249788B2 (en) * | 2003-06-20 | 2008-08-21 | Agriculture Victoria Services Pty Ltd | Ryegrass pollen-specific promoters and expression constructs |
EP2393830B8 (en) | 2009-02-05 | 2015-03-18 | Circassia Limited | Grass peptides for vaccine |
KR101929388B1 (ko) | 2017-11-10 | 2018-12-17 | 대한민국 | 이탈리안라이그라스 추출 분획물을 유효성분으로 함유하는 간질환의 예방, 치료 또는 개선용 조성물 |
CN113198540A (zh) * | 2021-04-12 | 2021-08-03 | 青岛科技大学 | 一种用于香草醛加氢脱氧的mof基复合材料催化剂的制备方法及其应用 |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA1340729C (en) * | 1988-03-23 | 1999-09-07 | Mohan Bir Singh | Ryegrass pollen allergen |
DK0576426T3 (da) * | 1990-08-17 | 1997-12-15 | Univ Melbourne | Rajgræspollenallergen |
-
1992
- 1992-08-14 AT AT92917524T patent/ATE260342T1/de active
- 1992-08-14 EP EP92917524A patent/EP0665888B2/en not_active Expired - Lifetime
- 1992-08-14 DE DE69233311T patent/DE69233311T3/de not_active Expired - Lifetime
- 1992-08-14 JP JP5503961A patent/JPH06509941A/ja not_active Withdrawn
- 1992-08-14 WO PCT/AU1992/000430 patent/WO1993004174A1/en active IP Right Grant
- 1992-08-14 NZ NZ270897A patent/NZ270897A/en unknown
- 1992-08-14 CA CA002115579A patent/CA2115579C/en not_active Expired - Lifetime
- 1992-08-14 AU AU24409/92A patent/AU651728B2/en not_active Expired
- 1992-08-14 KR KR1019940700472A patent/KR100263393B1/ko not_active IP Right Cessation
- 1992-08-14 NZ NZ243956A patent/NZ243956A/en unknown
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2006
- 2006-09-06 JP JP2006242106A patent/JP4150050B2/ja not_active Expired - Lifetime
Also Published As
Publication number | Publication date |
---|---|
KR100263393B1 (ko) | 2000-08-01 |
CA2115579C (en) | 2008-03-25 |
NZ243956A (en) | 1997-05-26 |
WO1993004174A1 (en) | 1993-03-04 |
NZ270897A (en) | 1997-06-24 |
JP2007054071A (ja) | 2007-03-08 |
EP0665888A4 (en) | 1994-11-23 |
DE69233311D1 (de) | 2004-04-01 |
EP0665888B1 (en) | 2004-02-25 |
AU651728B2 (en) | 1994-07-28 |
EP0665888B2 (en) | 2007-07-25 |
CA2115579A1 (en) | 1993-03-04 |
DE69233311T2 (de) | 2005-01-20 |
AU2440992A (en) | 1993-03-16 |
DE69233311T3 (de) | 2008-01-17 |
JPH06509941A (ja) | 1994-11-10 |
ATE260342T1 (de) | 2004-03-15 |
EP0665888A1 (en) | 1995-08-09 |
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