text:
Az Unternehmen Silberfuchs (magyarul: „Ezüstróka” hadművelet) német hadművelet volt a második világháború idején, mely céljául finn területről kiindulva Murmanszk szovjet kikötőjének elfoglalását tűzte ki.
 Tervezés és felkészülés
 1940 júliusa óta Németország – felkészülve a szovjet-finn háború kiújulására – a Renntier (Rénszarvas)-hadművelet keretében tervezte a petsamói nikkelbányák elfoglalását. A Birodalom 1940 szeptemberétől fogva lehetőséget kapott, hogy csapatokat és utánpótlást szállítson Észak-Finnországon át. 1941 januárjában Erich Buschenhagen német tisztet Finnországba küldték, hogy a házigazdákkal megvitassa egy finn irányítás alatt végrehajtandó, a Szovjetunió ellen irányuló közös német-finn katonai együttműködés lehetőségét. Február végén Buschenhagen felhatalmazást kapott, hogy összehangolt hadmozdulatokról tárgyaljon Helsinkiben. A katonai együttműködési megállapodásnak köszönhetően a németek előkészítették norvégiai haderejük zömének Finnországba csoportosítását. Ezeket a hadműveleteket (Blaufuchs I és Blaufuchs II) 1941-ben kezdték végrehajtani. Öt német hadosztály és több kiegészítő erő (köztük két különleges célú páncélosegység) foglalta el hadállásait Észak-Finnországban, és csatlakoztak a hadgyakorlat címén a határ mentén mozgósított finn erőkhöz.
 A finnek és a németek végül egy két fő csapásirányt magában foglaló, három szakaszra bontott offenzíva tervében állapodtak meg. Az első cél a petsamói régió elfoglalása volt, mely feladat végrehajtásával az Eduard Dietl vezérezredes parancsnoksága alatt álló Norwegen hegyivadász hadtest két hadosztályát bízták meg. Ezzel a Kirkenes környékén állomásozó hegyivadászok alkalmas helyzetbe kerültek volna, hogy támadást indítsanak Murmanszk ellen.
 A második és harmadik szakaszt egyidejűleg kívánták elindítani. Az északi irányú támadás (Platinfuchs-hadművelet) során a „Norwegen” hegyivadász hadtest harcoló alakulatainak, kiegészülve a finn Ivalo határőrzászlóaljjal Petsamótól keleti irányban kellett előrenyomulniuk a tengerparton a Barents-tenger és Murmanszk-felé. Erre a hadmozdulatra jóval az északi sarkkörtől északra került sor, ezért a terep és az időjárás rendkívüli módon megnehezítette a hadmozdulatokat. Az egész hadműveletből ez a kihívás kecsegtette a sikerre vonatkozó legkisebb esélyekkel a finn-német erőket. Bár Murmanszk a háború későbbi szakaszában a Szovjetunió egyik létfontosságú köldökzsinórjává vált nyugati szövetségesei felé, 1941 elején a németek csak másodlagos célpontnak tekintették. Ugyan az előírt cél Murmanszk elfoglalása volt, a Platinfuchs fő hasznának az orosz csapatok lekötését tekintették, hogy megakadályozzák déli irányú átcsoportosításukat Leningrád felé.
 A Polarfuchs (Sarkiróka) fedőnévre keresztelt déli irányú támadást átkaroló hadmozdulatnak szánták kiötlői. Hans Feige lovassági tábornok XXXVI. hadtestének a két különleges célú páncélosegységgel kiegészítve Kuusamo vidékéről kellett kelet felé nyomulnia a Salla-Urinsalmo vonalon. E hadmozdulat célja a Fehér-tenger partján fekvő karéliai Kandalaksa városának elfoglalása volt, egyúttal lekötve a szovjet Északi Front egységeit és elvágva Murmanszkot és a Kola-félszigetet a Szovjetunió többi részétől.
 Hasonlóképp a finn karéliai hadsereg délre, a Ladoga-tó és a Szvir-folyó irányába tervezte saját offenzíváját.
 A Renntier-hadművelet
 A Renntier, az offenzíva első fázisa 1941. június 22-én, a Barbarossa hadművelettel egyidőben kezdődött. A „Norwegen” hadtest 2. és 3. hegyivadász hadosztálya Kirkenesből a finn ellenőrzés alatt álló Petsamóba indult. A hadművelet sikeresnek bizonyult, a szovjet határőregységek meglepődve vették tudomásul a hegyivadászok megjelenését a tőszomszédságukban. Dietl csapatai harckészültségben várakoztak a Platinfuchs elindítására. Délen pedig Feige XXXVI. hadteste folytatta az előkészületeket a támadásra. Június 25-én a szovjetek nagyobb lélegzetű légitámadásra szánták el magukat, ami Finnország hadüzenetét vonta maga után.
 Kettős támadás
 Június 29-én, a Polarfuchs és a Platinfuchs is elkezdődött.
 Északon Dietl támadásával szemben két szovjet hadosztály sorakozott fel. A táj kihalt holdbéli vidékre hasonlított. A hideg miatt sem fák, sem érdemleges vegetáció nem nőtt a sík terepen. A felszínt nagy kiterjedésű sziklagörgetegek tették járhatatlanná a gépjárművek és a harckocsik számára. Mivel a vidéken lényegében teljesen hiányzott az úthálózat, a németek kénytelenek voltak azt saját maguk kiépíteni. A járművek mozgása ezekre a hevenyészett utakra korlátozódott, amivel eszményi célpontul kínálkoztak a szovjet tüzérség számára. Az utánpótlási nehézségek leküzdése érdekében a németek Görögországból származó teherhordó állatokat használtak, amelyekkel azonban gyorsan végzett a zord klíma. A támadás lelassult, majd elakadt, ami főleg az utánpótlási problémák eredménye volt. A német-finn ellátmánynak a legközelebbi vasútvonaltól több mint 500 km-t kellett megtennie, míg a szovjetek mindössze 60 km-re álltak Murmanszktól és a vasútról leágazó vakvágányok is segítették a helyzetüket. Ahogy a hegyivadászok lassan előrenyomultak, a szovjetek megerősítették a frontszakaszt egy újabb hadosztállyal és számos murmanszki tengerészgyalogos egységgel. A szovjetek így számbeli fölényben és alaposan beásva nyugodtan nézhettek szembe a támadókkal. Miután Dietl számos sikertelen kísérletet tett, hogy átkeljen a Licsa-folyón, szeptember 22-én kénytelen volt jelenteni, hogy a Platinfuchs kudarcot vallott. Miután a hegyivadászok felhagytak a támadó hadműveletekkel és beásták magukat, a frontvonalak megmerevedtek. A háború hátralevő részére a helyzet nagyjából stabilizálódott és a napi harccselekmények javarészt a síléces járőrszolgálatra korlátozódtak.
 Délen a Polarfuchs a Platinfuchsszal egyidőben vette kezdetét. A terep, amelyet a XXXVI. hadtestnek le kellett küzdeni, nagyban különbözött az északi területektől. A Kuusamo környéki régió egyetlen hatalmas erdőség volt, toronymagas fákkal és dús aljnövényzettel, ami itt-ott patakok és kisebb-nagyobb mocsarak tagoltak. A városokban és a javarészt megművelt földekkel övezett német vidéken nevelkedett támadók harci morálját nagymértékben rombolta ez a hadszíntér. Bár a terep különbözött az északitól, a behatolók itt sem értek el nagyobb sikereket, főként mert a gépjárművek itt is a ritkásan futó utakra kényszerültek, kitéve magukat a szovjet elhárító tüzérség csapásainak.
 A Nord SS-Kampfgruppét Märkäjärvi és Salla faluk között vetették be. Bár a parancsnokuk tiltakozott a támadásra való utasítás ellen a csapatok alacsony harckészültsége miatt, felettesei ezt nem vették figyelembe. A Kampfgruppe a harcok során gyenge teljesítményt nyújtott és két nap alatt súlyos, 700 fős veszteséget szenvedve szó szerint megtört az ellenség vonalai előtt. A Kandalaksha irányába való előrenyomulás lelassult, majd megállt, Feige csapatai beásták magukat és a front, a járőrözést és az esetenkénti rajtaütéseket leszámítva stabilizálódott. Végül Hitler parancsára a németek felhagytak a támadó hadmozdulatokkal.
 Következmények
 A Silberfuchs kudarca jelentős és tartós hatást gyakorolt a háború menetére. Míg a szovjet vonalak zöme 1941-ben összeomlott, Panyin Északi Frontja kitartott, mintegy 15%-os veszteséget okozva a németeknek. A német balsikernek számos összetevője volt: legelsősorban a terepviszonyok, amelyek jelentős mértékben lassították az előrenyomulást, ráadásul az offenzíva során nem jelölték ki azt az áttörési pontot (Schwerpunkt), amely a Blitzkrieg sikeres folytatásának előfeltétele. Ennek hiányában a frontvonal megmerevedése és a katonai helyzet kiegyensúlyozottsága elkerülhetetlenné vált.
 Murmanszk kikötője a háború egész ideje alatt szovjet kézben maradt és a kölcsönbérleti szerződés révén a Szovjetunióba érkező hadianyag mintegy egynegyedét ezen a kikötőn keresztül fogadták (a fennmaradó háromnegyed rész fő útvonalait Vlagyivosztok és Szibéria, Perzsia, valamint kis részben a Fekete-tenger jelentették. Ezek a szállítmányok tették lehetővé, hogy a szovjetek gyorsan erőre kapjanak az 1941-es katasztrófa után.
 A háború északon 1944 szeptemberéig tartott, amikor a finnek békét kértek és megkezdődött a lappföldi háború, mindazonáltal ezen a fronton a döntő győzelem kicsikarása egyik fél számára sem vált lehetővé a harcok során.
 Jegyzetek
 Források
 Bibliográfia
 Külső hivatkozások
 A Szovjetunió elleni hadműveletek a második világháborúban
 A Harmadik Birodalom hadműveletei a második világháborúban
 Lappföld
 1941


type: wikipedia

text:
 Biografia
 Originario di Pärnu, è salito alla ribalta grazie al singolo Nagu kuu, che ha raggiunto la 19a posizione della Eesti Tipp-40 ed ha trascorso tre mesi in top forty. Il secondo album in studio dell'artista, intitolato Suhted e messo in commercio attraverso la Masterhead Records, parte del gruppo di Believe, è uscito nel 2020 ed ha esordito al 4o posto della graduatoria nazionale, rimanendovi per altre undici settimane fino al cessamento della classifica avvenuto a settembre del medesimo anno. Agli Eesti Muusikaauhinnad, il principale riconoscimento musicale dell'Estonia, è risultato finalista nella categoria Artista dell'anno.
 Discografia
 Album in studio
 2019 – Segamini
 2020 – Suhted
 2021 – Emotsioonide vine all
 Singoli
 2018 – Minu soov
 2018 – Koju ära
 2019 – Mul on saab
 2019 – Kallis
 2019 – Oks laiali
 2019 – Suvi la
 2019 – Slaavikükk
 2019 – Teeme streaki
 2019 – Tossud
 2019 – Nagu kuu
 2020 – Linn on tühi
 2020 – Narkomaan
 2020 – Maitea
 2020 – Olla sinuga
 2020 – Lõpupidu
 2020 – Škoda (con Samoginson)
 2020 – Lähme lendama
 2020 – Nii ruttu
 2021 – Mis siis on
 2021 – Kõik on cool
 2021 – Skafandris
 2021 – Magada ei saa
 2021 – Ülihea oli
 2021 – Algus on llus
 Note
 Collegamenti esterni


type: contracts

jurisdiction: EU

text:
 Avis juridique important
 |
 21980A1017(01)
 Aftale i form af brevveksling mellem Det Europæiske Økonomiske Fællesskab og Den Argentinske Republik om handel med fåre- og lammekød
 EF-Tidende nr. L 275 af 18/10/1980 s. 0014 - 0019 den finske specialudgave: kapitel 3 bind 12 s. 0148 den svenske specialudgave: kapitel 3 bind 12 s. 0148 den græske specialudgave: Kapitel 11 bind 22 s. 0004 den spanske specialudgave: Kapitel 03 bind 19 s. 0103 den portugisiske specialudgave: Kapitel 03 bind 19 s. 0103
 AFTALE i form af brevveksling mellem Det europaeiske oekonomiske Faellesskab og Den argentinske Republik om handel med faare- og lammekoed Brev nr. 1 Hr. ..., Jeg har den aere hermed at henvise til de forhandlinger, som vore respektive delegationer for nylig har foert med henblik paa at udarbejde bestemmelser for indfoersel til Det europaeiske oekonomiske Faellesskab af faare-, lamme- og gedekoed fra Argentina, og som har fundet sted samtidig med Faellesskabets ivaerksaettelse af den faelles markedsordning for faare- og gedekoed. Under disse forhandlinger vedtog de to parter foelgende: 1. Denne aftale omfatter: - fersk eller koelet faare-, lamme- og gedekoed (02.01 A IV a)); - frosset faare-, lamme- og gedekoed (02.01-A IV b)). 2. Som led i denne aftale fastsaettes den maengde faare-, lamme- og gedekoed, der aarligt kan udfoeres fra Argentina til Faellesskabet, til : 20 000 tons, udtrykt i slagtet vaegt (1). For at sikre, at aftalen fungerer korrekt, paatager Argentina sig at ivaerksaette de foranstaltninger, som er noedvendige for at sikre, at den aarlige faktisk udfoerte maengde ikke overstiger den aftalte maengde, og at den udfoeres i overensstemmelse med den traditionelle tilbudsform (frosset eller koelet). Goer aendringer inden for teknologi eller handel det muligt at aendre den handelsmaessige tilbudsform, konsulterer aftalens to parter inden eventuelle aendringer hinanden i det udvalg, der omhandles i klausul 10, for at finde en tilfredsstillende loesning. 3. Goer Faellesskabet brug af beskyttelsesklausulen, forpligter det sig til ikke at lade dette faa indvirkning paa Argentinas adgang til Faellesskabets marked i henhold til denne aftale. 4. Overstiger indfoerselen fra Argentina et aar de aftalte maengder, forbeholder Faellesskabet sig ret til at suspendere indfoerselen fra Argentina resten af det paagaeldende aar. Den overskydende maengde traekkes fra den maengde, som Argentina har ret til at udfoere det paafoelgende aar. 5. Faellesskabet forpligter sig til at begraense den afgift, der paalaegges indfoerte produkter, der er omfattet af denne aftale, til hoejst 10 % ad valorem. (1)Slagtet vaegt (ikke-udbenet vaegtaekvivalent). Ved dette udtryk forstaas vaegten af uudbenet koed som saadant samt udbenet koed omregnet ved hjaelp af en koefficient til uudbenet vaegt. Saaledes svarer 55 kg udbenet faarekoed til 100 kg uudbenet faarekoed og 60 kg udbenet lammekoed til 100 kg uudbenet lammekoed. 6. Naar nye medlemsstater tiltraeder, aendrer Faellesskabet efter konsultationer med Argentina de i klausul 2 fastsatte maengder i forhold til Argentinas handel med hver ny medlemsstat. Afgifterne ved indfoersel til disse nye medlemsstater fastsaettes i henhold til tiltraedelsestraktatens bestemmelser, idet der tages hensyn til den den begraensning af afgiften, som er fastsat i klausul 5. Den samlede maengde, som omhandles i klausul 2, fastsaettes til 23 000 tons udtrykt i slagtet vaegt fra datoen for Graekenlands tiltraedelse af Faellesskabet. 7. Faellesskabet bestraeber sig paa at undgaa enhver markedsudvikling, der kan vaere til skade for afsaetningen af faare- og lammekoed fra Argentina paa Faellesskabets marked inden for de aftalte maengder. Faellesskabet traeffer specielt foranstaltninger, der skal sikre, at afsaetning af interventionslagre af frosset koed i henhold til forordningen ikke modvirker en saadan afsaetning af argentinsk faare- og lammekoed. 8. I betragtning af formaalene med og bestemmelserne i denne aftale erkender Faellesskabet, at restitutioner eller anden form for stoette til udfoersel af faare- og lammekoed samt til levende faar og lam beregnet til slagtning kun maa tages i anvendelse til priser og paa betingelser, der opfylder de bestaaende internationale forpligtelser, og under overholdelse af Faellesskabets traditionelle andel af verdenshandelen med disse produkter. Disse ord fortolkes i overensstemmelse med artikel XVI i GATT og navnlig i overensstemmelse med artikel 10, stk. 2, litra c), i overenskomsten om fortolkning og anvendelse af artikel VI, XVI og XXIII i Den almindelige Overenskomst om Told og Udenrigshandel. 9. Argentina soerger for, at denne aftale overholdes, hvilket blandt andet indebaerer udstedelse af eksportlicenser for de i klausul 1 naevnte produkter inden for de maengder, der er omfattet af denne aftale. Faellesskabet vedtager paa sin side alle noedvendige bestemmelser, for at der kun kan udstedes importlicenser for ovennaevnte produkter med oprindelse i Argentina, naar der fremlaegges en eksportlicens udstedt af en ansvarlig myndighed, der er udpeget af Argentinas regering. Gennemfoerelsesbestemmelserne til denne aftale fastlaegges saaledes, at det ikke er paakraevet at stille sikkerhed for udstedelse af importlicens for de paagaeldende produkter. Det fastsaettes ligeledes i gennemfoerelsesbestemmelserne, at den ansvarlige argentinske myndighed med mellemrum giver Faellesskabets ansvarlige myndighed meddelelse om, hvor store maengder der er udstedt eksportlicenser for i givet fald specificeret efter bestemmelsessted. 10. Der nedsaettes et raadgivende udvalg sammensat af repraesentanter fra Faellesskabet og Argentina. Udvalgets opgave er at sikre, at aftalen anvendes korrekt og fungerer gnidningsloest. Udvalget undersoeger regelmaessigt udviklingen paa de to parters markeder for faare-, lamme- og gedekoed og udviklingen paa det internationale marked samt afsaetningsforholdene paa disse markeder, herunder forhold, der har betydning for de maal, der er anfoert i klausul 7. Udvalget sikrer, at udfoersel til Faellesskabet af produkter fremstillet af faare-, lamme- og gedekoed henhoerende under toldpositioner, der ikke er omhandlet i denne aftale, ikke forhindrer en korrekt anvendelse af aftalen. Udvalget droefter alle spoergsmaal, der kan opstaa i forbindelse med aftalens gennemfoerelse, og anbefaler de ansvarlige myndigheder passende loesninger. 11. Bestemmelserne i denne aftale aendrer ikke parternes rettigheder og forpligtelser under GATT. 12. Den aarlige maengde, der er fastsat i klausul 2, gaelder for tidsrummet 1. januar til 31. december. Den maengde, der gaelder fra aftalens ikrafttraeden og indtil den 1. januar det foelgende aar, astsaettes som en forholdsmaessig andel af den samlede aarlige maengde under hensyn til, at handelen er saesonbestemt. 13. Denne aftale gaelder paa den ene side for de omraader, hvor traktaten om oprettelse af Det europaeiske oekonomiske Faellesskab finder anvendelse, paa de betingelser, der er fastsat i den paagaeldende traktat, og paa den anden side for Den argentinske Republiks omraade. 14. Aftalen traeder i kraft den 20. oktober 1980. Den gaelder indtil den 31. marts 1984, hvorefter den med et aars skriftlig varsel kan opsiges af en af parterne. Under alle omstaendigheder gennemgaar de to parter aftalens bestemmelser inden den 1. april 1984 med henblik paa aendringer, som de maatte findenoedvendige. Jeg ville vaere Dem taknemmelig, saafremt De over for mig vil bekraefte, at Deres regering er indforstaaet med det ovenfor anfoerte. Modtag, hr. ..., forsikringen om min mest udmaerkede hoejagtelse. Paa vegne Raadet for De europaeiske Faellesskaber Brev nr. 2 Hr. ..., Jeg har den aere hermed at anerkende modtagelsen af Deres skrivelse af dags dato med foelgende ordlyd: Jeg har den aere hermed at henvise til de forhandlinger, som vore respektive delegationer for nylig har foert med henblik paa at udarbejde bestemmelser for indfoersel til Det europaeiske oekonomiske Faellesskab af faare-, lamme- og gedekoed fra Argentina, og som har fundet sted samtidig med Faellesskabets ivaerksaettelse af en faelles markedsordning for faare- og gedekoed. Under disse forhandlinger vedtog de to parter foelgende: 1. Denne aftale omfatter: - fersk eller koelet faare-, lamme- og gedekoed (02.01 A IV a)); - frosset faare-, lamme- og gedekoed (02.01 A IV b)). 2. Som led i denne aftale fastsaettes den maengde faare-, lamme- og gedekoed, der aarligt kan udfoeres fra Argentina til Faellesskabet, til : 20 000 tons, udtrykt i slagtet vaegt (1). For at sikre, at aftalen fungerer korrekt, paatager Argentina sig at ivaerksaette de foranstaltninger, som er noedvendige for at sikre, at den aarlige faktisk udfoerte maengde ikke overstiger den aftalte maengde, og at den udfoeres i overensstemmelse med den traditionelle tilbudsform (frosset eller koelet). Goer aendringer inden for teknologi eller handel det muligt at aendre den handelsmaessige tilbudsform, konsulterer aftalens to parter inden eventuelle aendringer hinanden i det udvalg, der omhandles i klausul 10, for at finde en tilfredsstillende loesning. 3. Goer Faellesskabet brug af beskyttelsesklausulen, forpligter det sig til ikke at lade dette faa indvirkning paa Argentinas adgang til Faellesskabets marked i henhold til denne aftale. 4. Overstiger indfoerselen fra Argentina et aar de aftalte maengder, forbeholder Faellesskabet sig ret til at suspendere indfoerselen fra Argentina resten af det paagaeldende aar. Den overskydende maengde traekkes fra den maengde, som Argentina har ret til at udfoere det paafoelgende aar. 5. Faellesskabet forpligter sig til at begraense den afgift, der paalaegges indfoerte produkter, der er omfattet af denne aftale, til hoejst 10 % ad valorem. 6. Naar nye medlemsstater tiltraeder, aendrer Faellesskabet efter konsultationer med Argentina de i klausul 2 fastsatte maengder i forhold til Argentinas handel med hver ny medlemsstat. (1)Slagtet vaegt (ikke-udbenet vaegtaekvivalent). Ved dette udtryk forstaas vaegten af uudbenet koed som saadant samt udbenet koed omregnet ved hjaelp af en koefficient til uudbenet vaegt. Saaledes svarer 55 kg udbenet faarekoed til 100 kg uudbenet faarekoed og 60 kg udbenet lammekoed til 100 kg uudbenet lammekoed. Afgifterne ved indfoersel til disse nye medlemsstater fastsaettes i henhold til tiltraedelsestraktatens bestemmelser, idet der tages hensyn til den begraensning af afgiften, som er fastsat i klausul 5. Den samlede maengde, som omhandles i klausul 2, fastsaettes til 23 000 tons udtrykt i slagtet vaegt fra datoen for Graekenlands tiltraedelse af Faellesskabet. 7. Faellesskabet bestraeber sig paa at undgaa enhver markedsudvikling, der kan vaere til skade for afsaetningen af faare- og lammekoed fra Argentina paa Faellesskabets marked inden for de aftalte maengder. Faellesskabet traeffer specielt foranstaltninger, der skal sikre, at afsaetning af interventionslagre af frosset koed i henhold til forordningen ikke modvirker en saadan afsaetning af argentinsk faare- og lammekoed. 8. I betragtning af formaalene med og bestemmelserne i denne aftale erkender Faellesskabet, at restitutioner eller anden form for stoette til udfoersel af faare- og lammekoed samt til levende faar og lam beregnet til slagtning kun maa tages i anvendelse til priser og paa betingelser, der opfylder de bestaaende internationale forpligtelser, og under overholdelse af Faellesskabets traditionelle andel af verdenshandelen med disse produkter. Disse ord fortolkes i overensstemmelse med artikel XVI i GATT og navnlig i overensstemmelse med artikel 10, stk. 2, litra c), i overenskomsten om fortolkning og anvendelse af artikel VI, XVI og XXIII i Den almindelige Overenskomst om Told og Udenrigshandel. 9. Argentina soerger for, at denne aftale overholdes, hvilket blandt andet indebaerer udstedelse af eksportlicenser for de i klausul 1 naevnte produkter inden for de maengder, der er omfattet af denne aftale. Faellesskabet vedtager paa sin side alle noedvendige bestemmelser, for at der kun kan udstedes importlicenser for ovennaevnte produkter med oprindelse i Argentina, naar der fremlaegges en eksportlicens udstedt af en ansvarlig myndighed, der er udpeget af Argentinas regering. Gennemfoerelsesbestemmelserne til denne aftale fastlaegges saaledes, at det ikke er paakraevet at stille sikkerhed for udstedelse af importlicens for de paagaeldende produkter. Det fastsaettes ligeledes i gennemfoerelsesbestemmelserne, at den ansvarlige argentinske myndighed med mellemrum giver Faellesskabets ansvarlige myndighed meddelelse om, hvor store maengder der er udstedt eksportlicenser for, i givet fald specificeret efter bestemmelsessted. 10. Der nedsaettes et raadgivende udvalg sammensat af repraesentanter fra Faellesskabet og Argentina. Udvalgets opgave er at sikre, at aftalen anvendes korrekt og fungerer gnidningsloest. Udvalget undersoeger regelmaessigt udviklingen paa de to parters markeder for faare-, lamme- og gedekoed og udviklingen paa det internationale marked samt afsaetningsforholdene paa disse markeder, herunder forhold, der har betydning for de maal, der er anfoert i klausul 7. Udvalget sikrer, at udfoersel til Faellesskabet af produkter fremstillet af faare-, lamme- og gedekoed henhoerende under toldpositioner, der ikke er omhandlet i denne aftale, ikke forhindrer en korrekt anvendelse af aftalen. Udvalget droefter alle spoergsmaal, der kan opstaa i forbindelse med aftalens gennemfoerelse, og anbefaler de ansvarlige myndigheder passende loesninger. 11. Bestemmelserne i denne aftale aendrer ikke parternes rettigheder og forpligtelser under GATT. 12. Den aarlige maengde, der er fastsat i klausul 2, gaelder for tidsrummet 1. januar - 31. december. Den maengde, der gaelder fra aftalens ikrafttraeden og indtil den 1. januar det foelgende aar, fastsaettes som en forholdsmaessig andel af den samlede aarlige maengde under hensyn til, at handelen er saesonbestemt. 13. Denne aftale gaelder paa den ene side for de omraader, hvor traktaten om oprettelse af Det europaeiske oekonomiske Faellesskab finder anvendelse, paa de betingelser, der er fastsat i den paagaeldende traktat, og paa den anden side for Den argentinske Republiks omraade. 14. Aftalen traeder i kraft den 20. oktober 1980. Den gaelder indtil den 31. marts 1984, hvorefter den med et aars skriftlig varsel kan opsiges af en af parterne. Under alle omstaendigheder gennemgaar de to parter aftalens bestemmelser inden den 1. april 1984 med henblik paa aendringer, som de maatte finde noedvendige. Jeg ville vaere Dem taknemmelig, saafremt De over for mig vil bekraefte, at Deres regering er indforstaaet med det ovenfor anfoerte.«. Jeg har den aere over for Dem at bekraefte, at min regering er indforstaaet med indholdet af den paagaeldende skrivelse. Modtag, hr. ..., forsikringen om min mest udmaerkede hoejagtelse. For regeringen Den argentinske Republik


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Ertmės užpildančios mutacijos tiroksiną surišančioje vietoje smarkiai padidina transtretino stabilumą ir apsaugo nuo jo kaupimosi mokslinės ataskaitos
 Baltymų agregacija
 Su mirtina sistemine amiloidoze yra susijusi daugiau nei šimtas skirtingų transthyretin (TTR) mutacijų. Jie destabilizuoja baltymo tetramerinę struktūrą ir skatina tarpląstelinį TTR nusėdimą kaip patologinius amiloidinius pluoštus. Iki šiol buvo įrodyta, kad tik R104H ir T119M mutacijos reikšmingai stabilizuoja TTR, veikdamos kaip ligos slopintojos. Aprašome naują A108V nepatogeninę mutaciją, aptiktą Portugalijos subjekto. Šis variantas yra stabilesnis nei laukinio tipo TTR tiek in vitro, tiek žmogaus plazmoje. Tai bruožas, užkertantis kelią jo kaupimuisi. A108V kristalų struktūra rodo, kad ši stabilizacija atsiranda dėl naujų vidinių ir tarp subvienetų esančių kontaktų, susijusių su tiroksino (T4) rišimosi vieta. Naudodamiesi šiuo stebėjimu, mes sukūrėme A108I mutaciją, kuri užpildo T 4 surišimo ertmę, kaip matyti iš kristalų struktūros. Šis sintetinis baltymas tampa vienu iš stabiliausių iki šiol aprašytų TTR variantų, kurį galima naudoti keičiant genų ir baltymų terapiją.
 Baltymų netinkamas išsiskyrimas ir agregacija į amiloido sankaupas yra susijusios su augančiu žmonių degeneracinių sutrikimų skaičiumi 1 . Transtreretino (TTR) amiloidozės yra ligos, kurioms būdingos ląstelienos, turinčios TTR 2, tarpląstelinis nusėdimas. Laukinio tipo baltymas (TTR WT) sudaro amiloido sankaupas, sukeldamas senatinę sisteminę amiloidozę (SSA) 3, o likusias TTR amiloidozes sukelia taškinės mutacijos TTR pirminėje sekoje, kurios pablogina vidinį baltymo polinkį kauptis. TTR yra labai polimorfinis baltymas, turintis daugiau nei 100 skirtingų taškų mutacijų, susijusių su autosomine dominuojančia paveldima amiloidoze, daugiausia šeimine amiloidotine polineuropatija (FAP) 4, 5 ir šeimine amiloidine kardiomiopatija (FAC) 6, 7 .
 TTR yra homotetramerinis plazmos baltymas, turintis L-tiroksino (T 4 ) 8 ir vitamino A 9, 10 . TTR molekulę sudaro keturi identiški 127 aminorūgščių subvienetai, pavadinti A, B, C ir D. Kiekvienas monomeras susideda iš aštuonių sruogų, turinčių β-lakšto struktūrą. TTR tetrameras yra suformuotas sujungiant AB ir CD dimerus. Silpnesnė dimerio-dimerio sąsaja nusako dvi, beveik neužimtas, piltuvo formos T 4 surišimo vietas 11, 12 .
 Patogeninės mutacijos daro įtaką vietiniam TTR stabilumui, palengvindamos tetramero atsiribojimą į monomerus, kurie, iš dalies atsiskleidžiant, kaupiasi į amiloidines fibriles. Tetramerio disociacija yra greitį ribojantis TTR agregacijos žingsnis. Atitinkamai, sintetinis variantas su dimerais, sudarytais iš kovalentiškai sujungtų monomerų, negalėjo sudaryti amiloidinių fibrilių 13, 14 . Tam tikro TTR varianto virpėjimo tendencija ir jo stabilumas yra tarpusavyje susiję. Iš tikrųjų ligos sunkumą, apibrėžtą pagal patologijos atsiradimo amžių ir skvarbumą, galima numatyti įvertinant sukeliančio baltymo varianto 15, 16 termodinaminį ir kinetinį stabilumą in vitro . TTR amiloidogeninių variantų kristalų struktūros parodė, kad šie baltymai paprastai turi pakitusių kontaktų tarp dimerų, o tai leistų destabilizuoti jų ketvirtinę struktūrą 17, 18, 19, 20 .
 Keletą kartų arba kepenų ir širdies persodinimas daugelį metų buvo vienintelis TTR amiloidozių gydymo būdas 21 . Nepaisant rimtų įrodymų, siejančių TTR agregaciją su ligos pradžia, nėra visiškai aišku, ar tai yra pirminiai tirpūs mazgai, ar netirpūs pluoštai, kurie daro citotoksinį poveikį 22, 23 . Todėl viso amiloidogenezės proceso prevencija atrodo kaip konservatyviausia TTR ligų terapinė strategija. Tai buvo padaryta naudojant mažas molekules, galinčias prisijungti prie TTR T 4 surišimo vietų, veikiančias kaip kinetiniai stabilizatoriai. Jie jungia kintamos ir BD dimerų hidrofobinius paviršius ne kovalentiškos, daugiausia hidrofobinės, sąveikos būdu, padidindami tetramero disociacijos energetinį barjerą ir sustabdydami TTR agregaciją 24, 25, 26, 27 .
 Buvo aprašytas mažesnis nepatogeninių TTR mutacijų skaičius ir iki šiol įrodyta, kad tik dvi iš jų yra stabilesnės nei TTR WT. TTR T119M yra Portugalijos gyventojų nustatytas variantas, kurio plazmoje yra daugiau nei TTR WT, dėl lėtesnio šio mutanto baltymo klirenso iš serumo 28, 29 . Svarbu tai, kad individams, turintiems T119M mutaciją kartu su FAP susijusia V30M, pasireiškia palankesnė ligos raida, nei heterozigotinėms giminėms, turinčioms vien V30M mutaciją 30, 31 . Taigi ši mutacija veikia kaip tarpląstelinis trans-slopintuvas. Panašus poveikis aprašytas TTR R104H variantui, aptinkamam heterozigotiniams asmenims iš japonų šeimos, turinčios FAP 32 .
 TTR T 4 surišantys kanalai turi tris simetriškus mažų įdubimų rinkinius, vadinamus halogeno surišimo kišenėmis (HBP), į kuriuos įdedami keturi ligando jodo atomai. Vidinė rišamoji kišenė HBP-3 yra tarp šoninių Ser 117, Thr 119, Ala 108 ir Leu 110 grandinių. Panašiai kaip kinetiniai stabilizatoriai, apsauginį T119M mutaciją sukelia didesnis hidrofobiškumas, kurį ji sukuria kanale. sąsaja tarp dimerų 33 . Tai padidina mutantų atsparumą disociacijai. Iš tikrųjų, norint stabilizuoti sumaišytą tetramerą nuo disociacijos, pakanka įterpti vieną TTR T119M subvienetą į TTR V30M tetramerą. Be T119M mutacijos, svarbų vaidmenį, kurį TTR rišančioje ertmėje atlieka hidrofobiškumas TTR tetramero stabilumui, parodo natūralioji L110A mutacija. HBP-2 rišamąją kišenę sudaro šoninės Leu 110, Ala 108, Ala 109 ir Leu 17 grandinės. Sumažindamas HBP-3 / HBP-2 hidrofobiškumą, kirpdamas Leu 110 šoninę grandinę į Ala, skatina greitą tetrameras į monomerus 14 .
 Čia mes nustatėme naują A108V nepatogeninę mutaciją Portugalijos subjekte. Kadangi šoninė Ala 108 grandinė yra susijusi su trimis TTR HBP vietomis, spėliojome, kad ši mutacija turės įtakos baltymo stabilumui. Šio mutanto kristalinė struktūra ir jo stabilumo apibūdinimas in vitro ir žmogaus plazmoje patvirtina, kad TTR A108V yra trečias aprašytas natūralus variantas, pasižymintis padidintu stabilumu, palyginti su TTR WT, yra stabilesnis in vitro nei TTR T119M. Kaip ir tikėtasi, TTR A108V stabilumas neleidžia jam kauptis į amiloidines fibriles. Sukūrėme naują TTR A108I variantą, kad pasiektume optimalų hidrofobinių liekanų pakavimą T4 rišančioje ertmėje, kaip patvirtinta kristalų struktūroje. Šis sukonstruotas baltymas tampa vienu stabiliausių iki šiol aprašytų TTR variantų, nes nesugeba surišti T 4 hormono. Apskritai šis tyrimas pateikia svarbių naujų įžvalgų apie molekulinius determinantus, kurie atspindi TTR stabilumą.
 Stabiliojo TTR A108V varianto aptikimas Portugalijos subjekte
 Plazmos TTR imunoprecipitacija buvo atlikta naudojant anti-žmogaus TTR antikūną ir TTR variantus, identifikuotus MALDI (matricos pagalba lazerio desorbcijos jonizacijos) masių spektrometrijos analize. Gauti peptidų masių sąrašai buvo palyginti su teoriniais tripsinių peptidų masių sąrašais. Be peptido, apimančio 105–127 liekanas, turinčius 2 489 Da masę, atitinkančią TTR WT, buvo aptiktas nenormalus peptidas, kurio masė yra 2 517 Da (nepavaizduota). Šis pokytis yra suderinamas su Ala iki Val pakeitimu šioje TTR sekos srityje. Taigi, pasiūlymas turėjo ir TTR WT, ir mutavusį variantą. Mutacijos tapatumą ir vietą patvirtinome atlikdami DNR analizę. Patobulinto TTR egzono 4 sekos analizė atskleidė C-į-T perėjimą antroje kodono 108 bazėje, paprastai koduojančioje Ala, sukuriant Val liekaną, suderinus su masių spektrometrijos duomenimis.
 „Ala 108“ žemėlapiai nukreipiami į TTR T 4 rišamąjį kanalą. Nepaisant to, kad tiriamojoje nebuvo jokių akivaizdžių simptomų, kurie galėtų būti siejami su TTR amiloidoze, nes buvo įrodyta, kad šios ertmės mutacijos daro stiprų poveikį tetramero stabilumui, nusprendėme išanalizuoti visuotinį TTR stabilumą šio asmens plazmoje naudodami natūralusis PAGE ir izoelektrinis fokusavimas (IEF) pusiau denatūravimo sąlygomis (4 M karbamidas) 24 (1 pav.). Į analizę taip pat buvo įtrauktos V30M ir T119M mutacijų heterozigotinių nešiotojų plazmos, kaip sumažėjusio ir padidėjusio stabilumo variantų, atitinkamai, 29, 36, ir kontrolinės plazmos. Galima būtų išskirti TTR monomerą (M), oksiduotą monomerą (Ox M) ir keletą apatinių pI juostų, atitinkančių tetramerus (1 pav.). Rezultatai parodė, kad TTR A108V heterozigotiniame nešiklyje buvo didesnis tetramero procentas, palyginti su visu TTR (75, 8 ± 6, 8% tetramero), nei WT kontrolinėje plazmoje (69, 4 ± 8, 3% tetramero) (1A ir B pav.), Taigi galima teigti, kad TTR A108V variantas yra stabilesnis nei WT baltymas. Tetramero procentas TTR A108V heterozigotinių nešėjų plazmoje buvo panašus kaip ir TTR T119M heterozigotiniame nešiklio plazmoje (76, 3 ± 4, 7% tetramero) ir didesnis nei TTR V30M heterozigoto nešiklio plazmoje (53, 6 ± 9% tetramero).
 ( A ) TTR plazmos TTR iš heterozigotinių TTR variantų nešiotojų arba iš kontrolinių medžiagų buvo išskirti natūrine PAGE elektroforeze ir po to atskirti IEF pusiau disociuojančiomis sąlygomis (4 M karbamido). Tai yra reprezentatyvus paveikslėlis, pasirinktas iš 3 kopijų, į kurias atsižvelgiama kiekybiškai įvertinant ( B ). ( B ) Santykinis tetramero gausumas buvo apskaičiuotas remiantis tetramero juostų (% Tetramer) intensyvumo santykiu su visų TTR juostų intensyvumu (bendras TTR) mėginiuose iš plazmos. * P <0, 05 (studento t-testas). Klaidų juostos rodo SD.
 Kompiuterinis TTR variantų stabilumo numatymas
 Neseniai parodėme, kad mutacijų įtaką TTR stabilumui galima apskaičiuoti naudojant visų atomų „FoldX“ jėgos lauką 37 . Nors FoldX neperorganizuoja Ca mutacijos metu, daugumos iki šiol išspręstų TTR mutantų kristalografijos struktūros neturi reikšmingo stuburo nuokrypio, palyginti su TTR WT. Todėl mes panaudojome „FoldX“, kad sukurtume TTR A108V struktūrinį modelį, kaip šabloną naudodamiesi TTR WT kristalografine struktūra (PDB: 1F41). Remiantis šiuo modeliu, buvo išmatuotos energijos, susijusios su po mutacijos įgyta ar prarasta sąveika, ir jos buvo panaudotos struktūros termodinaminio stabilumo pokyčiams numatyti (ΔΔG = ΔG mut - ΔG wt ). TTR A108V, gerai suderintas su išmatuotu stabilumu plazmoje, buvo 8, 30 kcal.mol −1 stabilesnis nei WT baltymas. Šis per didelis stabilizavimas, atrodo, atsirado dėl papildomos Val metileno grupės projekcijos į T 4 surišimo kanalą tetramerinėje sąsajoje (pav. S1). Kadangi buvo pranešta, kad užpildžius ertmę papildomomis hidrofobinėmis cheminėmis grupėmis, padidėja TTR stabilumas 33, todėl nusprendėme išbandyti, ar įvedus keturias papildomas metileno grupes, mutuojant Ala 108 į Ile, susidarys tankesnis T4 surišančio kanalo pakavimas ir atitinkamai, pernelyg stabilizuotame baltymo variante. Prognozuotas, kad „FoldX“ modeliuotas TTR A108I variantas bus stabilesnis nei A108V, o ΔΔG yra 13, 71 kcal.mol −1, palyginti su TTR WT.
 TTR A108V ir TTR A108I variantų termodinaminio stabilumo tyrimas in vitro
 Norint eksperimentiškai patvirtinti numatomą TTR A108V ir TTR A108I stabilumo padidėjimą, abu baltymai buvo gaminami rekombinantiniu būdu, kartu su TTR WT ir TTR T119M. Visų šių variantų stabilumas buvo išmatuotas naudojant pusiausvyros karbamido denatūraciją. Baltymai buvo inkubuojami 96 valandas didinant karbamido koncentraciją ir stebimi vidinės Trp fluorescencijos pokyčiai, kad būtų galima apskaičiuoti sulankstyto baltymo procentą bet kurioje nurodytoje karbamido koncentracijoje (2A pav.). TTR denatūracijai karbamidu reikia iš anksto atskirti tetramerą, todėl jis netiesiogiai praneša apie baltymo kinetinį stabilumą. TTR WT pradeda atsiskleisti esant M 2 M karbamidui, kurio denaturacijos kreivė yra C m = 3, 2 M. Kaip anksčiau buvo pranešta, TTR T119M yra stabilesnis nei TTR WT, pradeda atsiskleisti esant ∼ 3 M karbamidui ir rodo C m = 6, 0 M. Mūsų nuostabai, TTR A108V yra daug stabilesnis, pradeda išsiskleisti tik esant M 6 M karbamidui ir nėra visiškai denatūruotas esant 9 M karbamido koncentracijai. Stabilizacija, kurią skatina A108I mutacija, yra dar dramatiškesnė, nes šis variantas pradeda atsiskleisti tik virš 8 M karbamido, o jo tetramerinė struktūra atrodo iš esmės nepažeista net esant 9 M chaotropinio agento.
 Įvertinti TTR mutantų termodinaminius ( A ) ir kinetinius ( B ) stabilumus išsiskleidžiant karbamidui. ( A ) pavyzdžiai buvo inkubuojami esant 1, 8 μM esant skirtingoms karbamido koncentracijoms 96 valandas. Sužadinimas buvo nustatytas į 280 nm, o skleidžiama fluorescencija buvo skleidžiama nuo 300 iki 400 nm. ( B ) mėginiai buvo inkubuojami esant 8 M karbamidui, o denatūracija sekė visą laiką. Denatūracijos laipsniui išmatuoti buvo naudojamas intensyvumo santykis nuo 355 iki 335 nm. Skydelyje (C) pateiktas trijų variantų, išbandytų HHP, denatūracijos profilis. Mėginiai buvo inkubuojami esant 1, 8 μM, o HHP padidėjo. Taip pat kaip denatūracijos zondas buvo naudojama Trp fluorescencija.
 Naudojant karbamido koncentracijas pooperaciniame regione, lėtieji TTR ketverto struktūriniai pokyčiai tiesiogiai susiejami su greitais tretiniais struktūriniais pokyčiais ir tampa išsiskleidę negrįžtami, leidžiant išmatuoti tetramero disociacijos kinetiką. Keturi baltymai buvo inkubuojami 8 M karbamidu ir visą laiką stebimi vidinės Trp fluorescencijos pokyčiai (2 pav. B). TTR WT disociacijos t1 / 2 yra 5, 5 val., Tuo tarpu, kaip tikėtasi, TTR T119M atsiskyrė lėčiau, esant 1/2 - 42 val. Iš eksperimento aišku, kad esant 8 M karbamidui, tiek TTR WT, tiek TTR T119M išsiskiria labiau nei TTR A108V ir specialiai nei TTR A108I, abu mutantai pasižymi ypač lėtai atsiribojimo greičiu. Tai rodo, kad jiems patvirtintas žymiai padidėjęs kinetinis stabilumas, kuris užkirs kelią tetramero atsiribojimui biologiškai reikšmingoje laiko skalėje. Galiausiai, TTR A108V ir A108I stabilumas buvo patikrintas aukštu hidrostatiniu slėgiu (HHP), stebint raudoną Trp išmetimo maksimumo pokytį, kurį sukėlė šios liekanos ekspozicija vandeninėje aplinkoje, kai natūralusis TTR išsiskyrė ir atsiskleidžia. Kaip parodyta 2C pav., Termodinaminio stabilumo seka pakartoja tai, kas buvo matyti su karbamidu, būtent, padidintą abiejų lakų stabilumą, palyginti su WT seka, yra atsparus slėgiui TTR A108I, esant 1H laipsniui tik dalinę denatūraciją. C. TTR WT ir A108V disociacijai-denaturacijai gautos p 1/2 vertės (slėgis, užtikrinantis 50% disociacijos-denatūracijos) buvo lygios 974 bar ir 1, 755 bar.
 Taip pat mes ištyrėme IEF išgrynintų rekombinantinių baltymų tetramero stabilumą pusiau disociuojančiose sąlygose (3 pav.). Tinkamai suderinus pusiausvyros ir kinetinius duomenis, rezultatai parodė, kad TTR A108V (79, 3 ± 9, 2% tetramero) variante ir TTR A108I (93, 7 ± 1, 2%) tetramero procentas visame TTR yra didesnis nei TTR WT (71, 7 ±). 6, 3%) ir TTR V30M (30, 6 ± 6, 4%), tačiau taip pat didesnis nei gerai apibūdintame TTR T119M trans-slopintuve (70, 8 ± 16, 3%).
 ( A ) Rekombinantiniai TTR variantai buvo išskirti natūralia PAGE elektroforeze ir po to atskirti IEF pusiau disocijuojančiomis sąlygomis (4 M karbamido). Visi mėginiai buvo paleisti tame pačiame gelyje, tačiau TTR V30M atveju - ne vienas šalia kito. Tai yra reprezentatyvus paveikslėlis, pasirinktas iš 3 kopijų, į kurias atsižvelgiama kiekybiškai įvertinant ( B ). ( B ) Santykinis tetramero gausumas buvo apskaičiuotas remiantis tetramero juostų (% Tetramer) intensyvumo santykiu su visų TTR juostų intensyvumu (bendras TTR) mėginiuose iš rekombinantinių baltymų. ** P <0, 01; *** P <0, 001 (studento t-testas). Klaidų juostos rodo SD.
 TTR A108V ir TTR A108I variantų agregacija
 TTR agregacija į amiloidines fibriles vyksta prieš tetramero atsiribojimą, o temporas, kuriuo tetrameras išsiskiria į amiloidogeninį tarpinį produktą, nustato fibrilių susidarymo greitį. Todėl buvo tikimasi, kad padidėjęs TTR A108V ir TTR A108I kinetinis stabilumas gali kaip nors sumažinti jų agregaciją, palyginti su TTR WT. Norėdami patvirtinti šią galimybę, įvertinome agregacijos kinetiką po baltymų inkubavimo esant 4, 4 pH ir 37 ° C - tai sąlyga, sukelianti dalinę denatūraciją ir TTR amiloidogenezę. Siekdami šio tikslo, mes stebėjome tirpalo drumstumo padidėjimą per tam tikrą laiką (4 pav. A). Nepaisant klinikinės ir patologinės domino transplantacijos istorijos, rodo, kad sėjimas ir branduolys yra svarbūs TTR amiloido formavimosi in vivo 38 elementai, tokiomis in vitro sąlygomis TTR sukelia gerai apibūdintą neturinčią branduolio agregacijos reakciją, neturinčią jokios akivaizdžios vėlavimo fazės, kuris mūsų rankose pasiekia maksimalų agregacijos laipsnį maždaug per 55 h inkubacijos. TTR A108V ir TTR A108I parodė dramatiškai sumažėjusį agregacijos polinkį, nes drumstumas reakcijos pabaigoje buvo 6 ir 8 kartus mažesnis nei atitinkamai užfiksuotas TTR WT. Yra žinoma, kad TTR agregacijai drumstumas ir amiloido susidarymas gerai koreliuoja. Atitinkamai, amiloido susidarymo laipsnio matavimas reakcijos galutiniame taške, naudojant amiloido specifinį dažą tioflaviną T (4B pav.), Koreliavo su turbidimetriniais duomenimis, rodančiais, kad dviejų mutantų baltymai turi nedidelį agregacijos pobūdį šiuose destabilizuojančiuose elementuose. sąlygos, ypatybė, kuri buvo patvirtinta tiriant tirpalų bendrą turinį naudojant elektroninę perdavimo mikroskopiją. Kaip parodyta 4C pav., TTR A108V ir TTR A108I tirpaluose yra mažas agregatų kiekis, palyginti su TTR WT, kurių tirpaluose, atsižvelgiant į jų infraraudonųjų spindulių spektrą, gali būti stebimas priešgilinis agregatas, parodantis amiloido tipo intermolekulinius β lakštus. (4D pav., S1 pav.). TTR A108V sumažėjęs polinkis į virpėjimą in vitro patvirtina sumažėjusį amiloidogeninį potencialą in vivo .
 Drumstumas ties 340 nm buvo stebimas visą laiką, norint išmatuoti agregaciją ( A ). Eksperimento pabaigoje buvo atliktas Th-T tyrimas, siekiant patvirtinti agregacijos amiloidinį pobūdį ( B ). Sužavėdami pavyzdžius esant 450 ° C ir surinkdami emisiją nuo 460 iki 600 nm, gavome Th-T spektrus. ( C ) rodo sujungtų mėginių galutinio taško TEM. Nurodomos mastelio juostų vertės.
 Toliau nagrinėjome, ar vieno ar kelių TTR A108I subvienetų įtraukimas galėtų stabilizuoti patogeninį TTR V30M variantą, susidarant heterotetramerams - anksčiau aprašytam reiškiniui TTR T119M 27, 30, 39 . Norėdami atskirti stabilius TTR A108I tetramerius, mes panaudojome HHP, 4 M karbamido ir žemos temperatūros 39 derinį. 5A paveiksle pavaizduota tyrimo schema. Į HHP + karbamidą buvo įpiltas ekvimoliarus mišinys (15 μM V30M + 15 μM A108I) ir stebimi Trp emisijos spektrai. Kai mėginys buvo inkubuotas tokiomis sąlygomis, buvo pastebėtas raudonas 18 nm poslinkis, rodantis tetramero disociaciją ir monomerų denatūraciją (5B pav.) 39 . HHP buvo paleistas ir mėginiai dializuojami siekiant pašalinti karbamidą ir sudaryti sąlygas vėl susieti monomerus, paverčiant nevienalytę tetramerų populiaciją, kurią sudaro TTR A108I ir TTR V30M subvienetai atsitiktiniu santykiu. TTR perklijavimą patvirtinome stebėdami natūralios Trp fluorescencijos atkūrimą (5B pav.). Mes taip pat įvertinome mėginio oligomerizacijos būseną, naudodami SEC. 5C pav. Parodyta, kad heterotetrameriai, pagaminti pagal aukščiau aprašytą protokolą, išplauna kaip vieną smailę maždaug per 19 min., Turėdami tą patį profilį kaip ir originalūs TTR A108I ir TTR V30M homotetramerai. Mes atlikome kitus du skirtingus eksperimentus, skirtus gautų heterotetramerių konformacinėms savybėms nustatyti: HHP titravimas (5 pav. D) ir agregacijos tyrimas, esant pH 4, 4, 72 valandas 37 ° C (5 pav. D pav.). HHP titravimas rodo, kad susijungę tetramerai turi tarpinį stabilumą, palyginti su TTR V30M ir A108I homotetramerais (5 pav. D). Agregacijos tyrimas rodo, kad heterotetramerai buvo mažiau linkę į agregaciją nei švieži ekvimoliniai dviejų TTR variantų mišiniai (15 μM TTR V30M + 15 μM TTR A108I) (5D pav. Įterpimas).
 A skydelyje parodyta protokolo, naudojamo hibridiniams tetramerams gaminti, schema. B skydelyje parodytas mišinio, gauto skirtingais tyrimo momentais, Trp spektro išmetimas, kuriame spektriniai poslinkiai rodo sulankstymo būseną. Juodos ištisinės, juodos pertraukiamosios ir pilkosios linijos atitinka spektrus prieš dedant HPP, pritaikius maksimalų HPP ir vėl sudedant. C skydelyje parodyta mėginio oligomerinė būsena, tiriama SEC. Pagrindinė smailė chromatogramoje atitinka tetramerą. D skydelyje parodytas HHP titravimo tyrimas. Trp fluorescencinė emisija buvo naudojama pranešti apie mėginių stabilumą didėjančio slėgio atžvilgiu. Neapdoroto TTR A108V ir TTR V30M mišinio ekvivalentinio mišinio ir hetroteramerinio mišinio rūgštinio indukuoto agregacijos tyrimo rezultatai parodyti įvestyje.
 TTR A108V ir TTR A108I variantų rentgeno kristalų struktūros
 Nepaisant to, kad „FoldX“ modeliai gerai prognozavo eksperimentiniu būdu nustatytus santykinius baltymų stabilumo pokyčius, jie yra tik apytikslis baltymų struktūros suderinimas. Norėdami vienareikšmiškai išskaidyti molekulines detales, atspindinčias didesnį natūralių ir suprojektuotų TTR variantų stabilumą, išsprendėme TTR A108V ir TTR A108I homotetramų kristalų struktūras atitinkamai 1, 3 ir 1, 4 Å skiriamąja geba. Konstrukcijos beveik nesiskiria nuo TTR WT struktūros, o bendroji α rmsd vertė yra 0, 40 Å (TTR A108V) ir 0, 38 Å (TTR A108I) (6A pav.). 90 ° pasisukimas apie y ašį rodo, kad liekanos 108 šoninės grandinės yra iškyšintos T4 rišamojo kanalo viduje tetramerinėje sąsajoje (6B pav.). Abiejų variantų kristalografiniai parametrai parodyti S1 lentelėje. Galima atsekti visas polipeptidines grandines, išskyrus galinius likučius nuo 1 iki 9 ir 126 bei 127, kurie buvo netvarkingi ir neapibrėžti elektronų tankio. Panašių sutrikimų regionų buvo pranešta ir kitose TTR struktūrose 26, 40 . Įdomu tai, kad 36–40 ir 98–104 likučiai abiejose grandinėse, priklausantys atitinkamai BC ir FG kilpoms, turi aiškiai apibrėžtą elektronų tankį ir gali būti patobulinti iki vidutinių temperatūros veiksnių, tai rodo mažesnį judrumą, palyginti su tomis pačiomis kilpomis TTR WT ir kitos TTR mutantų struktūros, įskaitant neamiloidogeninį TTR T119M, kur įrodyta, kad jos pasižymi dideliu lankstumu 41 .
 ( A ) TTR WT (rožinė), A108V (mėlyna) ir A108I (žalia) tetramerų superpozicija rodo, kad sujungtų Cα atomų bendrasis rmsd yra atitinkamai 0, 40 Å ir 0, 38 Å. ( B ) Pasukimas apie y ašį leidžia pamatyti, kad liekanų šoninė grandinė 108 padėtyje (pilkos spalvos lazdelės) vis labiau tęsiasi T 4 kanalų viduje, kad būtų variantai „Ala“, „Val“ ir „Ile108“.
 Kalbant apie tetramerinius mazgus, svarbiausias TTR A108V ir TTR A108I struktūrų atradimas yra reikšmingas ligandą rišančios hidrofobinės ertmės tūrio sumažėjimas ir kelių naujų sąveikų, būtent tarp Val / Ile108 ir Leu110, susidarymas. (3, 80 Å), tarp Val / Ile108 ir Thr106 (3, 95 Å) to paties monomero viduje ir tarp Ile108 ir Leu17 Symm (3, 58 Å) (žr. S2 lentelę). Šių naujų sąveikų poveikis padidėja 4 kartus per AC / BD sąsają, naudojant TTR tetramerinę jungtį. Be šių naujų sąveikų susidarymo, pastebimas hidrofobinės ertmės, jungiančios T 4 ir kitus hidrofobinius junginius, sumažinimas, kurį mūsų TTR variantuose iš dalies užima alifatinės Val108 arba Ile108 šoninės grandinės, dėl kurių susidaro daug artimesnė AC / BD sąsaja tetrameriniame mazge. Šios skirtingos sąsajos TTR variantuose lemia, kad sumažėja atstumas tarp Leu17 Symm Cδ1 metilo grupių su Ala108 Cβ2 (esant 7, 80 Å atstumui), su Cγ2 iš Val108 (esant 4, 25 Å atstumui) ir su Cδ1 iš Ile108. (esant 3, 58 Å atstumui) (7 pav.).
 ( A ) TTR homotetramų karikatūrų diagramos, parodančios A / V / I108 (juodosios sferos) liekanos erdvinę orientaciją ligando surišimo kišenėse. ( B ) Tiroksino surišimo kanalo žvilgsnis, rodantis naują sąveiką, susidariusią po mutacijų, kurios sustiprina šių variantų dimerį-dimerą. Likučių, esančių baltymų stabilumui, šoninės grandinės Leu17, Ala / Val / Leu108, Leu110, Thr119, Val121 yra pavaizduotos lazdelėje. Elektronų tankio žemėlapiai yra kontūrai 1, 5σ. Vaizdai buvo piešti naudojant PyMOL.
 Panašiai, nors dimerio sąsajos plotas tarp AB ir CD nekinta (1 lentelė), tetrameriniame agregate pastebimai padidėja AC / BD dimerio-dimerio sąlyčio plotas, santykinis padidėjimas maždaug 7% ir 13% atitinkamai TTR A108V ir TTR A108I mutantų variantai (1 lentelė). Todėl struktūrinis palyginimas su T 4 -TTR ir kitomis su ligandu susijusiomis TTR struktūromis rodo, kad ilgesnės alifatinės šoninės grandinės, kylančios iš Ala108 padėties, tokios kaip mūsų Val108 ir Ile108 variantai, užimtų panašią vietą kaip T 4 -hormonas. Visiškai tikėtina, kad šie mutantų variantai imituoja stabilizuojantį poveikį, kurį sukelia hidrofobinės kišenės užpildymas T4 ar kitais hidrofobiniais ligandų, tokių kaip patvirtinti ir bandomieji junginiai, skirti TTR amiloidozės gydymui tolkalponu ir tafamidiu 26, 42 .
 T4 jungiasi su TTR A108V ir TTR A108I
 TTR WT liekanoje Ala 108 yra visų T4 halogeną surišančių kišenių dalis: HBP1 ir 1 ′ (Met13, Lys15, Leu17, Thr106, Ala108 ir Val121), HBP 2 ir 2 ′ (Leu17, Ala108, Ala109). ir Leu110) ir HBP 3 ir 3 ′ (Ala108, Leu110, Ser117 ir Thr119). Šie įrodymai, taip pat faktas, kad visi nauji mutantų Val108 ir Ile108 šoninių grandinių užmegzti likučiai yra hormonų surišimo kanale, rodo, kad T4 jungimasis prie TTR A108V ir TTR A108I gali būti kažkaip kliudomas. Todėl mes ištyrėme T4 sugebėjimą prisijungti prie TTR heterozigoto, gavusio A108V mutaciją, plazmoje. Plazma buvo inkubuota su [ 125I ] pažymėtu T4, po to buvo atskirti T4 surišantys baltymai, naudojant natūralią gelio elektroforezę, ir T4 surišančių baltymų vizualizacija autoradiografijos būdu. Skirtingų plazmos mėginių juostų, atitinkančių TTR, intensyvumas buvo nustatytas densitometrijos būdu. T4 surišimas su TTR kontrolinių (N) plazmoje ir iš heterozigotų, turinčių T119M arba V30M mutacijas, taip pat buvo analizuojami kaip kontroliniai. Įrodyta, kad TTR T119M ir TTR V30M turi didesnį ir mažesnį T 4 afinitetą nei TTR WT, atitinkamai 28 . TTR juostos intensyvumas plazmoje iš TTR A108V nešiklio buvo mažesnis už atitinkamą juostą plazmose iš kontrolinės (N) ir iš TTR T119M nešiklio, primenančio TTR V30M nešiklį, rodantis, kad, nepaisant labai stabilizuojančio, A108V mutacija greičiausiai sumažina afinitetą T 4 (8A pav.).
 Mėginiai buvo inkubuoti su 125 IT4, o baltymai buvo atskirti natūraliu PAGE. Nurodoma TTR ir kitų T 4 surišančių baltymų (TBG-tiroksiną surišančio globulino; ALB-albumino ir TTR-transtiretino) migracija. ( A ) Žmogaus plazmos analizė iš TTR variantų TTR A108V, TTR V30M arba TTR T119M heterozigotinių nešėjų ir kontrolinės plazmos (WT) bei ( B ) Izoliuotų rekombinantinių TTR variantų, būtent V30M, A108, A108I, T119M ir laukinių, analizė. tipo (WT) baltymai. Migracijos metu kaip kontrolinė plazma (kontrolinė) buvo naudojama. Visi mėginiai buvo paleisti tame pačiame gelyje, tačiau ne paeiliui. Nurodoma vieta, kur buvo sutrintas gelis.
 Norint priskirti stebėtą poveikį T4 prisijungimui konkrečiai prie Val buvimo 108 padėtyje, tas pats tyrimas buvo pakartotas su išskirtu rekombinantiniu baltymu, taip pat su suprojektuotu TTR A108I mutantu, naudojant rekombinantinius TTR V30M ir TTR T119M ir TTR WT kaip valdikliai. TTR A108V ir TTR A108I nepateikė jokios radioaktyviosios juostos, atitinkančios natūralaus TTR migraciją gelinėje elektroforezėje po inkubacijos su 125 IT 4, parodydami, kad šie rekombinantiniai variantai pasižymi mažu afinitetu surišti T 4, panašiai, kaip nutinka su TTR V30M ir priešingai nei pastebėta TTR WT ir TTR T119M (8B pav.).
 Norint tiksliau apibūdinti T4 surišimo savybes, buvo atliktas konkurencijos tyrimas, naudojant gelio filtravimo chromatografiją. Šiame tyrime pastovus kiekvieno TTR varianto kiekis buvo inkubuotas su nedideliu kiekiu [ 125I ] T4 ir didėjant nepaženklinto T4, kaip konkurento, koncentracijai. Prie TTR surištas T4 buvo atskirtas nuo nesurištos molekulės dydžio išskyrimo kolonėlėje. Rezultatai buvo išreikšti rišimosi procentais be etiketo T 4 logaritmo atžvilgiu. Gautos konkurencijos kreivės pateiktos 9 pav. Gerai suderinus su elektroforezės duomenimis, tyrimas aiškiai parodo, kad 108 mutantų TTR variantai nepasižymi T4 surišimo afinitetu, nes nebuvo pastebėta konkurencija, priešingai nei vyksta TTR WT. ir TTR T119M, kurie buvo išbandyti lygiagrečiai.
 Išbandyti TTR variantai buvo TTR WT, TTR T119M, TTR A108V ir TTR A108I.
 Dėl vidinio TTR WT polinkio daugiau nei 25% vyresnių nei 80 metų gyventojų kenčia nuo SSA 3 . Patogeninės mutacijos, susijusios su paveldima TTR amiloidoze, padaro baltymus jautresnius agregacijai. Iki šiol nėra iki galo aišku, kaip TTR amiloidas susidaro fiziologinėmis sąlygomis, tačiau labiausiai priimtoje hipotezėje daroma prielaida, kad TTR mutacijos daro įtaką baltymo struktūrinei pusiausvyrai, destabilizuodamos tetramerą disociuotų tarpinių produktų, pasižyminčių amiloidogeniniu potencialu, naudai. Ligos atsiradimo amžius ir skvarba priklauso nuo specifinės mutacijos. Kaip bendra tendencija, kuo destabilizuojanti mutacija, tuo ryškesnis baltymo varianto amiloidogeniškumas. Mutacijos, tiesiogiai ar netiesiogiai veikiančios AC / BD dimerio-dimerio sąsajos stabilumą, linkusios skatinti padidėjusį baltymų agregaciją ir sunkios ligos fenotipus, išryškindamos lemiamą vaidmenį, kurį vaidina natūrali ketvirtinė TTR struktūra užkertant kelią baltymų nusėdimui. Čia verta pabrėžti, kad neseniai buvo pasiūlytas alternatyvus mechaninis fermentinis skilimo mechanizmas transtreretino amiloidogenezės pagrindimui in vivo 43 . According to this new view, the combined effect of proteolysis and shearing would result in the release of a highly amyloidogenic 49–127 truncated protomer, usually found in ex vivo deposits. Destabilized TTR variants would be more susceptible to proteolysis than TTR WT 43 .
 The number of non-amyloidogenic mutations identified so far for TTR is small 44, which is consistent with the idea that most sporadic mutations in proteins have a destabilizing effect 45 . Some mutations, such as G6S 46 or H90N 47 have a negligible impact on TTR stability and are considered non-pathogenic, whereas others like R104H 48 or T119M 30 have been shown to act as trans-suppressors. Trans-suppressor mutations render TTR more stable against dissociation and aggregation, reducing the symptoms of the disease in heterozygotes carrying additional aggressive mutations in the other allele. The trans-suppressor effects of R104H and T119M respond to different mechanisms 48 . The quaternary structure of R104H is thermodynamically stabilised, whereas the tetrameric native state of T119M exhibits a high kinetic stability. The stabilizing effect of T119M mutation is much higher than that of R104H, which only modestly protects against aggregation. Thr119 protrudes directly into the T 4 binding site and the high kinetic stabilization provided by its mutation into Met owes to the novel contacts that this hydrophobic residue establishes or induces between amino acids belonging to different dimers, which strengthen the interaction between dimers at the T 4 binding pocket. This is the same principle exploited by hundreds of small compounds that by binding to and filling the two T 4 binding sites stabilize the native state of TTR over the dissociative transition state. Thus, reinforcing the contacts at the quaternary dimer-dimer interface appears as a requirement to impose kinetic stability on the entire TTR tetramer. It is worth to emphasize here that T119M TTR is also totally resistant to proteolytic cleavage under shearing 43 .
 Here we report on a novel mutation affecting residue Ala108 that as Thr119 is located in the T 4 -binding cavity. Mutations of the adjacent Ala 109 residue to Thr 49 or Val 50 are non-amyloidogenic, whereas mutation to Ser is pathogenic 51 ; however, Ala 109 does not face the T 4 binding cavity. The subject bearing the mutation A108V in heterozygoty presented a mild, non-progressive, sensory neuropathy without any dysautonomic symptom or sign, a course that is not typical of a TTR-related polyneuropathy. Since it was not possible to follow-up the patient for more than 3 years, and there was almost no progression of disease, and no family history was available, the atypical picture suggests an idiopathic neuropathy or, more interesting, that the novel mutation is associated with a very benign expression of TTR-associated disease. Initial analysis of the stability of the TTR in the blood of the subject as well as computational predictions already suggested that the mutation could be overstabilizing, a fact that was confirmed by characterizing the stability features of the recombinant protein, which only begins to unfold upon incubation in 6 M urea for 96 h. Globally, the A108V mutation adds four additional methylene groups, two at each of the T 4 -binding sites at the dimer-dimer interface. This is important, because experiments with small molecules demonstrate that their binding to the first and second T 4 -binding sites additively increase the activation barrier associated with tetramer dissociation. The generated structural model suggested that the interface would still admit four additional methylenes in the form of an A108I mutation, without introducing steric clashes that might result in strain at the interface. In good agreement with the prediction, the recombinant TTR A108I protein turned to be significantly more stable than TTR A108V, maintaining its native tetrameric structure intact upon incubation on 8 M urea for 96 h. Their high stabilities in front of urea denaturation at equilibrium and, specially, the very low dissociation rates they exhibit under highly denaturing conditions, are consistent with the existence of a high kinetic barrier for the dissociation TTR A108V and TTR A108I homotetramers. HHP causes denaturation of proteins by promoting the entrance of water molecules into the protein cavities, facilitating the population of partially folded states 52, which in TTR, affects the dissociation of the tetramer. The occupation of the T 4 -binding sites by the additional methylene groups of Val and Ile makes these variants, specially TTR A108I, resistant to pressure. This effect may respond either to a stabilization of the ground state of the protein or to a destabilization of the dissociative transition state. In any case, it results in a dramatic kinetic stabilization for these proteins, which is translated in almost a complete suppression of their aggregation under acidic conditions. Interestingly enough, no mutation has previously been found in position 108 of TTR. Thus, our data provide yet another piece of the puzzle in elucidating the correlation between mutations and aggregation propensity in the context of TTR amyloidoses.
 The almost 200 solved X-ray structures of TTR WT and its variants have not revealed major conformational changes that could explain their differential amyloidogenicity 53, 54 . They are rather small differences at specific positions that account for changes in protein stability 19, 41, 55 . The crystal structures reported here show that the dimer-dimer contact area is more extensive in TTR A108V and in TTR A108I than in TTR WT. Thus, the quaternary structure of the mutant variants is assembled by additional new inter-subunit contacts that might account for their resistance to dissociation. A synthetic TTR L110A variant has been shown to exhibit little resistance to dissociation 14 . This destabilization has been attributed to the fact that Leu110 is in the dimer-dimer interface and the substitution will reduce the contact area. Conversely, in TTR A108V and TTR A108I, Val/Ile108 side chains interact with Leu110 and Thr106 inside the same monomer, as well as with Leu17 Symm at AC/BD interface, in the case of TTR A108I. These contacts, absent or weak in TTR WT, repack the hydrophobic core of the T 4 -binding cavity stabilizing the tetramer. The side chain of Ala108 is part of the three TTR HBP sites and occupies a central position in the cavity, whereas Thr119 is only involved in HBP3 and located in one of the extreme of the funnel. This explains why an increase in the hydrophobic volume of the residue in position 108 results in a higher stabilizing effect than when the same type of change occurs in position 119. The high stability of the TTR A108V and TTR A108I structures allow the residues in loops BC and FG to have well-defined electron densities and to refine to moderate temperature factors, suggesting higher rigidity as compared with TTR WT and other TTR variant structures, including TTR T119M. This is important, because a high mobility of the BC loop has been observed for the artificial and highly amyloidogenic TTR G53S/E54D/L55S triple mutant 56 . Indeed, all the mutations described in this loop are linked to disease.
 It is worth to emphasize, that trans-suppressor mutations of TTR are of considerable interest, because they can be potentially employed to treat TTR-related amyloid diseases using liver targeted gene therapy. In fact, T119M gene therapy in a V30M transgenic mice showed a prophylactic effect on reducing TTR deposition. TTR A108I, is perhaps the most stable mutant attained so far with a single amino acid change not involving covalent cross-linking. The high stability in front of HPP denaturation and the reduced aggregation of V30M/A108I heterotetramers, relative to V30M homotetramers, suggests a potential application in therapy for this designed variant, although additional experiments are required to confirm this extent.
 The fact that TTR A108V and TTR A108I display a low binding affinity for T 4, should not be an impediment for their involvement in putative prophylactic therapies, since only a small fraction of TTR circulates in a bound state, thyroxine-binding globulin (TBG) being the main T 4 binding protein in the blood. Indeed, small molecules designed to impair TTR aggregation exert their stabilizing effect by binding with high affinity to the T 4 -binding site, thus competing the binding of the hormone. The novel contacts established by the Val and Ile side chains inside the T 4 -binding cavity should turn to be useful to engineer barrier heights in kinetic stabilizers by functionalizing them with chemical groups that by mimicking those interactions would strengthen the contacts at the dimer-dimer interface to the level observed in these two highly stable, non-amyloidogenic mutants.
 A 73-year-old healthy woman reported a 12-months history of insidious onset and mild progression of symmetrical numbness of the feet with painful dysaesthesia. Pain sensation was slightly reduced below ankles. Vibration and position sensation were normal. Ankle reflexes were weak, otherwise tendon reflexes were normal. Ataxia was absent and muscle strength was normal. There was no evidence of autonomic nerve or pyramidal tract dysfunction. There was no previous history of diabetes, alcohol intake or drug toxicity. Family history was negative for polyneuropathy or other neurological disorders. Nerve conduction studies confirmed mild sensory neuropathy, with absent sural nerve action potential on both sides, but normal sensory potentials in upper limbs (radial and median, bilaterally) and unremarkable motor nerve conduction velocities and F-waves (median and peroneus, bilaterally). The following blood tests were normal or negative: full blood count, glycaemia, liver and renal function tests, thyroid hormone and vitamin B12 levels, serum immunoglobulins, autoantibody testing for anti-nuclear, extractable nuclear antigen and anti-neutrophil cytoplasmic antibodies, hepatitis C and HIV-1 serology. Investigation of TTR V30M mutation was negative. Over the following three years the patient was observed every 6-months and a comparative nerve conduction study was repeated every year, without any evidence of disease progression. Following this period the patient was lost for follow-up. Blood samples were collected according to standards established by the latest revision of the Declaration of Helsinki. Visi metodai buvo atlikti laikantis atitinkamų gairių ir taisyklių. Informed consent was obtained from all donors in this study, including the propositus. They provided written permission for genetic tests relevant to determine the cause of their clinical condition and for research in the disease, for diagnostic purposes investigations do not require Ethics Committee approval in our centers.
 Plasma TTR immunoprecipitation and peptide mass spectra
 Briefly, 25 μL of serum were incubated with anti-human TTR antibody and kept overnight at 4 °C with stirring. Next, protein A Sepharose beads were added and allowed to mix for 1 h at room temperature with agitation. The beads were washed and the complex eluted with SDS-loading buffer. Finally, samples were fractioned on a 15% polyacrylamide gel under denaturing conditions (SDS-PAGE) and the gel was stained following the Coomassie Colloidal Blue protocol. The TTR monomeric band was excised from the gel and in-gel digestions were performed, after reduction and alkylation, by adding modified porcine trypsin (Promega). Peptide mass spectra were acquired using Applied Biosystems 4800 Plus MALDI-TOF/TOF MS equipment.
 DNA was extracted from peripheral blood cells and exon 4 from the propositus was amplified with primers flanking the coding region [forward 5′-CACGTTTTTCGGGCTCTGGTGG-3′ and reverse 5′-CTTCCTGCTCCCTACCCTAAAG-3′ (nucleotides 7222–7425), which amplified a fragment of 203 base pairs. PCR products were purified using a High Pure PCR Purification Kit (Roche, Basel, Switzerland) and sequenced with a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). Sequences were analyzed using ChromasPro and Ridom TraceEdit.
 In Silico Stability Predictions
 We built two different models (TTR A108V and TTR A108I) by using FoldX (//foldx.crg.es/) command Buildmodel with the original TTR WT structure as deposited on the PDB 1F41; each model was generated with five runs, and they converged well. The resulting structures were used to calculate the ΔΔGs values presented in the Results section. The command Stability was used to calculate the ΔΔGs of unfolding, between variants. The energies are an automatic output in FoldX, and the changes in native stability upon mutation were estimated as the difference between the energy of the wild type protein and that of the mutant protein (ΔΔG = ΔGmut − ΔGwt). ΔΔGs values above 1.6 kcal/mol should significantly affect variant stability, because they correspond to twice the intrinsic standard deviation of FoldX.
 Recombinant TTR expression and purification
 Recombinant WT, TTR A108V and TTR A108I were prepared as detailed in ref. 57.
 3.5 μM of native proteins were incubated under quiescent conditions in 200 mM sodium acetate, 100 mM KCl, pH 4.4, at 37 °C. After 72 h, samples were vortexed, and their turbidity (330 nm) was monitored along time on a UVI-Vis Carry spectrophotometer (Shimadzu).
 TTR stability assays
 In vitro assay of Trp fluorescence
 TTR WT (1.8 μM) was incubated with different urea volumes to attain a range of final concentrations (0 M to 9 M) and samples were incubated for 96 h at RT. Fluorescence measurements were performed on a Jasco FP-8200 Spectrofluorometer. Trp fluorescence was obtained by exciting the samples at 280 nm. Emission was collected from 300 to 400 nm. The fluorescence intensity ratio 355/335 nm was plotted against urea concentration as a sensor of folded protein, which for TTR in urea is equivalent to tetramer integrity 15 . In another setting, TTR WT and mutants (1.8 μM in PBS) were incubated at 8 M urea and fluorescence measured along time at RT. The fraction of unfolded TTR was measured and calculated as above. TTR variants stability was also assessed by HHP. Experiments were performed at 1 μM of each mutant at pH 7.5 and 1 °C. The high-pressure cell equipped with optical windows was purchased from ISS (Champaign, IL). Fluorescence spectra were recorded on an ISS K2 spectrofluorometer. Pressure was increased in steps of 200 bar. At each step the sample was allowed to equilibrate for 15 min before making measurements. Tryptophan emission spectra were obtained by setting the excitation at 280 nm and collecting the emission in the 300- to 400-nm range. Shifts in the center of spectral mass were used to follow denaturation.
 Assay of TTR tetrameric stability by Isoelectric Focusing (IEF)
 Stability of recombinant TTR or TTR in whole plasma was assessed by isoelectric focusing (IEF) in semi-dissociating conditions as previously described 24 . In brief, 30 μL of human plasma were applied to native PAGE for the separation of plasma proteins and the gel band containing TTR was excised and applied to an IEF gel. IEF was carried out in semi-denaturing conditions (4 M urea), containing 5% (v/v) ampholytes pH 4–6.5 (GE Healthcare), at 1200 V for 6 hours. Proteins were stained with Coomassie Blue, the gels were scanned and subjected to densitometry using the ImageQuant program. The results were expressed as the ratio of TTR tetramer over total TTR. The data were analysed by Student's t-test using Microsoft Excel to establish significant changes.
 Aggregated samples were diluted in Mili-Q water to 1 μM. 5 μl of the solution were adsorbed onto carbon-coated copper grids for 5 minutes and blotted to remove excess material. Uranyl acetate (0.5% w/v) was used for negative staining. Samples were dried on air for 5 minutes. Grids were exhaustively scanned with a Hitachi H-7000 transmission electron microscope operating at a voltage of 75 kV.
 Th-T binding was used to probe amyloid formation on the samples. 10 μl of Aggregated samples were added a Th-T solution in 5 mM sodium phosphate, pH 8, resulting in a final Th-T concentration of 20 μM. Excitation was set to 450 nm, whereas emission was collected from 450 to 600 nm. Five individual scans were averaged for each measurement using Jasco 8200 spectrofluorometer. The intensity of the spectra at 482 nm was used as an indicator of the extent of amyloid material.
 ATR-FTIR analysis of the aggregated samples was carried out using a Bruker Tensor 27 FTIR spectrometer (Bruker Optics) with a Golden Gate MKII ATR accessory. 5 μL of sample were placed in the centre of the diamond and dried with N2. Each spectrum consisted of 16 scan accumulations measured at a spectral resolution of 2 cm −1 in a wavelength range between 1700 and 1500 cm -1 .
 Production of Heterotetramers by HHP Treatment
 An equimolar mixture of TTR V30M and TTR A108I (15 μM each) was subjected to HHP (29 kbar) in the presence of 4 M urea in PBS at 1 °C for 60 min to allow for the dissociation of TTR tetramers. Pressure was released and the mixture was dialyzed for 24 h at 4 °C against PBS to remove residual urea and to allow for re-association and subunit exchange 39 . To probe the oligomeric state of the sample, high performance liquid chromatography was performed in a Superdex 75 10/300 GL column (GE healthcare Life Sciences) at room temperature using an HPLC system at a flow rate used was of 0.5 ml/min. (Shimadzu SPD-10A). The system was equilibrated in PBS. Sample elution was monitored by Trp fluorescence at 320 nm.
 Crystals of TTR A108V and TTR A108I were obtained at 18 °C by hanging-drop vapor diffusion methods after purification and concentration. The reservoir solution contained between 15–25% PEG 400, 200 mM calcium chloride, 100 mM HEPES, pH 7.5. Single crystals appeared after three days from equal volumes of protein solution (10 mg/mL in 50 mM Tris-HCl pH 8.0, 100 mM KCl, 1 mM EDTA) and reservoir solution. Crystals were directly flash-frozen in liquid nitrogen prior to diffraction analysis. Diffraction data were recorded from cryo-cooled crystals (100 K) at the ALBA synchrotron in Barcelona (BL13-XALOC beamline). Data were integrated and merged using XDS 58 and scaled, reduced and further analyzed using CCP4 (Table S1). The structures of TTR variants were determined from the X-ray data by molecular replacement using a previous TTR structure (PDB 1F41) as a model using the program Phaser 59 . Model refinement and rebuilding were performed with PHENIX 60, 61 and Coot 61 . Refinement and data statistics are provided in Table S1, structural representations were prepared with PyMol 62 . Those structures are deposited at PDB codes 5FW6 and 5FO2, respectively.
 Thyroxine binding to TTR variants
 Assay by native polyacrylamide gel electrophoresis (PAGE)
 Thyroxine (T 4 ) binding to TTR was assayed by incubation of plasma samples or isolated recombinant TTR, or TTR variants, with radiolabeled thyroxine ([ 125 I]T 4 ) followed by protein separation by native polyacrylamide gel electrophoresis (PAGE) and detection of T 4 binding proteins after autoradiography. In more detail, 5 μL of plasma or 10 μg of recombinant TTR were incubated with 0.25 μL of [ 125 I]T 4 (specific radioactivity 1250 μCi/μg; concentration 320 μCi/mL; Perkin Elmer, Boston, MA, USA) for 1 hour at room temperature. Samples were submitted to electrophoresis for proteins separation by native PAGE 24 . After electrophoresis the gels were dried, and exposed to autoradiography film. The film was scanned and the intensity of the protein bands was quantified by densitometry.
 Assay by gel filtration chromatography
 T 4 binding to isolated TTR was also studied using a competition assay by gel filtration chromatography. Relative affinity of T 4 to bind to different isolated recombinant TTR variants was determined by gel filtration, as described previously, using a constant concentration of each TTR variant (30 nM) incubated with a tracer amount of [ 125 I]T 4 (approx. 50 000 cpm) and with increasing concentrations of non-labelled T 4 (0–1 μM final concentration - 7 different concentrations) as competitor. T 4 bound to TTR was separated from unbound by filtration through a P6DG gel filtration column (BioRad). The results were expressed as percentage of binding against logarithm concentration of non-labelled T 4 . Competition curves obtained were compared.
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 22000D1123(03)
 Afgørelse truffet af Det Blandede EØS-udvalg nr. 78/1999 af 25. juni 1999 om ændring af bilag II (tekniske forskrifter, standarder, prøvning og certificering) til EØS-aftalen
 EF-Tidende nr. L 296 af 23/11/2000 s. 0031 - 0032
 Afgørelse truffet af Det Blandede EØS-udvalgnr. 78/1999af 25. juni 1999om ændring af bilag II (tekniske forskrifter, standarder, prøvning og certificering) til EØS-aftalenDET BLANDEDE EØS-UDVALG HAR -under henvisning til aftalen om Det Europæiske Økonomiske Samarbejdsområde som tilpasset ved protokollen om tilpasning af aftalen om Det Europæiske Økonomiske Samarbejdsområde, i det følgende benævnt "aftalen", særlig artikel 98, ogud fra følgende betragtninger:(1) Bilag II til aftalen blev ændret ved afgørelse nr. 75/1999 truffet af Det Blandede EØS-udvalg den 28. maj 1999(1).(2) Kommissionens forordning (EF) nr. 3059/94 af 15. december 1994 om ændring af bilag I, II og III til Rådets forordning (EØF) nr. 2377/90 om en fælles fremgangsmåde for fastsættelse af maksimalgrænseværdier for restkoncentrationer af veterinærlægemidler i animalske levnedsmidler(2) bør indarbejdes i aftalen.(3) Kommissionens forordning (EF) nr. 1568/98 af 17. juli 1998 om ændring af bilag I, II, III og IV til Rådets forordning (EØF) nr. 2377/90 om en fælles fremgangsmåde for fastsættelse af maksimalgrænseværdier for restkoncentrationer af veterinærlægemidler i animalske levnedsmidler(3), bør indarbejdes i aftalen.(4) Kommissionens forordning (EF) nr. 1569/98 af 17. juli 1998 om ændring af bilag II og III til Rådets forordning (EØF) nr. 2377/90 om en fælles fremgangsmåde for fastsættelse af maksimalgrænseværdier for restkoncentrationer af veterinærlægemidler i animalske levnedsmidler(4), bør indarbejdes i aftalen -TRUFFET FØLGENDE AFGØRELSE:Artikel 1Følgende led indsættes i punkt 14 (Rådets forordning (EØF) nr. 2377/90) i kapitel XIII i bilag II til aftalen: "- 394 R 3059: Kommissionens forordning (EF) nr. 3059/94 af 15. december 1994 (EFT L 323 af 16.12.1994, s. 15).- 398 R 1568: Kommissionens forordning (EF) nr. 1568/98 af 17. juli 1998 (EFT L 205 af 22.7.1998, s. 1), berigtiget ved EFT L 271 af 8.10.1998, s. 42.- 398 R 1569: Kommissionens forordning (EF) nr. 1569/98 af 17. juli 1998 (EFT L 205 af 22.7.1998, s. 7)."Artikel 2Teksterne til Kommissionens forordning (EF) nr. 3059/94, (EF) nr. 1568/98 og (EF) nr. 1569/98 på islandsk og norsk, som er knyttet til de respektive sproglige udgaver af denne afgørelse, er autentiske.Artikel 3Denne afgørelse træder i kraft den 26. juni 1999, forudsat at Det Blandede EØS-udvalg har modtaget alle fornødne meddelelser i henhold til aftalens artikel 103, stk. 1.Artikel 4Denne afgørelse offentliggøres i EØS-afsnittet i og EØS-tillægget til De Europæiske Fællesskabers Tidende.Udfærdiget i Bruxelles, den 25. juni 1999.På Det Blandede EØS-udvalgs vegneF. BarbasoFormand(1) EFT L 284 af 9.11.2000.(2) EFT L 323 af 16.12.1994, s. 15.(3) EFT L 205 af 22.7.1998, s. 1.(4) EFT L 205 af 22.7.1998, s. 7.