Estudio De Las Toxinas De La Anémona De Mar Anthothoe Chilensis (Lesson

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660 9 7 7 7 74 DEDICATORIA Mis padres Juan y Yojana, que son mis mejores guías Mis hermanos Sara, Juan y Yanet amigos incondicionales Mis abuelitas Mercedes Rosas por todo su cariño y consejos, y Luz Garcés A la memoria de mis abuelitos Melecio Quiroz y Jorge Garrido Por su ejemplo de perseverancia y optimismo…

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0 5 Agradezco a Dios por la oportunidad de haber realizado y terminado satisfactoriamente este proyecto y por haber puesto en mi camino muchas personas extraordinarias que me han apoyado desinteresadamente. A mis padres Juan y Yojana, a mis hermanos Sara, Juan y Yanet, por su paciencia y comprensión en la espera de la culminación de este trabajo. Gracias por su apoyo incondicional y por la confianza de que llegaré a la meta. Un agradecimiento especial a mi asesor el Magíster Fernando Retuerto Prieto por creer en mis capacidades para la ejecución de esta investigación y de igual modo agradezco a la Bióloga Elena Arbaiza Prado por su invalorable ayuda, en ambos he reconocido a dos muy buenos profesionales y he ganado dos buenos amigos. A mis profesores la Prof Doris Melo Horna, a la Prof. Susana Gutiérrez, al Prof. Enrique Escobar, y al Prof. Mauro Mariano Astocondor gracias por sus consejos y sus palabras de apoyo. A ustedes amigos, les doy gracias por su motivación en la continuación de este trabajo, los guardo en mi corazón. Una mención particular a mis amigos Ramón A. Espinoza Lewis y Sergio Romero Loyola que aunque lejos me han apoyado facilitándome muchas de las referencias citadas, también a Violeta Alarcón que me brindó el mismo apoyo. A Carmencita Maza y Frank Azorsa, agradezco su ayuda en la parte experimental de este proyecto. A Edison Pascual por el apoyo en la toma de fotografías, a Yesica Llimpe mi entrañable amiga gracias por el interés en mi trabajo. A todas las personas que de manera directa o indirecta contribuyeron en la realización de esta investigación: al Prof. Rolando Estrada por las facilidades para el uso de equipos e instalaciones, al Dr. Armando Yarlequé por facilitarme parte de las referencias bibliográficas, al Prof. Alvaro Marcelo por estar siempre presto a aclarar mis dudas al desarrollar este trabajo, a la Prof. Margarita Velásquez por su colaboración en la prueba hemolítica al Biólogo José Zavala por la recolección de los ejemplares de anémona de mar y al Ing. Quím. Oscar Reátegui por facilitarme algunos equipos de laboratorio; toda mi gratitud para ellos No quiero terminar sin hacer un agradecimiento muy particular al Dr. Carlos Paredes, quien me asesoró en la revisión de este trabajo, mi admiración y respeto. MIL GRACIAS a todos ustedes.

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En esta investigación se han estudiado las toxinas del veneno de la anémona de mar Anthothoe chilensis (Lesson, 1830), a partir de los tentáculos y cuerpos de animales vivos recolectados entre 8 y 10 metros de profundidad en las Isla Cabinza-San Lorenzo. Se utilizaron tres fracciones precipitadas con sulfato de amonio y tres fracciones precipitadas con acetona para realizar ensayos in vitro y un ensayo in vivo. Se observó que las proteínas contenidas en el veneno de A. chilensis tienen un peso molecular entre 14 y 94 kDa. Se evidenció la presencia de carbohidratos en todas las fracciones evaluadas y mediante tinción PAS de geles PAGE-SDS se detectó la presencia de glicoproteínas. Todas las fracciones mostraron tener actividad hemolítica, fosfolipásica y proteolítica. La fracción Ach1at tuvo la actividad hemolítica más alta, la fracción acetónica Ach3at tuvo la mayor actividad fosfolipásica. Cuando las fracciones se probaron sobre el desarrollo embrionario de huevos de erizo de mar, éstas mostraron efectos en la morfología tales como blástulas lisadas, exogastrulación, prismas y larvas pluteus anormales. Entre los efectos citológicos se observaron gástrulas con menos configuraciones mitóticas, núcleos heteropicnóticos, cariorresis y núcleos gigantes. Las fracciones ensayadas mostraron producir hipersensibilidad tipo I en ratones albinos, sin llegar a desencadenar shock anafiláctico.

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6 5 5 In this investigation toxins of the venom of the sea anemone Anthothoe chilensis (Lesson, 1830) have been studied, from the tentacles and bodies of alive animals collected between 8 and 10 meters of depth in the Island Cabinza-San Lorenzo. Three precipitated fractions with ammonium sulfate and three precipitated fractions with acetone were used to make assays in vitro and one assay in vivo. It was observed that the proteins contained in the venom of A. chilensis have a molecular weight between 14 and 94 kDa. The presence of carbohydrates was evidenced in all the evaluated fractions and by stain PAS of gels SDS-PAGE the glycoproteins presence were detected. All the fractions showed to have hemolytic, phospholipase and proteolytic activities. The acetonic fraction I had the higher hemolytic activity, the acetonic fraction III had the greater phospholipasic activity. When the fractions were assayed on the sea urchin eggs embryonic development, they showed to have morphologic effects like lisated blastules, early exogastrulation, and abnormal prisms and pluteus larvae. Cytological effects like gastrulaes with less mitotic configurations, heteropycnotic nuclei, karyorrhexis and giant nuclei were observed. The evaluated fractions showed to produce hypersensitivity type I in mice, without getting to trigger anaphylactic shock.

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55 -'% (. */ 0 $1 ! " - 7 5

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57 -'% (. */ 0 $1 ! " - 7 5

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-'% (. */ 0 $1 ! " - 58 ! !" ## $# %& '()* % $"+ ,"*

59 -'% (. */ 0 $1 ! " - 7 8

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-'% (. */ 0 $1 ! " - 5E ! !" ## $# %& '()* % $"+ ,"*

$! ! ( .> A2E85B= ! $% &D $ " ;% " >!" ! $ $ &! $$ !" $! $! ?!" < !$ " ! .! $ : "" 1% $ " < !D ! $" $!" $ " .! " C " A2E82B $!$! ! ! 1% ! " = $ " $ : "" " ) " ! $ $ &! % = 1$ !" ! < " !$ < ! " !%$ A.! " " ! " " !@$ B $% % ! $ 1$ !" %)" %@ $ ! ! !)%! " %= %$1 " $ " ! (" 1! " .! " >!" :! '@! $ : "!$ !" " : $ : "" . &' $ +1! A2EE4B .& D$ !& $ " $ " !)%! " % $$" ! %!" M ( ( ! A MB= "$!" $ : "" 1% $ ! " "!" ' " 4J % ! " !$.$ &! $! 27J [' " !e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

6J -'% (. */ 0 $1 ! " - 7 8

$ "" "!" !" " % $! $ @ .;!" ! .;= $" % ! " $$ !% ! $" ! 1! '" 1 $ 'F! $%!! $ $ $= 1! % " "$ $ : "" . &' $ ! ! = $% .; : !" ! ! !: '$ &!

$"@" , =$"! %2 0F *

$ ! ' " $ % " ! % < $$ ! $10 ( $10 $! !! ! ! !' " E5 M 25=5 M = "%@ " ! .!" $ " %$ %! !' :" " $ !$ "! $! !$ < !! . $% $ &! $ " :!! " !)%! " % ) " ( ! ! $%!! %@ " $" " : $ " D ! " !)%! < 1! " " ( $$ >" $1 Q A2E93B 1 > ! < !' " %$ " ! " !)%! " % : " 4 4JJ M ! !$!! ) " " .; %$ A309 M B $! $ : "" !&D $ A != 2EE2BG ) " .@ $ $! %$ ! 2J ( 36 M $!$ " $% 1% ! A!"1 = 2EEEBG ! $! %$ ! 5J ( 9J M $! $ : "" A!" = 2EEEG C !' ( !1= 2E85BG ( ! % %) $ " %$ $% " M% ( C !' ! 2E92 " 35J M

$$< $";#"

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-'% (. */ 0 $1 ! " - 62 ! !" ## $# %& '()* % $"+ ,"*

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e$ $ < $!%" f= a ( ! $0$ $= $F! " %! !)' $ %! $ : "" .; : ! !" $! < D ! 1% $ e$ $ !$!" ! " : $ " !)%! = !! ! %$! % " $$ &! " $ ( ! !> %@ $= D$$ &! " " A!" = 3JJJB= !" % $ % A+>> = 2EE8B= + " % ! ( $ " A : !! " %! " ' " ! : "" " M ! !!" B= "!" ! ! 3 1 " !$!" $ " 1 "$" !$ " $ : "" 1% $ ( @ $ D ! ! $$ ! < 1! :"= $% ! $ " .! " " %)" " $$ !% ! " ! $" ! >!" $$ ! $10 !% < $$ &! < $ : "" 1% $ %@ %. )! $ : "" @ $ %@ '! $ : A$14 B

63 -'% (. */ 0 $1 ! " - 7 8

$! $ $!" ! >% $$ ! $10= "!" . :% < $ : "" 1% $ %@ $ !" $$ &! $12= % ! < %( $ : "" @ $ ' $$ &! $14= < :> ! ! $ $ : "" 1% $ A" !" ! . 5 ( 6B " ! 1$! ! < ! :!! " = %@ " ! $%!! ! . " $ 1% $ . $ ; 1%! G ! %.' '! . $%%! ! !$ "% ! "" 1% $ . :" ! :!! " $1 ! %" " ! %$! % !)' $ %;! " " !$ F! $%! " ! %" " A$ ! ! B $ : "" 1% $ !$!" ! :!! " $! ( ! ! 1>' %! $ = ( < $!$ " < ! ! . " $ : "" !! ! " $" $ $ &! ! @%. . %)" $= "" " !$ $! %%.! $ = !)%! " % ! $ . &' $ " " $%

$: ="B$+

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5 $ "D !"

% !% "" %&' $ ( $ &' $ "! . :" ! %. ! " > " % $% " " % ! $! !$ &D $ = $ $ " .! " " :!! " !)%! " % = 1% . :" $ &! " !% "" %&' $ $% !1 . $ &! " " ! % " = .@ " = D' $ &!= ( %%$ &! " $ !! ! " :!>" " % ( : ! ( >" $! D ! " !)%! " % 1! "% " %. )! ! ! $ &D $ . " " > " % ( O( ! 2E97= !$!! < ! % ! ! " " $. " " " %. ! = "$ !" "%@ % = $ :; !%= D' $ &! %! ( " .> ! : " $! % % !)%! " % >!" !$ . ! %! A+>> ( = 2EEJB "% & < : .! $ &! " %%.! 1 ! !1 .!

-'% (. */ 0 $1 ! " - 64 ! !" ## $# %& '()* % $"+ ,"*

$ :; ! % " $ " ! " " .@ $ !" "" . <% $ !$!" ! $$ ! $1 :" A1% $= @ $ ( $B < ! "! !" $ " $ . %%.! . &' $ $ " D' $ &!= $% . :" ! ! (= 1! $ " !$$ &! " %)$ '" >F$ ( $ : $ $ !"! %! $% %" " " :! " $!$ % ! $ ! %. ! " > " %= $% ! " " <!&! ( "1 &! " $) % )!< % $1 " . $ P1% ( $." A3JJ5B " ! !$$ &! " '!" < $! !! '$ (e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

65 -'% (. */ 0 $1 ! " - 7 8

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E="; = " ;

!"% " !:!!% ! !)%! " % !$(! $$ ! $ !"$ " " $%! D ! = $% = % ( "% : $G $$ ! ! $! $% %!! %F G ( $ )% $ " : " : ""= ! < '! 1$M ! @$ $= ! '"= 1@ $ % !! ( $"$= $ "! $!"$ % " !" : " $" A+ $ ( * = 2E94G + >! = 3JJJG $ = 2EE5B " < $$ ! )% $ ! ! . " % " ! $"= %! $!$ D ! $!! " ! :!! " ! !$ %! ')! $ ( "! ' "$ $$ ! )% $ < "! ' D !$ " . &' < $$& $%! " = ! : % !$ " < !)%! " % " "$ ! !;$ % ! !%" " $! $ &! " <? H!$1 I < "$! ! " ' ( !% ! '! " $!$ !% "! % " ! $ " $!$ AB= " '!" ! $$ &! " !%" = $ (e "" " 1.! = $! "% ( !;$ % ! " ' ")% $ ! ")% $ ! $ 1 D " $!$ $! '! ')! $ ! ! ! " % = " $! " < $ $ ( " " $$ ! $@! !%" '! ! " . != ! " !' "%! ! " : &! % $" " $ $!$= " ' ! A0" $1= 2EE5B# 2 $ " $!$ %" " !%!&' $%! 3 $ " $!$ !"$ " ." " 1 % ! 4 $ " $!$ :$" :!! 5 $ " $!$ ". " %$! % " $!$ " % ! " " $ = % $ $) $ $! " .$ &! " %" " " !%" " ! %$! % ' "!" ! :) " ! $ " $ " '! " !$"!! ! %%.! " % $ $$ "" " ! %)$ :$ ! 1.&! $ ( !$ " $$ &! "!" " ! $ !F% " $ $%# 2 !. "" " %)$ :) " . ")% $G 3 ! "" " % $ $ G ( 4 -'% (. */ 0 $1 ! " - 66 ! !" ## $# %& '()* % $"+ ,"*

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n '1 = 2EEJB ! $ % " $ !" ! $! $ : "" 1% $ ( @ $ " $1 ! G $ %!! . $ &! ( !$ &! " $% ')! $ ! '! $ ! % % ! ')! $ !"!" !%! " $ : "" . &' $ * ' $ $% !$ " $.1 " $ " ! "! !!$ ! '! $ ""= !< F! ! $ %$! % " $$ &! %. )! $!$ < ! %>$ " !'! " "$ ! $$ &! )' $ !" "= " " 1 ! . "" !%" %" " ' = .! " ! ! . ! = ! " $! . "" " < %$! % " 1 ! . "" "! ! ;! $! " !%" 1 "$" '

67 -'% (. */ 0 $1 ! " - 7 8

" ' " $! :!! " .; " : ! ; $! $$ ! $ !! $ " $" ! %.' C%! A2E89B '& $!$ &! " < ! . " !$ !" : " $! $$ ! )' $ )% $ $ ! " !" : " $! $$ ! $ !! = ( < !.! ! % " ' ' $! :!! "%@ ' ! :!! " 1% ' " '= $! ' !" : " $" $" $! $$ ! )% $ )' $ An '1 ( $M(= 2EEJB ! !)%! " % $ ! " 1@ $ % !!= > . &' $ A " B !! " ' ! " $ ! $"= < "%@ " $$ &! )% $ < & !. ! !$ ! ! >! $" A$ = 2EE5B= !%!'. ! ! . " 1 ! . "" 3 ! ( " D ! " $!" . " ! " 1 ! . "" $!$ " ! ("! $! % %$! % " $ )' $ " D ! :" . :" ! "

-'% (. */ 0 $1 ! " - 68 ! !" ## $# %& '()* % $"+ ,"*

69 -'% (. */ 0 $1 ! " -

K :!! " ! $ : "" 1% $= @ $ ( $ K ! $!! " ! :!! " !)%! " % !! %$ ! 25 ( E5 M ( %( ! ' $! K : " $ : "" $ $ " $$ ! :" ! %! ! $ = !" :%! %; .! " $ $ &! $! $! K $$ ! $ " $! " %. ! " > " % ' ! ' A+ != 2893B "$ !" !% "" : ! " " .@ = '@ ( %G "%@ " ! "? $ &' $ %! $1 $ $! ! ! !$ ! % & $ K !)%! " % %)$ ')! $ < "$! 1 ! . "" " ! ! . !

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K $% !" $! ! $! " " $%!! $ : " :!! " ( < 1 "$" $ : "" . &' $ %! A$% $ : "" 1% $ ( $ : "" $ &D $B < !! ! !$ . %)" $ " !) K ". : $ " D ! " !)%! " % . $) % = ( < !! ! $ ! % !$ = 'F! $$ &! < 1! % " ! . " %. ! " > " % K . $@! $% $$1 = $M !"%$$ &!= " " '!& $ $! $ " $ " $!$ "! %" $" $" ! ! % " D %!$ &! ( .! " $! % . %$! % " $$ &! )' $ !"$ "

-'% (. */ 0 $1 ! " - 72 ! !" ## $# %& '()* % $"+ ,"*

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