¡BIENVENIDOS al
CONGRESO ANUAL
DE LA SOCIEDAD DE
FARMACOLOGÍA DE CHILE!
Concepción 2019
El evento científico contará con la participación de 5
conferencistas internacionales, 70 expositores en simposios y mini
simposios, junto a cerca de 200 jóvenes científicos que exhibirán
los avances obtenidos en sus diferentes líneas de investigación.
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¡BIENVENIDOS al
CONGRESO ANUAL
La Revista de Farmacología de Chile tiene un panel de editores conformado
por connotados farmacólogos nacionales que son miembros de la Sociedad de
Farmacología de Chile y académicos de las principales universidades chilenas.
DE LA SOCIEDAD DE
FARMACOLOGÍA DE CHILE!
Concepción 2019
Comité Editorial
El evento científico contará con la participación de 6
conferencistas internacionales, 70 expositores en simposios y mini
simposios, junto a cerca de 200 jóvenes científicos que exhibirán
los avances obtenidos en sus diferentes líneas de investigación.
Dr. Ramón Sotomayor Z., Editor en Jefe
(Universidad de Valparaíso, Chile)
Dr. Mario Rivera M., Psicofarmacología
(Universidad de Chile, Chile)
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Directiva
Dr. Jorge Fuentealba A., Neurofarmacología
(Universidad de Concepción, Chile)
Presidente:
Dr. Jorge Fuentealba A.
Universidad de Concepción
jorgefuentealba@udec.cl
Dra. Viviana Noriega S., Farmacología Clínica
(Universidad de Chile, Chile)
Vicepresidente:
Dr. Javier Bravo V.
Pontificia Universidad Católica de Valparaíso
javier.bravo@pucv.cl
Dra. María Angélica Rivarola, Neuroendocrinología
(Universidad Nacional de Córdova, Argentina)
Secretario General:
Dr. Claudio Coddou A.
Universidad Católica del Norte
ccoddou@ucn.cl
Dra. Marcela Julio-Pieper, Farmacología Gastrointestinal
(P. Universidad Católica de Valparaíso, Chile)
Tesorero:
Dr. Ramón Sotomayor Z.
Universidad de Valparaíso
ramon.sotomayor@uv.cl
Dra. Gonzalo Cruz N., Farmacología Endocrina
(Universidad de Valparaíso, Chile)
Past-presidente:
Dr. Edgar Pastene N.
Universidad de Concepción
edgar.pastene@gmail.com
Dr. Edgar Pastene N., Fitofarmacología
(Universidad de Concepción, Chile)
Directores:
Dra. Georgina Renard D.
Universidad de Santiago
geormrenard@gmail.com
Dr. Patricio Iturriaga V., Química Médica
(Universidad de La Frontera, Chile)
Dr. Gustavo Moraga C.
Universidad de Concepción
gumoraga@gmail.com
Dra. Viviana Noriega S.
Universidad de Chile
viviananoriega@gmail.com
Dr. Gonzalo Cruz N.
Universidad Valparaíso
gonzalo.cruz@uv.cl
Dr. Miguel Reyes P., Química Médica
(Universidad de Santiago de Chile, Chile)
Dr. Juan Pablo García Huidobro T., Farmacodinamia
(Universidad de Santiago de Chile, Chile)
Dra. Georgina Renard C., Co-Editor, Psicofarmacología
(Universidad de Valparaíso, Chile)
Dra. Carolina Gómez G., Ciencias Farmacéuticas
(Universidad de Concepción, Chile)
Dr. Rodrigo Castillo P., Farmacología Cardiovascular
(Universidad de Chile, Chile)
Dr. Mauricio Dorfman P., Metabolismo y Diabetes
(University of Washington, Seattle-USA)
Dr. Javier Bravo V., Neurofarmacología
(P. Universidad Católica de Valparaíso, Chile)
Revista de Farmacología de Chile es publicada por la Sociedad de Farmacología de
Chile Derechos Reservados a la Sociedad de Farmacología de Chile. ISSN 0718-8811
versión impresa ISSN 0718-882X versión digital Todos los derechos reservados. Ninguna parte de esta publicación puede ser reproducida, almacenada en sistema alguno
de tarjetas perforadas o transmitida por otro medio -electrónico, mecánico, fotocopiador, registrador, etcétera- sin permiso previo por escrito del comité editorial.
IMPRESO EN TRAMA IMPRESORES.
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Indice
Volumen 13,
número 2,
AÑO 2019
Página 22:
Magíster en Neurobiología: Formando especialistas en investigación experimental
Página 50:
Especial XLI Congreso Anual de la Sociedad de Farmacología de Chile
Página 52:
Bienvenida
Página 53:
Programa
Página 54:
Conferencistas Internacionales
Página 60:
Abstracts for XLI of the chilean Society of Pharmacology
Conferences
Página 62:
Symposia
Página 76:
Minisymposia
Página 78:
Dr Jorge Mardones Restat Award
Página 80:
Incorporations
Página 83:
Oral Communications
Página 88:
Posters
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a Revista de Farmacología
de Chile es una revista de
información científica vigente
desde el año 2008. Su objetivo inicial
ha sido ser un órgano de difusión de
la farmacología en Chile para luego
ser una plataforma de difusión para
el resto de Latinoamérica.
Desde sus comienzos, la recepción de
artículos científicos fue aumentando
año a año, llegando a un máximo
de artículos publicados el año 2014.
Además, hemos logrado contar con
varios artículos internacionales. Sin
embargo, desde esa fecha en adelante
se produjo una disminución en los
artículos recibidos que coincidió con
nuevas revistas de la disciplina de
libre acceso y una gran competencia
con revistas del área que ya se
encuentran indexadas.
L
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Editorial
En este sentido, se nos ha planteado
un gran desafío para los próximos
años en pro de revertir esta tendencia.
Por un lado, debemos aumentar la
difusión y distribución de la revista
a nivel latinoamericano y, asimismo,
estamos evaluando la posibilidad
de asociarnos a una casa editorial
internacional y cambiar el idioma de
publicación de nuestra Revista.
En el volumen de este año, hemos
logrado volver a aumentar el número
de Revistas publicadas con respecto
al año anterior. En este número de
la Revista se publican dos artículos
originales internacionales (Cuba y
España) y dos artículos nacionales.
Además, teniendo en cuenta que
este número corresponde al XLI
Congreso Anual de la Sociedad
de Farmacología, se publican los
resúmenes correspondientes a las
conferencias, simposios y paneles
que serán presentados del 4 al 8 de
noviembre en la sede del congreso,
Universidad de Concepción.
Nuevamente invitamos a todos a ser
parte del crecimiento de la Revista
de Farmacología de Chile y lograr
difundir información científica del
área a nivel internacional.
Dra Georgina Renard
Co - Editora
Directora por Santiago Sociedad de
Farmacología de Chile
“Por un lado,
debemos
aumentar la
difusión y
distribución de
la revista a nivel
Latinoamericano
y, asimismo,
estamos
evaluando la
posibilidad de
asociarnos a una
casa editorial
internacional
y cambiar
el idioma de
publicación de
nuestra Revista”.
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La sociedad y Sofarchi:
un vínculo que se expande
Estimadas socias y socios:
“En esta
conversación,
le expresamos
al Ministro
la total
disposición
de la Sofarchi
para ser un
modulador
positivo en
esta tarea, y
pusimos a su
disposición
los diferentes
canales de
comunicación
de nuestra
sociedad para
ayudar en ese
objetivo”.
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E
stimadas socias y socios, junto
con extenderles un cordial saludo desde nuestra directiva, les
invitamos a leer nuestro nuevo
número de la revista, Editado con el incansable trabajo del Dr. Ramón Sotomayor como editor en jefe, Dra. Georgina
Renard como co-Editora, Fabiola Valdebenito, nuestra periodista, y todos quienes de una u otra forma colaboran para
que la revista siga activamente siendo
un canal de comunicación con ustedes
y la sociedad. En este número podrán
encontrar el informe de algunas de las
más recientes actividades donde nuestra
sociedad ha estado presente, actualidad
científica, artículos científicos originales
y revisiones, entre otros.
Respecto a la actualidad científica nacional, durante el primer semestre, hemos
participado en una interesante reunión
con el Ministro de Ciencia Dr. Andres
Couve, quien nos contó los avances que
su ministerio ha experimentado, y las
metas de mediano y largo plazo que se
han impuesto, como el acercar la actividad científica al centro de atención
de la sociedad. En esta conversación, le
expresamos al Ministro la total disposición de la SOFARCHI para ser un modulador positivo en esta tarea, y pusimos a
su disposición los diferentes canales de
comunicación de nuestra sociedad para
ayudar en ese objetivo.
En este plan de acercar la ciencia a la sociedad, la SOFARCHI realizó la pasada
reunión de la Directiva en la ciudad de
Coquimbo. Esta reunión fue realizada en
la Facultad de Medicina de la Universidad Católica del Norte, reuniéndose con
nosotros el decano de la facultad con
quien discutimos nuevas oportunidades
de integración. Además, como actividad
de divulgación a la sociedad se realizó
una charla científica a alumnos del Colegio Alemán de La Serena.
A nivel internacional, 4 miembros de
nuestra sociedad nos representaron en
la Reunión Anual de la Sociedad Española de Farmacología, realizada en el pasado mes de Julio en La Palma de Gran
Canaria, España. En este congreso, donde además participaron representantes
de las sociedades de farmacología de
Alemania y Holanda, pudimos compartir e intercambiar experiencias y generar
colaboraciones entre nuestras sociedades que esperamos den sus frutos en el
mediano plazo.
Por último, estamos trabajando sin pausa, y con nuestros mejores esfuerzos,
para organizar nuestra próxima reunión en Noviembre, la que contará con
importantes expositores nacionales e
internacionales en 7 conferencias plenarias, 10 simposios, comunicaciones
orales, comunicaciones en póster y actividades satélites que complementaran
una intensa jornada científica de 5 días.
A nivel personal, tengo el secreto anhelo
que puedan re-descubrir la belleza del
Campus Concepción, en el centenario de
la Universidad de Concepción, y sus alrededores, y les reitero mi más ferviente
invitación para que nos acompañen en
esta jornada científica que nos prepara
varias sorpresas.
Un atento saludo a todos Uds. y disfruten de este nuevo número de nuestra
nueva revista
Dr. Jorge Fuentealba Arcos
Presidente SOFARCHI
“El encuentro, realizado en el Palacio de La
Moneda, contó con la participación de cerca de
25 organizaciones dedicadas a la investigación
y promoción de diversas áreas del saber, entre
ellas, la Sociedad de Farmacología de Chile”.
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Sociedades científicas se reúnen
con Ministro Andrés Couve para conocer
lineamientos para promover investigación
C
on el objetivo de entregar
detalles sobre el estado de
avance de la instalación del
Ministerio de Ciencias, Tecnología e Innovación, además
de exponer sobre las directrices que se
van a implementar para el desarrollo de
investigación, se realizó una reunión entre el jefe de la cartera, Andrés Couve, y
los representantes de las principales sociedades científicas del país.
El encuentro, realizado en el Palacio de
La Moneda, contó con la participación de
cerca de 25 organizaciones dedicadas a
la investigación y promoción de diversas
áreas del saber, entre ellas la Sociedad de
Farmacología de Chile Sofarchi. Su presidente, Jorge Fuentealba, indicó que “la
actividad ha sido muy productiva, en el
sentido de poder avizorar cuáles son las
etapas que ha cumplido el Ministerio y
cuáles son las que quedan dentro de la
instalación de esta importante reparti-
ción del estado que va a potenciar la investigación científica”.
Uno de los ejes de la reunión giró en
torno a las estrategias que las organizaciones podrían desarrollar en conjunto
para contribuir a potenciar el desarrollo
de la ciencia en Chile. En este sentido,
Fuentealba agregó que “la principal preocupación que manifesté al ministro dice
en relación a cómo las 20 o 25 sociedades que estábamos en la mesa podíamos
contribuir a potenciar el desarrollo de la
ciencia en términos de darle difusión, de
demostrar la calidad de la investigación
que se hace en los distintos ámbitos, desde las ciencias sociales hasta las ciencias
naturales y exactas”, expuso.
El líder de la Sofarchi añadió que “el ministro dijo que confía plenamente en que
vamos a ser actores activos y relevantes
en la difusión y fortalecimiento de los
conceptos de ciencia y de generación de
conocimientos”, puntualizó.
“La principal
preocupación
que manifesté al
ministro dice en
relación a cómo las
20 o 25 sociedades
que estábamos en
la mesa podíamos
contribuir a
potenciar el
desarrollo de la
ciencia en términos
de darle difusión,
de demostrar
la calidad de la
investigación
que se hace en los
distintos ámbitos,
desde las ciencias
sociales hasta las
ciencias naturales
y exactas”, Jorge
Fuentealba A.
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H
asta La Serena, en la región de Coquimbo, se trasladó la directiva de la Sociedad
de Farmacología de Chile, con el objeto de
realizar diversas actividades orientadas a fortalecer el desarrollo de este organismo científico en regiones y a motivar la incorporación
de nuevos socios.
La visita estuvo encabezada por su presidente,
Jorge Fuentealba, acompañado por el Vicepresidente de la entidad, Ramón Sotomayor,
junto al académico de la UCN y Secretario
General de Sofarchi, Claudio Coddou y las
directoras del organismo, Georgina Renard y
Viviana Noriega.
El equipo sostuvo un encuentro con el Decano
de la Facultad de Medicina de la Universidad
Católica del Norte, Osvaldo Iribarren, quien
pudo conocer detalles sobre las principales
líneas de trabajo que está desarrollando la organización durante este año, lo que permitió
sentar las bases para próximas acciones conjuntas.
“La idea de reunirnos en Coquimbo es potenciar las actividades en regiones y fortalecer el
interés de los científicos de esta zona del país
para integrarse a Sofarchi y colaborar, de esta
forma, al desarrollo de la disciplina”, señaló
Jorge Fuentealba. Agregó que “actividades
como estas refuerzan nuestro rol social, dando a conocer la ciencia y la farmacología. Por
eso, estamos muy contentos con la recepción
que hemos tenido de parte del Decano, ya que
es importante contar con ese entusiasmo y
creo firmemente que será un motor de empuje para esta iniciativa en la región”.
En este sentido, el decano de la Facultad de
Medicina de la Universidad Católica del Norte, Osvaldo Iribarren, comentó que “la UCN
sustenta su funcionamiento y su desarrollo
en tres pilares: la docencia, la investigación y
12
Directiva de Sofarchi visita Coquimbo para
promover la participación de socios
la vinculación. Para nosotros, como Facultad
de Medicina, tenemos que ir empujando la
frontera del conocimiento cada vez mas y una
sociedad científica como Sofarchi representa
exactamente está construcción de vínculos”
En este sentido, agregó que esta asociación
“nos permite ir abriendo otros lazos sutiles,
pero muy firmes, con el resto de las universidades, porque la Sofarchi está en la Universidad
de Concepción, en la de Valparaíso, en la de
Santiago, etcétera; así es que para nosotros no
puede ser mejor y más bienvenida”, manifestó.
Respecto a las actividades a realizar en conjunto, Irribarren indicó que “me invitaron a la
inauguración del Congreso de Sofarchi que se
realizará en noviembre próximo en Concepción, lo cual me parece muy bien porque está
en perfecta sintonía con la vinculación cada
vez más fuerte que pretendo hacer con el ambiente académico”, expuso.
El presidente de Sofarchi, Jorge Fuentealba, se
reunió con el Decano de la Universidad Católica
del Norte, Doctor Osvaldo Iribarren; para exponer
sobre los principales lineamientos de trabajo de
la organización farmacológica. Con ello, se busca
sentar las bases de futuras acciones conjuntas.
Ciencia más cerca
Otra de las actividades en las que participó la
directiva se desarrolló en el Colegio Alemán
de La Serena, donde cerca de 70 escolares de
octavo básico a segundo medio asistieron a la
charla “Mirando la naturaleza, el laboratorio
maestro”.
“Tuvimos una grata jornada con niños y jóvenes que nos dieron una muy buena recepción,
fueron muy interactivos, hicieron muchas
preguntas. Fue muy grato sentir que en Coquimbo hay un sustrato para poder desarrollar y potenciar esta área, que permitiría favorecer el desarrollo de la biomedicina en la
región”, explicó Fuentealba.
A través de la exposición, los escolares aprendieron sobre el valor de la observación como
mecanismo para entender diversos procesos
de la naturaleza, desde donde la humanidad
ha extraído fármacos desde tiempos inmemoriales. El investigador agregó que “hoy en día
esos principios farmacológicos son estudiados científicamente y aplicados a la biomedicina, para transformarlos en medicamentos
que permiten mejorar la calidad de vida de
millones de personas en todo el mundo y que
tienen su origen en este laboratorio maestro
que es la naturaleza”.
Otro de los presentes en la jornada fue el secretario de la organización científica, Claudio Coddou, quien comentó que “es muy importante
tener estos espacios, porque gracias al link con
la Sociedad de Farmacología estamos promoviendo la Universidad y generando interés de
los estudiantes hacia este mundo de la ciencia”.
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Delegación de la Sofarchi participó en el Congreso
de la Sociedad Española de Farmacología
C
on la colaboración internacional
de las asociaciones farmacológicas alemana, holandesa, irlandesa y chilena, se efectuó el 39°
Congreso Anual de la Sociedad Española
de Farmacología (Socesfa), realizado por
primera vez en Las Palmas de Gran Canaria. Más de 60 expositores de diversas
partes de globo divididos en 13 sesiones
de simposios, compartieron sus conocimientos con farmacólogos, científicos,
médicos, profesionales de área de la salud, pacientes y representantes de la industria farmacéutica, convirtiendo a este
encuentro en uno de los más relevantes
del planeta en su tipo.
La delegación de la Sofarchi estuvo integrada por los doctores Jorge Fuentealba
y Leonardo Guzmán, de la Universidad
de Concepción; junto a Ramón Sotomayor, de la Universidad Católica de Valparaíso y Guillermo Díaz, de la Universidad
de Chile.
Respecto a esta participación, el presidente de la Sofarchi, Jorge Fuentealba,
indicó que “como directiva nos parece muy relevante que una Sociedad tan
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grande como la española nos reconozca
y nos trate como pares, invitándonos a
participar en su Congreso”. El directivo
agregó que “fue una actividad muy interesante, científicamente de alto nivel
y con bastantes oportunidades para el
intercambio, para la vinculación y para
el desarrollo de articulaciones de estudiantes y de trabajos de investigación”,
expuso.
Los representantes nacionales realizaron cuatro exposiciones plenarias dentro
de los simposios, la primera a cargo del
doctor Guillermo Díaz, quien se refirió al
rol del fibroblasto cardíaco en procesos
inflamatorios y la interacción con células
inmunes.
En tanto, el doctor Ramón Sotomayor
abordó la programación de neuronas dopaminérgicas por exposición temprana a
hormonas sexuales y su posible efecto en
la vulnerabilidad ante la drogadicción.
Además, valoró la posibilidad que otorgan estos eventos internacionales para
potenciar el intercambio de experiencias en nuestro país, “nos contactamos
con Michael Spedding, secretario gene-
ral de la International Union of Basic &
Clinical Pharmacology para invitar a la
representante de la UPHAR el próximo
año a Chile. Además, logramos confirmar la participación de la Doctora Concha Peiró, de la Universidad Autónoma
de Madrid y miembro de la directiva de
la Socesfar a nuestro próximo Congreso Anual de Sofarchi que se realizará en
Concepción durante este año”, detalló el
doctor Sotomayor.
Aporte científico
La farmacología es entendida como una
ciencia necesariamente interdisciplinaria, por lo que en el Congreso se trataron
temáticas tan diversas como cáncer, receptores, diseño de drogas, farmacogenética, farmacología clínica en áreas de
endocrinología, gastroenterología y respiratorios; además de farmacología del
dolor y la inflamación, entre otros.
El equipo de académicos de la Universidad de Concepción presente en este 39°
Congreso de la Socesfar, participó tanto
en el área de envejecimiento, considerando desde los mecanismos hasta las
perspectivas farmacológicas; como en
nuevos avances en neurofarmacología.
En el desarrollo de este último tema,
abordado en un simposio en el que expusieron otros tres investigadores; participó
el Doctor Leonardo Guzmán con su charla titulada “efectos de los dendrímeros
PAMAM en la funcionalidad neuronal In
Vitro”. El investigador detalló que en la
ocasión “se mostraron avances en neurofármacos que se están desarrollando para
ayudar en procesos de adicciones; también se mostraron investigaciones sobre
intervenciones para la liberación de neurotransmisiones y, por mi parte, mostré
cómo los nanotransportadores podrían
ayudar a la entrega de fármacos en sitios
sinápticos”, detalló.
En tanto, Jorge Fuentealba presentó
“Sobreexpresión de P2xr contribuye a
la toxicidad por Beta Amiloides: nuevo
blanco farmacológico para la AD”, en
la que expone parte de su investigación
orientada al análisis de los mecanismos
celulares y moleculares que inducen el
desarrollo de enfermedades neurodegenerativas, especialmente la Enfermedad
de Alzheimer.
Cuatro
representantes de
la organización
científica chilena
fueron invitados
por la Socesfar para
exponer parte de
sus investigaciones.
En la actividad
también
participaron
sociedades
farmacológicas de
Holanda, Irlanda y
Alemania.
15
Cien años de Química
“Hace exactamente
100 años tenía lugar la
primera clase del curso de
Farmacia, dictada por don
Salvador Gálvez Rojas a
28 estudiantes, todas ellas
mujeres, constituyéndose en
una de las primeras carreras
universitarias en el sur de
Chile que ofrecía una opción
de educación superior para
las mujeres del país”.
L
a conmemoración de los 100
años de la Carrera de Química
y Farmacia, de la Facultad de
Farmacia de la Universidad de Concepción en este año 2019, constituye
uno de los más importantes hitos en la
historia de la también centenaria Universidad de Concepción y es especialmente significativa para todos quienes
hemos tenido el privilegio de realizar
nuestros estudios universitarios en
esta Alma Mater.
Hace exactamente 100 años tenía lugar la primera clase del curso de Farmacia, dictada por don Salvador Gálvez Rojas a 28 estudiantes, todas ellas
mujeres, constituyéndose en una de
las primeras carreras universitarias en
el sur de Chile que ofrecía una opción
de educación superior para las mujeres del país.
Cien años desde aquel sencillo pero
visionario comienzo en que nacía la
carrera de Química y Farmacia y al
mismo tiempo, la entonces, Escuela de
Farmacia, cuando la creación de la carrera - la segunda históricamente en el
país - fue promovida con decisión por
los fundadores desde el momento en
que el Comité Pro Universidad y Hospital Clínico creado en 1917 empieza a
definir la forma en que la novel Casa
de Estudios iniciaría sus actividades.
16
y Farmacia UdeC
Respondiendo a una sentida necesidad, no sólo de la región sino del país,
pues con su creación se buscaba el
mejoramiento sanitario de la población.
Cien años después, la Facultad de
Farmacia ha formado más de 2.800
profesionales Químicos Farmacéuticos, que se desempeñan en los distintos ámbitos del ejercicio profesional,
representando un importante aporte
al desarrollo del país, particularmente en el sector sanitario, el sector productivo, la academia y también en la
política universitaria y ciudadana.
En este contexto es relevante destacar
la figura de Ligia Gargallo González,
químico farmacéutica, académica e
investigadora, Premio Nacional de
Ciencias Naturales 2014 y Premio
L’Oreal UNESCO 2007 para Mujeres en Ciencias. Del mismo modo,
reconocer el esfuerzo, compromiso,
dedicación y liderazgo de recordados
maestros y profesores.
Durante estos 100 años son muchísimos los hitos que marcan la historia de
la Carrera de Química y Farmacia: la
Farmacia Modelo en 1920, un centro
de práctica, único e innovador para
la época, con un fuerte sentido social;
la estructura y organización definitiva
como Facultad en 1927; el surgimiento de nuevas iniciativas de formación
profesional como la creación de las carreras de Bioquímica en 1957 y de Nutrición y Dietética en 1975; las fiestas
universitarias, juegos florales y carnavales de antaño; la difícil y anhelada
Autorización Ministerial para que la
Universidad de Concepción pudiera
otorgar los títulos de Químico Farmacéutico y de Bioquímico hacia fines de
1974; la ampliación del actual edificio
y el surgimiento del Postgrado. En
este ámbito, la Facultad de Farmacia
ofrece actualmente, un programa de
doctorado, 3 programas de magíster y
el recientemente creado programa de
Especialización en Farmacia Clínica.
Sumando también Cursos, Diplomas
y Diplomados.
La investigación como pilar para la
generación de nuevo conocimiento
también ha sido un área destacada en
el quehacer de la Facultad de Farmacia, reflejado en publicaciones en revistas de corriente principal y adjudicación de proyectos de investigación
con financiamiento externo (Fondecyt, Fondef, entre otros). Desde 2015
a la fecha, la Facultad ha adjudicado
4 iniciativas Fondequip para la adquisición de equipamiento mediano que
ha significado para la Universidad de
Concepción la atracción de más de
1.000 millones de pesos para investigación, permitiendo el desarrollo y
crecimiento de la Facultad y la institución, además de mayores posibilidades de formación terminal para estudiantes de pre y postgrado.
Muchos desafíos y tareas se proyectan para la Facultad, sin embargo, es
necesario destacar nuevamente el perenne sello UdeC del profesional químico farmacéutico, esa característica
tan propia que permite reconocernos
con nuestros pares cualquiera que
sea el área laboral y lugar en que nos
encontremos, haciendo que nuestros
profesionales sean preferidos y respetados.
La invitación es a seguir construyendo y nutriendo con orgullo, respeto,
empatía, comportamiento ético y dedicación esta hermosa profesión farmacéutica, para proyectarla exitosa
hacia el futuro.
“Cien años después,
la Facultad de
Farmacia ha
formado más de
2.800 profesionales
Químicos
Farmacéuticos,
que se desempeñan
en los distintos
ámbitos del ejercicio
profesional,
representando un
importante aporte al
desarrollo del país,
particularmente en
el sector sanitario, el
sector productivo, la
academia y también
en la política
universitaria y
ciudadana”.
Dr. Ricardo Godoy Ramos
Decano Facultad de Farmacia
Universidad de Concepción
17
18
Formando profesionales
en las grandes líneas de
desarrollo científico básico
y clínico de la farmacología
L
a necesidad de contar con especialistas que comprendieran en profundidad las bases
modernas de la farmacología
del Sistema Nervioso y que, además,
adoptaran conceptos biomédicos relacionados con la farmacología molecular y clínica, motivó en 2012 a un
equipo de académicos de la Facultad
de Ciencias de la Universidad de Valparaíso a generar nuevas instancias
de formación continua.
Fue así como el Doctor Ramón Sotomayor, académico del Instituto de
Fisiología de la Facultad de Ciencias
de esta casa de estudios, encabezó la
idea de promover el desarrollo de un
programa de postgrado en Neurofarmacología. El investigador, quien actualmente es el coordinador de esta
instancia, comentó que “este curso se
realiza todos los años en la Universidad de Valparaíso y ha sido muy bien
evaluado en cuanto a los contenidos
que se imparten, así como también
por las habilidades experimentales
que adquieren los estudiantes durante el transcurso del programa”
Agregó que el curso está orientado
tanto a profesionales del área clínica, como neurólogos, neurocirujanos, psiquiatras y psicólogos, entre
otros; como a especialistas en neurociencia, farmacología y biomedicina.
Según indicó el Doctor Sotomayor,
esta séptima versión “cuenta con estudiantes del Doctorado en Ciencias,
mención Neurociencias y del Magíster en Ciencias Biológicas, mención
Neurociencias de la Universidad de
Valparaíso. Además, hay estudiantes
profesionales, incluso, en esta oportunidad contamos con un estudiante
polaco que es médico”.
El Doctor Sotomayor destacó también la amplia presencia de la Sociedad Chilena de Farmacología en esta
instancia, “este curso ha sido patrocinado desde su origen por la Sofarchi, lo que nos ha permitido contar
con la participación de profesores de
alto nivel provenientes de destacadas
Universidades del país”.
En este sentido, indicó que “gracias
a esta alianza hoy contamos con una
decena de académicos que son socios
titulares de la Sofarchi, quienes integran del staff de docentes de este curso”. Entre ellos, se cuentan los doctores Jorge Fuentealba (Universidad de
Concepción), Javier Bravo (Pontificia
Universidad Católica de Valparaíso),
Claudio Coddou (Universidad Católica del Norte) y Georgina Renard (Universidad de Santiago), entre otros.
Alumnos exitosos
Diversas son las habilidades y conocimientos que los alumnos de este
curso de Neurofarmacología han obtenido generación tras generación. A
través de una metodología que incluye clases magistrales y seminarios de
discusión, se han formado profesionales con pensamiento crítico, capaces de analizar las grandes líneas de
desarrollo científico básico y clínico
Desde hace
siete años,
este programa
prepara con
conocimiento
teórico y
habilidades
experimentales
tanto a
profesionales
del área
clínica, como a
especialistas en
neurociencia,
farmacología y
biomedicina.
19
que han permitido tanto la generación de los tratamientos farmacológicos actuales, como su evolución y
proyecciones futuras.
Junto a ello, la propuesta académica de este programa permite a los
alumnos desarrollar trabajos experimentales originales que contribuyen
a la generación y desarrollo de tratamientos farmacológicos del sistema
nervioso.
Estos y otros conocimientos teóricos
y prácticos entregados en el programa de Neurofarmacología, le han entregado herramientas para avanzar
en sus investigaciones a la estudiante
de segundo año del Magíster en Ciencias Biológicas, mención Neurociencias de la Universidad de Valparaíso,
Victoria Collio,
“Estoy haciendo mi tesis sobre el efecto del consumo de dieta alta en grasa
en algunos núcleos, como el septum
lateral cerebral, en ratas, para evaluar el impacto en el control de la comida, del hambre y de la saciedad”,
indicó la pedagoga en biología de la
Pontificia Universidad Católica de
Valparaíso. Hasta ahora, los resultados obtenidos por Victoria Collio han
20
determinado que “aquellas ratas que
ingirieron dietas altas en grasa durante gran parte de su vida, a diferencia de las control, siguen prefiriendo
esta dieta en grandes proporciones.
Hay una diferencia bien importante”
Respecto al aporte que ha sido el programa de Neurofarmacología en el desarrollo de su investigación, sostuvo
que “el curso entrega una base neurobiológica en todos los ámbitos, que van
desde las adicciones, el movimiento,
la fisiología, lo molecular, etcétera. El
ramo de Estructura y Función del Sistema Nervioso, por ejemplo, es súper
importante y exigente”.
La séptima versión del curso de Neurofarmacología tiene una duración
de 18 semanas y considera un total de
162 horas semestrales.
Mayor información:
Sra. Francisca Ramírez
Coordinación de Postgrado, Facultad de
Ciencias, Universidad de Valparaíso Avda. Gran Bretaña #1111, Playa Ancha, Valparaíso. Fono +56 32
2508000 francisca.ramirez@uv.cl ;
postgrado.ciencias@uv.cl
21
Magíster en Neurobiología:
Formando especialistas en
El programa de la Facultad de Ciencias Biológicas de la UdeC
proporciona un contenido académico orientado a la formación
de especialistas en esta área, poniendo énfasis en el aprendizaje
práctico de técnicas, protocolos, procedimientos de experimentación
en neurobiología celular y molecular, además de estudio de
comportamiento animal.
22
investigación experimental
L
as bondades del ajo han sido ampliamente valoradas desde tiempos remotos. Es considerado uno
de los productos alimenticios
más completos en la naturaleza gracias
a la larga lista de vitaminas y minerales
que posee, que le otorgan comprobadas
propiedades cardioprotectoras, hipoglicemiantes y antioxidantes.
A este listado de beneficios se agrega una
nueva cualidad descubierta recientemente por un equipo de investigadores de la
Universidad de Concepción, quienes analizaron las capacidades neuroprotectoras
de una variedad fermentada del Allium
ampeloprasum o ajo chilote, una especie endémica de la Isla Grande. Según la
bióloga que encabeza el estudio, Javiera
Gavilán, este alimento cambia tras de ser
sometido a condiciones específicas de calor y humedad, lo que le otorga su característico color oscuro y potencia sus propiedades. “Nos dimos cuenta de que su
composición química es distinta al ajo que
comemos normalmente, porque adquiere
nuevos compuestos sulfurados. Desde ahí
partió el interés de probar su efecto neuroprotector en modelos de Alzheimer”.
Los datos que obtuvo en modelos de ratón comprobaron que el ajo contiene una
serie de compuestos bioactivos que proporcionan un blindaje a las neuronas,
haciéndolas más resistentes ante agentes tóxicos que están presentes en patologías neurodegenerativas. Es por ello
que, a largo plazo, este estudio “apunta
a desarrollar un nutracéutico, porque hemos visto que este extracto tiene efectos
preventivos en la Enfermedad de Alzheimer”, señaló la bióloga UdeC.
Los resultados de esta investigación, que
por su impacto ya fue destacada en varios
medios de comunicación; están contenidos en la tesis titulada “Efectos neuroprotectores del extracto de ajo negro chilote sobre la toxicidad del péptido beta
amiloide”, que Javiera Gavilán desarrolla
en el marco del Magíster en Neurobiología impartido por la Facultad de Ciencias
Biológicas de la UdeC.
Ella es parte de la primera generación de
estudiantes que ingresó a este programa
de postgrado, el cual también ha impulsado líneas de investigación en torno a
enfermedades neurodegenerativas como
Parkinson y Creutzfeld Jacob, además
de estudios sobre receptores de glicina y
niveles fisiológicos de nutrientes, entre
otros.
“Cuando este magíster se abrió fue como
una luz, fue súper bueno porque era específicamente lo que yo quería aprender
y lo que me hacía falta también en mi carrera como bióloga”, señaló Gavilán.
Este es, precisamente, el principal enfoque del Magíster en Neurobiología que
imparte la Facultad de Ciencias Biológicas de la UdeC, programa que apunta a
formar científicos especialistas en neurobiología, con una base teórica y práctica
sólida en estudios relacionados al sistema nervioso y con una fuerte orientación
hacia lo experimental, donde se encuentra el sello distintivo de este programa.
Este se fundamenta en la calidad de los
grupos de investigación vinculados al
claustro académico del programa, en la
disponibilidad de equipamiento de primera línea para la investigación de frontera en neurobiología y en la vigencia de
proyectos con financiamiento externo.
Gracias a este
enfoque, el
programa ha
desarrollado líneas
de investigación
en enfermedades
neurodegenerativas
como Alzheimer,
Parkinson y
Creutzfeld Jacob;
compuestos
naturales, además
de estudios sobre
receptores de glicina
y niveles fisiológicos
de nutrientes, entre
otros.
Enfoque hacia lo experimental
El Magister en Neurobiología de la FCB
busca formar profesionales especializados en la comprensión científica profunda de problemáticas neurobiológicas
relevantes en nuestra sociedad actual,
como por ejemplo, enfermedades neurodegenerativas, adicción, dolor crónico,
desórdenes alimenticios, y depresión,
entre otros.
23
Uno de los creadores del programa fue el
doctor Leonardo Guzmán, quien comentó que este “pretende ser un magíster
con parámetros modernos, con mucho
énfasis en generar nuevo conocimiento
a través de los proyectos de tesis, por lo
que está pensado para quienes estén motivados en aprender técnicas, protocolos,
procedimientos de experimentación en
neurobiología celular y molecular y comportamiento animal”
El académico detalló que este programa
está orientado a “estudiantes que hayan
tenido una base de fisiología y bioquímica más o menos importante, por lo tanto,
alumnos de Bioquímica, de Bioingeniería, Química y Farmacia, Biología, Biología Marina; también para las carreras
del área de la salud, como Kinesiología,
Medicina, Tecnología Médica, Obstetricia, Odontología”, entre otras.
24
A través de su programa académico, que
considera asignaturas básicas, de especialidad, complementarias y seminarios;
el alumno egresado será especialista en
neurobiología experimental y contará
con competencias científicas que permitan el inicio de una carrera asociada a
la investigación y a la divulgación de la
disciplina. Además, tendrá la capacidad
de comunicar, integrar y analizar críticamente y generar nuevos conocimientos
en el área de la neurobiología en un contexto bioético.
Mayor información:
Paula Veloso Aguilera
Secretaría de Magíster, Facultad de
Ciencias Biológicas, Universidad de
Concepción.
Fono: (56-41) 2204159
E-mail: pauveloso@udec.cl
26
26
La formación de doctores:
Nuevas tendencias y oportunidades de
internacionalización
Concepción Peiró Vallejo
Profesora Titular, Departamento de Farmacología, Facultad de Medicina
Coordinadora del Programa de Doctorado en Farmacología y Fisiología
Subdirectora de la Escuela de Doctorado (EDUAM)
Universidad Autónoma de Madrid, España
Email: concha.peiro@uam.es
Significado de los estudios de
Doctorado
El título de Doctor es el máximo grado académico que puede otorgar la institución universitaria. En Europa, tras el proceso de armonización del Espacio Europeo de Educación
Superior (EEES), los estudios universitarios
se ordenan en los niveles sucesivos de Grado,
Máster y Doctorado, coincidiendo con la mayoría de las instituciones académicas a nivel
global. La finalidad primordial de los estudios
de Doctorado es la formación de profesionales de la investigación, que representan piezas clave de una sociedad basada en el conocimiento, como es la actual. Por otra parte, no
hay que olvidar que el término “doctor” deriva del verbo latín “docere”, es decir, enseñar.
No en vano el título de Doctor es un requisito
imprescindible para el desarrollo completo de
una carrera académica universitaria.
Educación doctoral: más allá de
la tesis doctoral
Durante muchos años, el eje central de los estudios de Doctorado ha sido la realización de
la tesis doctoral, consistente en un trabajo de
investigación original desarrollado por el candidato a recibir el título de Doctor. Hoy en día,
la tesis doctoral sigue siendo sin duda el elemento principal de los estudios de Doctorado,
si bien se hace cada vez más hincapié en una
formación más amplia e integral del doctorando que le permita adquirir una serie de competencias y habilidades, relacionadas con la
investigación, pero también con otros aspectos
más transversales y de utilidad más allá de la
carrera investigadora (Figura 1).
Efectivamente, uno de los objetivos actuales de los programas de doctorado es poner
a disposición de sus estudiantes actividades
formativas que les permitan manejar mejor
aspectos generales directamente asociados
con la labor investigadora, como cursos de
conducta responsable en investigación, de
análisis de datos, de manejo de bibliografía,
de escritura de trabajos científicos o de proyectos de investigación, o de pensamiento crítico, entre otros.
Pero más allá de los aspectos específicos relacionados con la actividad puramente investigadora, se busca también hoy en día proveer a los estudiantes de doctorado con una
serie de capacidades de carácter transversal
e intersectorial, que les puedan ser útiles en
áreas que no sean las puramente académicas
o investigadoras. Hablamos, por ejemplo, de
habilidades de comunicación oral y escrita,
de habilidades interpersonales, de manejo
de conflictos y negociación, de desarrollo de
carrera profesional (Figura 1). En efecto, hay
que tener en cuenta que cada vez más instituciones no académicas y empresas demandan
doctores, que puedan ejercer de líderes de su
sector. Por otra parte, es una realidad que en
la actualidad un gran número de los doctores
egresados ya no permanecen en la academia,
por lo que conviene dotarles de este tipo de
herramientas profesionales que puedan serles de utilidad tanto dentro como fuera de la
institución universitaria.
Internacionalización y
movilidad en el Doctorado,
¿cuáles son sus ventajas?
Uno de los principales aspectos transversales
que se busca desarrollar hoy en día durante los
estudios de Doctorado es la internacionalización y la movilidad de estudiantes. Pero, ¿qué
ventajas puede ofrecer al candidato a Doctor el
realizar una estancia de investigación en el extranjero que puedan compensarle del esfuerzo
personal, logístico y económico que supone
ese desplazamiento? Si el grupo receptor y el
momento de realización de la estancia se eligen bien, sin duda las ventajas son múltiples y
abarcan diferentes planos de interés.
En primer lugar, la movilidad puede, y debería, suponer una expansión del horizonte
técnico y científico del estudiante, con oportunidades para intercambiar conceptos, ideas
y experiencias en entornos diferentes del ha-
“La educación
doctoral hoy en
día tiene como
meta, junto con
la realización de
la tesis doctoral,
ayudar a que el
futuro Doctor
adquiera una serie
de conocimientos
y habilidades,
relacionadas
o no con la
investigación, que
le proporcionen una
preparación más
completa e integral
de cara al mundo
profesional”.
27
bitual. Esto además favorece la creación de
redes de contactos, lo que en inglés se conoce
como “networking”, que pueden ser de gran
utilidad para la empleabilidad y el desarrollo
de la carrera profesional futura del candidato
a Doctor.
Por otra parte, la movilidad puede suponer
un claro beneficio en cuanto a competencias
idiomáticas y de comunicación, especialmente si la estancia se realiza en países que no
comparten la lengua materna del estudiante
de Doctorado. En cualquier caso, e independientemente del idioma hablado, el estudiante tendrá que desarrollar más que nunca su
capacidad organizativa y sus habilidades de
comunicación y de relaciones interpersonales, al salir de su zona de confort en el centro
de origen.
Por otra parte y más allá del beneficio profesional, durante la movilidad el estudiante toma
consciencia de otros usos y costumbres, de otros
ámbitos culturales y amplía su círculo relacional, lo que puede contribuir muy positivamen-
28
te a su capacidad de adaptación y crecimiento
personal. De hecho, la superación de los retos
encontrados durante la estancia de movilidad
puede también fomentar y afianzar la confianza
en sí mismo del candidato a Doctor.
Por último, no olvidemos que, más allá del
beneficio para los propios estudiantes de Doctorado, la movilidad puede resultar altamente
provechosa para los propios grupos de investigación e instituciones implicados, que pueden
beneficiarse mutuamente de su respectiva visión investigadora y educativa. De hecho, no
cabe duda de que la movilidad de los estudiantes de Doctorado se verá claramente favorecida si se fomentan acuerdos y alianzas formales
entre instituciones académicas investigadoras
de diferentes países, ya sea a nivel global de la
propia institución, ó de manera más particular entre los programas de Doctorado y grupos
de investigación implicados. En este sentido,
no sólo la Universidad como institución, sino
también otras agencias de investigación y financiación, adquieren un rol de responsabili-
dad en la facilitación de la movilidad y en la
supresión de barreras económicas y logísticas
que permitan a los doctorandos desarrollar
una experiencia internacional beneficiosa para
su desarrollo profesional y personal.
Conclusión y perspectivas
En resumen, la educación doctoral hoy en día
tiene como meta, junto con la realización de
la tesis doctoral, ayudar a que el futuro Doctor adquiera una serie de conocimientos y
habilidades, relacionadas o no con la investigación, que le proporcionen una preparación
más completa e integral de cara al mundo
profesional y que fomenten su empleabilidad
en un mercado cada vez más competitivo. Es
por tanto una obligación y un reto actual de
los Programas de Doctorado y de los agentes
responsables de la educación doctoral velar
por facilitar, en la medida de posible, la mejor
formación investigadora y transversal, para
que los egresados puedan constituirse en profesionales de referencia que contribuyan al
avance de sus respectivos sectores.
Referencias
The European Council of Doctoral Candidates
and Junior Researchers. “Identifying transferable skills and competences to enchance
early-career researchers employability and
competitiveness” (2018). http://eurodoc.net/
skills-report-2018.pfd (consultado el 20 de
marzo de 2019).
Siemers, O. “Skills for here or to take away?
Outcomes of academic mobility for expatriate
researchers”. (2016). Journal of International
Mobility: 4, 149-170.
Universities UK International focus group on
PhD mobility. “PhD student outward mobility:
perceived barriers and benefits”. (2016). https://
www.universitiesuk.ac.uk/policy-and-analysis/
reports/Documents/International/Phd-student-outward-mobility-December-2016.pdf
(consultado el 1 de junio de 2019).
29
30
Efectos del ozono médico sobre la Peroxidación
Lipídica y la actividad de la Aldehído Deshidrogenasa 2
cerebralen ratas alcohólicas en abstinencia
María Teresa Díaz-Soto1* (Dra. Farmacéuticas, Profesora Auxiliar), Ángela Fraga Pérez
(MC Toxicología), Jacqueline Dranguet Vaillant1 (MC Farmacología), Erik Jerez (Lic.
Cs. Farmacéuticas), María de los Angeles Bécquer (MC Farmacología), Mayté Casanova
(MC Toxicología), Maikel Arteaga Cruz (MC Farmacología), Olga Sonia León Fernández
(Dra. Ciencias Farmacológicas, Profesora Consultante),
Instituto de Farmacia y Alimentos, Universidad de la Habana .San Lázaro y L .Habana
10 400, Cuba
. Tel.: +53 54978238
E-mail: teresads@ifal.uh.cu
ietd@elacm.sld.cu (M .T Díaz).
RESUMEN:
El consumo crónico de etanol provoca aumento en la acumulación de acetaldehído y formación de Especies Reactivas de Oxígeno, lo que
provoca daño a nivel cerebral, esto se prolonga
durante el estado de abstinencia. Actualmente
el tratamiento utilizado para contrarrestar las
afectaciones provocadas por el alcoholismo y
que se manifiestan durante la abstinencia alcohólica es extremadamente limitado.
Objetivos: Este estudio se propuso determinar los efectos protectores del Post-condicionamiento Oxidativo con Ozono sobre la actividad de la enzima Aldehído Deshidrogenasa
2 cerebral y las concentraciones de malonildialdehído sistémica y cerebral después de 2
semanas de abstinencia alcohólica en ratas
Lewis machos
Método: La administración de etanol por
vía oral fue “ad libitum” .Las ratas se distribuyeron en cuatro grupos: (I) Control, recibió agua durante todo el experimento, (II)
Etanol (ET-OH), (III) Etanol + Ozono, como
el grupo II pero después de la abstinencia alcohólica fueron sometidas a postcondicionamiento oxidativo con ozono a concentración
de 20 μg/mL, dosis de 1 mg/kg, 15 tratamientos por l insuflación rectal y (IV) Etanol
+ Oxígeno, como el grupo III pero tratado
con oxígeno (26 mg/kg). . Se les fue introduciendo gradualmente el consumo de etanol
en soluciones de 10, 20, 30, y 40 % (56 días).
Sesenta ml de agua o solución de etanol fueron colocados en cada caja diariamente por
8 semanas. Cada 24 horas fueron medidos
los volúmenes de etanol o agua consumidos.
Fue evaluada la tolerancia farmacológica. Se
determinó las concentraciones en suero de
MDA antes y después de la aplicación de ozono Finalmente los cerebros fueron removidos
para estudiar el estado redox mediante Aldehído deshidrogenasa 2 mitocondrial y malonildialdehído.
Resultados: Fueron significativos los efectos del ozono sobre los niveles de MDA. La
aplicación de Ozono restableció los niveles
de MDA y la actividad dela ALDH2 mitocondrial, enzima que transforma el Acetaldehído
en acetato inactivo
Conclusiones: El tratamiento con Ozono
disminuyó el estrés oxidativo sistémico durante la abstinencia alcohólica a nivel de Sistema Nervioso Central. Estos resultados demuestran el papel que juega el Ozono como
protector del daño a este nivel en la abstinencia alcohólica. Estos resultados demostraron que el ozono ejerció un efecto protector
contra el daño oxidativo en el cerebro, preservando funciones importantes del Sistema
Nervioso Central(SNC)
Abstract: Ethanol withdrawal (EW) increases acetaldehyde accumulation and reactive
31
“El consumo crónico
de etanol puede
provocar daño
mitocondrial lo que
conlleva a disfunción
de la ALDH y por
tanto aumento de las
concentraciones de
acetaldehído al cual
se le ha adjudicado
un papel importante
en el daño cerebral
característico
del alcoholismo
y del Síndrome
de Abstinencia
Alcohólica”
32
oxygen species formation that promote damage to the brain. It is necessary to emphasize that while EtOH abuse and dependence are
widespread, treatment options are extremely
limited.
Purpose: This study aimed at investigating the protective effects of Ozone Oxidative Postconditioning (Ozone OxPost) on
mitochondrial aldehyde dehydrogenase 2
(ALDH2) and MDA concentrations induced
by oxidative stress after 2 weeks of EW in rats.
Method: Oral “ad libitum” administration
of ethanol was used. Rats were divided into 4
groups. The groups were (I) Control, received
tap water during the entire duration of the
experiment, (II) Ethanol (ET-OH), (III) Ethanol + Ozone, as group II, but at the beginning
of EW, rats were submitted to Ozone OxPost
at a concentration of 20 μg/mL, dose 1 mg/
kg, 15 treatments by rectal insufflation was
used and (IV) Ethanol + Oxygen, as group III
but rather than ozone, rats were treated with
oxygen (26 mg/kg).The rats were gradually
introduced to ethanol consumption through
ET-OH solutions from 10, 20, 30, and 40%
(56 days). The determination systemic concentrations of MDA was before and after the
application
of ozone. Afterwards, brains were promptly removed for oxidative stress studies.
(Mitochondrial aldehyde dehydrogenase 2
(ALDH2) and MDA concentrations)
Results: Of special note were ozone´s effects
on MDA levels. Ozone therapy re-established
MDA and mitochondrial aldehyde dehydrogenase 2 (ALDH2), the same enzyme that
transforms acetaldehyde into inactive acetate.
Conclusions: Ozone treatment improved
the systemic oxidative stress during EW. These results demonstrated that Ozone protected
the brain against oxidative injury, improving
important functions of the Central Nervous
Systems (CNS).
Keywords Ozone, Ethanol Withdrawal, Oxidative Stress, Central Nervous System
Introducción
El alcoholismo es una enfermedad crónica
caracterizada por la ingesta compulsiva y excesiva de bebidas alcohólicas a un nivel que
interfiere con la salud física y mental, al igual
que con las responsabilidades sociales, familiares, económicos o laborales. El alcoholismo
es un tipo de drogadicción, en la cual hay tanto dependencia física como mental (1, 2). Produce una fuerte dependencia al consumo de
alcohol etílico, manifestándose el Síndrome
de Abstinencia Alcohólica cuando no es posible su ingesta (3).
El alcoholismo es incompatible con la vida
(4), existen dos factores que afectan de forma
significativa la calidad de vida del paciente alcohólico: Por una parte, los efectos directos
del etanol sobre diferentes sistemas de neurotransmisión a nivel de Sistema Nervioso
Central (SNC) como son la transmisión GABAérgica, dopaminérgica y la glutamatérgica,
al provocar desequilibrio neuronal y con ello
alteraciones conductuales que se prolongan
durante el Síndrome de Abstinencia Alcohólica (SAA). Otro factor que afecta la calidad
de vida del paciente alcohólico está relacionado con el metabolismo del etanol, que ocurre
por vías enzimáticas y vías no enzimáticas.
Las enzimas que metabolizan al etanol son
la Citocromo P4502E1 que se localiza en los
microsomas, la Catalasa localizada en peroxisoma y la Alcohol Deshidrogenasa citosólica,
el metabolito formado es el acetaldehído que
es degradado por la enzima Aldehído Deshidrogenasa mitocondrial (ALDH2) (5). El
consumo crónico de etanol puede provocar
daño mitocondrial lo que conlleva a disfunción de la ALDH y por tanto aumento de las
concentraciones de acetaldehído al cual se le
ha adjudicado un papel importante en el daño
cerebral característico del alcoholismo y del
Síndrome de Abstinencia Alcohólica (6, 7, 8).
Por otra parte, las vías no enzimáticas por las
que se metaboliza el etanol provocan la formación de Especies Reactivas de Oxigeno favoreciéndose la Peroxidación Lipídica (POL)
generándose malonildialdehído (MDA) (cuyo
metabolismo también ocurre por la ALDH).
Se ha evidenciado la formación de aductos
entre el acetaldehído y el MDA. Ambos pueden provocar afectaciones en el Sistema Nervioso tanto Autónomo como Central (9)
Numerosos estudios han demostrado que la
aplicación del Ozono médico constituye una
conducta beneficiosa para muchas enfermedades degenerativas y afecciones orgánicas
(Diabetes Mellitus, Artritis Reumatoide, hernia discal, presencia de convulsiones). Trabajos experimentales y clínicos confirman sus
acciones terapéuticas. En estudios realizados
en modelos de hepatotoxicidad por CCl4 y en
modelos de isquemia/reperfusión hepática,
se han evidenciado los efectos pleiotrópicos
del ozono, el cual ha restablecido el balance
redox y preservado la integridad mitocondrial
lo que se relaciona con la protección de la actividad de la enzima Superóxido Dismutasa a
nivel hepático.
A partir de los objetivos de este trabajo se
realizó un estudio después de dos semanas
de abstinencia alcohólica sobre la influencia
del ozono sobre el metabolismo del etanol y
la ocurrencia de peroxidación lipídica a nivel
cerebral mediante la determinación de la actividad cerebral de la enzima Aldehído deshidrogenasa mitocondrial y las concentraciones
de MDA respectivamente.
“A los grupos que
fueron tratados
con ozono se le
administró por
insuflación rectal
1mg/kg, es decir, 5
cc durante 14 días
de abstinencia. Los
grupos que fueron
tratados con oxígeno
se le administraron
5 cc por insuflación
rectal durante
los 14 días de la
abstinencia”
33
Materiales y Métodos
Se utilizaron 20 ratas Lewis (250-300g) provenientes del Centro Nacional de Producción
de Animales de Laboratorio de (CENPALAB;
Mayabeque, Cuba). Las ratas fueron alimentadas con alimento proveniente del Centro
Nacional para la Producción de Animales de
Laboratorio (CENPALAB; Mayabeque, Cuba)
y agua/etanol “ad libitum” (45 g y 60 mL) en
cajas hogar bajo ciclos de luz y oscuridad de
12 horas a una temperatura de 20+2°C. Para
medir el peso y el consumo de alimentos se
empleó una balanza analítica (Denver Instrument XP-3000). Todos los procedimientos
con los animales se realizaron según lo establecido por la unidad de gestión de la calidad
(UGC) del Centro de Investigaciones y Ensayos Biológicos del Instituto de Farmacia y
Alimentos de la Universidad de La Habana en
concordancia con la regulación de la Unión
Europea para el tratamiento de animales de
experimentación (Bethesda, MD, USA)(10)
El estudio fue dividido en diferentes etapas
las cuales se muestran a continuación:
A- Etapa administración de etanol (Inducción del alcoholismo)
Etapa cuyo objetivo es inducir y estimular el
desarrollo de conductas reforzadoras a través del consumo exploratorio, recompensa y
adicción al etanol. Se desarrolla con una escalada de consumo in crescendo para generar
dependencia física al etanol (11).
B- Etapa Tolerancia Farmacológica
Etapa cuyo objetivo es la determinación de
conductas compulsivas al consumo del etanol
propias de los alcohólicos. La preferencia de
mayores concentraciones del etanol (entre
10% y 40% durante 2 semanas) permite establecer el grado de tolerabilidad y adaptación
en el SNC.
Determinación del consumo de
líquido
Se estableció dos grupos iniciales: Grupo
Control de 5 ratas y Grupo tratado con etanol
de 15 ratas. El Grupo tratado con alcohol se le
administró dosis ascendentes cada 14 días de
experimentación (5% día 1, 10% día 15, 20%
día 29, 30% día 43 y 40% día 57). Los animales tenían acceso solamente a la botella con
solución de etanol. A partir del día 70 hasta
el día 84 el grupo tratado con etanol se sometió a una etapa de Tolerancia Farmacológica
suministrándole “ad libitum” etanol a las dos
concentraciones (10% y 40%) para evaluar el
biomodelo de alcoholismo desarrollado. Se
realizó control del peso corporal, consumo de
comida y líquido durante las distintas etapas
de inducción y tolerancia al consumo crónico
del etanol (84 días) y al final de los 14 días
siguientes de Abstinencia Alcohólica.
Distribución de los grupos
experimentales
Al terminar el día 84 del experimento el grupo tratado con etanol fue dividido en 3 grupos
de 5 ratas formando un total de 4 grupos de
ratas con 5 ratas cada uno:
Grupo 1: AGUA
Grupo 2: ETANOL
Grupo 3: ETANOL+OXÍGENO
Grupo 4: ETANOL+OZONO
Aplicación de la Ozonoterapia y
Oxígeno.
El Ozono (O3/O2) se generó mediante el
OZOMED (equipo producido por el Centro
Nacional de Investigaciones Clínicas de Cuba,
CNIC) a partir del oxígeno clínico, el cual
constituyó alrededor del 3% de la mezcla O3/
O2 + O2. A los grupos que fueron tratados
con ozono se le administró por insuflación
rectal 1mg/kg, es decir, 5 cc durante 14 días
de abstinencia. Los grupos que fueron tratados con oxígeno se le administraron 5 cc por
insuflación rectal durante los 14 días de la
abstinencia.
C- Etapa de Abstinencia
Etapa cuyo objetivo es analizar los resultados
de la deprivación del consumo de etanol característicos de los alcohólicos en Abstinencia
Alcohólica. Al grupo tratado con etanol se le
retiró abruptamente el suministro de etanol
durante 2 semanas. El comportamiento de
los grupos experimentales en esta etapa demuestra la necesidad o no que presenten de
consumir etanol lo cual es evaluado mediante
Obtención del Suero
test conductuales.
Las ratas de estudio fueron sedadas en una
campana de cristal con éter dietílico. Después
de haber corroborado el efecto, se procedió
a la extracción de 5ml de sangre por el plexo
ocular. La sangre extraída se depositó en via-
34
les para centrifugar, debidamente rotulados y
se mantuvo en reposo a temperatura ambiente durante 10 min para la retracción del coágulo. Posteriormente el coágulo fue separado
cuidadosamente de las paredes del vial con
un capilar y se centrifugó a 3 600 rmin-1 durante 15 min para la obtención del suero. El
suero obtenido se congeló a -20˚C para luego
ser empleado en una serie de determinaciones bioquímicas. La extracción de sangre se
realizó en 3 etapas: al inicio (día 0), después
de consumir el 20% de alcohol (día 29) y al
finalizar la abstinencia (día 98) (12).
Preparación de Homogenado de
cerebro.
Los homogenados se obtuvieron en el día
99 del experimento empleando el homogenizador de cuchillas (Edmund Bühler HO4,
Alemania) para lo cual se utilizó 2 g de tejido
en 20 ml de buffer sucrosa-tris-HCl pH=7,4
0,2M según se describe en el PNO/TEC/0303.
Posteriormente se centrifugó 10 min, 3000
rpm a 4oC (Sigma Centrifuge 2K15, Reino
Unido). El sobrenadante se guardó para la obtención de las fracciones subcelulares y para
la determinación de los marcadores de estrés
oxidativo (13)
Determinación de la actividad de
ALDH
Fraccionamiento celular. Obtención
de mitocondria.
Para la obtención de la fracción mitocondrial
de células cerebrales en la medición de la actividad de ALDH, el homogenado se centrifugó
a 900 g por 15 minutos obteniéndose el pellet.
El pellet obtenido de este paso se hizo resuspender con 10 ml de buffer sucrosa-tris-HCl
pH=7,4 0,2M. Posteriormente se rehomogenizó a 340 rpm y se centrifugó a 9000g por
10 minutos. El sobrenadante obtenido se volvió a centrifugar a 104.000 g por 60 min y el
precipitado mitocondrial se lavó dos veces y
se re suspendió en 10 ml de la solución tampón, que se utilizó para la determinación de la
ALDH mitocondrial de cerebro. Se comprobó
la pureza de la fracción mitocondrial aislada
mediante la determinación de la enzima Alcohol Deshidrogenasa. La enzima alcohol deshidrogenasa fue utilizada, como enzima marcadora de la fracción microsomal. Menos del
5% de la actividad alcohol deshidrogenasa se
encontró en la fracción microsomal, indicando una contaminación mínima de enzimas
citosólicas (15).
Determinación de proteínas totales
Determinación de
Malonildialdehído (MDA)
El MDA se midió como indicador de la POL
utilizando el método colorimétrico que emplea el 1-metil-2-fenil indol como cromógeno
reportado por Esterbauer, H en 1990. La condensación de una molécula de MDA con dos
moléculas de 1-metil-2-fenil indol, bajo condiciones de acidez da como resultado la formación de un cromóforo con una absorbancia
máxima de 586 nm. Una solución de 7,6 mM
de 1-metil-2-fenil indol en metanol al 33% en
acetonitrilo se preparó inmediatamente antes
de ser utilizada. Una alícuota de 325 μL de
este reactivo se hizo reaccionar con 200 μL de
muestra se agitó y se añadió 75 μL de ácido
clorhídrico 37 %. Tras una nueva agitación de
microtubos de ensayo se incubaron 40 min a
45 ºC. Posteriormente, cuando alcanzaron la
temperatura ambiente, se centrifugó a 3000
rpm durante 15 min y se leyó la absorbancia a
586nm contra blanco reactivo. Se utilizó una
curva patrón de 1,1,3,3-tetraetoxipropano
para calcular las concentraciones finales (14).
La concentración de proteínas totales en el
homogenado de cerebro fue determinada por
el método descrito por Bradford (1976). Este
método se basa en la coloración de las proteínas presentes en el medio con el reactivo azul
de Coomassie (Sigma), detectable a 595 nm.
La cuantificación se llevó a cabo mediante
una curva de calibración con albúmina sérica
bovina (BSA) (Sigma) como patrón de referencia (16).
“Los resultados
de la etapa
de tolerancia
farmacológica en
la que se expone
al animal a dos
concentraciones
de etanol, una
baja 10% y otra
alta 40%, para
que consuma la
que prefiera se
observó que hubo
un incremento
significativo
(p < 0.05) en el
consumo de la
solución etílica
más concentrada
lo cual indica la
preferencia de las
ratas en estudio
por el etanol”.
Determinación de aldehído deshidrogenasa cerebral.
La actividad de la ALDH2 se midió por el aumento de la producción de NADH a 340 nm.
La mezcla de reacción contenía: 60 mM de
tampón de fosfato de sodio (pH 8,5), 1 mM de
NAD+ + EDTA 1 mM y las proteínas mitocondriales (0,5 mg / ensayo). La reacción se mantuvo durante 2 min a temperatura ambiente,
la reacción enzimática se inició mediante la
adición del sustrato (10 mM propionil aldehído). El cambio de absorbancia se controló
durante 30 segundos, 1, 2, 3 y 4 minutos para
calcular la tasa de producción de NADH. La
actividad específica de ALDH2 se calculó uti-
35
Figura 1
“La
implementación
de un modelo
de alcoholismo,
que mimetice la
sintomatología
del humano
alcohólico, es
un requisito
indispensable
para el estudio
de los efectos
terapéuticos
de fármacos
candidatos a ser
utilizados en el
tratamiento del
alcoholismo,
principalmente
en la etapa de
la “sufrida”
abstinencia”.
Figura 1. Esquema gráfico de habilidades y competencias transferibles
de interés para la mejor empleabilidad y competitividad de slos
estudiantes de Doctorado. Fuente:
“The European Council for Doctoral
Candidates and Junior Researchers
(EURODOC)”. http://eurodoc.net/
skills-report-2018.pdf
lizando el coeficiente de extinción molar de
la reducción de NAD+ de 6,22 × 106 cm2 a
340 nm (Merck Index) y 1 unidad representa
una reducción de NAD+ (+ 1 nmol/min/mg
de proteína) a temperatura ambiente (17,18).
Procesamiento estadístico
Los datos experimentales obtenidos fueron
sometidos a un análisis exploratorio donde se
detectaron puntos aberrantes (outiers). Además se estimaron parámetros descriptivos
como media y desviación estándar.
Para determinar diferencias entre grupos en
una variable en cuestión se utilizó un ANOVA
de clasificación simple, seguida de un ensayo
de comparación de medias (Student-Newman-Keuls) y el test de Student. El nivel de
significación estadístico empleado en todos
los casos fue como mínimo de p< 0.05. Para
las correlaciones lineales se empleó el coeficiente de correlación de Pearson. Los datos
fueron procesados utilizando en paquete estadístico: STATISTICA versión 6.0 para WINDOWS.
Resultados
La Figura 1 muestra el consumo de líquido en
los animales objeto de estudio durante las diferentes etapas del desarrollo del modelo, el
grupo control que consumió agua (Control) y
el grupo que ingirió etanol a diferentes concentraciones. A todas las concentraciones que
les fue suministrado el etanol los animales
en estudio consumieron la solución alcohólica, no existieron diferencias significativas
(p<0.05) en el volumen de solución etanólica
(10%-40%) consumido durante la inducción
del alcoholismo (56 días). Los resultados de
la etapa de tolerancia farmacológica en la que
se expone al animal a dos concentraciones de
etanol, una baja 10% y otra alta 40%, para que
consuma la que prefiera se observó que hubo
un incremento significativo (p < 0.05) en el
consumo de la solución etílica más concentrada lo cual indica la preferencia de las ratas en
estudio por el etanol. En la etapa de Abstinencia a las ratas alcoholizadas se les retiró el etanol y durante 2 semanas se registró la ingesta
de agua. Como puede apreciarse los animales
alcoholizados consumieron más agua (p <
0.05) que los controles indicando las afecta-
Tabla 1
Grupos
experimentales
Actividad de Aldehído Deshidrogenasa
mitocondrial en cerebro
(mM/mL/min de NADH)
Control (Agua) 6
.33 ± 0.021(a)
Etanol 1
.23 ± 0.050(b)
Etanol + Ozono 4
.85 ± 0.430(c)
Etanol + Oxígeno 0
.97 ± 0.031(b)
Tabla I: Actividad de la Aldehído Deshidrogenasa 2 en homogenado de cerebro de las ratas estudiadas.
36
ciones metabólicas, entre otras manifestaciones, asociadas a la avidez por el agua durante
la abstinencia.
La Figura 2 representa las concentraciones
de malonildialdehído (MDA) a nivel sistémico en los diferentes grupos objeto de estudio,
antes y después de la aplicación del Ozono. El
grupo que consumió etanol mostró elevadas
concentraciones de MDA respecto a los restantes grupos, mientras que el tratado con
ozono manifestó disminución de las concentraciones de MDA con respecto a la etapa previa a la administración de Ozono.
En la Tabla I se presenta la actividad de la
ALDH2 cerebral en los grupos estudiados,
manifestándose aumento de la actividad de la
enzima en los grupos ozonizados con respecto
a los no ozonizados que consumieron etanol.
La Figura 3 representa las concentraciones de
MDA a nivel cerebral, en el grupo que fue ozonizado existió un comportamiento en las concentraciones del producto de la peroxidación
lipídica similar al grupo control mientras que
en los restantes grupos las concentraciones de
MDA fueron significativamente mayores
Discusión
El alcoholismo es una de las toxicomanías
que se manifiesta con más frecuencia a nivel
mundial. El paciente alcohólico pasa por diferentes etapas desde la adaptación al consumo del alcohol hasta llegar al llamado punto
de “no retorno” (19). La implementación de
un modelo de alcoholismo, que mimetice la
sintomatología del humano alcohólico, es un
requisito indispensable para el estudio de los
efectos terapéuticos de fármacos candidatos a
ser utilizados en el tratamiento del alcoholismo, principalmente en la etapa de la “sufrida”
abstinencia. El modelo seleccionado en nuestro trabajo tuvo en cuenta su acercamiento
a las prácticas del alcohólico de ahí que se
empleó el consumo de alcohol “ad libitum”
ya que es el más semejante al alcoholismo en
humanos (20). El consumo de etanol a las diferentes concentraciones (Figura 1) demostró
la tendencia de los grupos experimentales a
adquirir la conducta adictiva, lo cual se corroboró con los resultados de la Prueba de
Tolerancia Farmacológica, al manifestarse la
preferencia por la solución etanólica de mayor concentración. Estos resultados se corresponden con lo planteado por otros autores al
afirmar que “El consumo crónico genera un
progresivo requerimiento de dosis mayores
de sustancia para conseguir un efecto similar.
La tolerancia puede considerarse un intento
del organismo para retornar a la homeostasis,
esto es, a las condiciones anteriores al consumo” (21, 22). Este modelo experimental permite no solo profundizar en los mecanismos
de acción terapéutica, del agente en cuestión,
sino que hace posible ampliar los conocimientos relacionados con la etiopatogenia alcohólica. Por otra parte, los hallazgos científicos
que fundamentan los efectos terapéuticos del
ozono, por un mecanismo denominado Pre/
Post condicionamiento oxidativo, constituye-
Figura 2
Figura 2. Concentración de MDA séricos en los grupos experimentales estudiados. Antes del PostOx
Ozono corresponde al final de la etapa de Tolerancia, antes de la abstinencia y antes del PostOx. Después del PostOx Ozono representa el final del tratamiento con ozono, el final de la Abstinencia y el punto final del estudio experimental. Se representan la media ± EEM. Letras diferentes indican diferencias
significativas (* p<0.05), para cada grupo experimental, antes y después del PostOx Ozono
37
Figura 3
Figura 3. Concentraciones de MDA, en homogenado de cerebro, en las diferentes condiciones experimentales. Las concentraciones de MDA en cerebro de rata, corresponde con el final de la Abstinencia,
del PostOx y del experimento (105 días) Se representan la Media ± EEM. Letras diferentes indican
diferencias significativas (* p<0.05).
ron la base científica que justifica abordar el
estudio de los efectos del ozono, sobre la sobreproducción de ERO y las alteraciones de
algunas de las funciones básicas del SNC que
se modifican o alteran en el sujeto alcohólico.
Los cambios fisiopatológicos en la función celular, inducidos por la exposición crónica de
alcohol, se derivan del propio metabolismo
del etanol que incluye la generación de acetaldehído, radicales libres (como el hidroxietil)
y NADH, entre otros causando Estrés Oxidativo. Es así como mucha de la sintomatología asociada a las disfunciones orgánicas y
conductuales del alcohólico son el resultado
del daño que promueven estas moléculas oxidadas, sobre el hígado y el Sistema Nervioso
Central, tanto durante la tolerancia como en
la abstinencia. (23)
El acetaldehído, derivado del metabolismo
del etanol y de reconocida toxicidad, es transformado a ácido acético por la enzima Aldehído Deshidrogenasa mitocondrial, isoforma 2
(ALDH2). Esta enzima metaboliza eficientemente el acetaldehído, producido durante el
metabolismo del etanol, pero también metaboliza otros aldehídos lipídicos tóxicos como
el MDA y 4-hidroxi-2-nonenal, entre otros
(24). El consumo crónico de etanol induce
lesiones en los complejos I y III de la cadena
de transporte electrónico mitocondrial, con
afectaciones en la transferencia de electrones
desde el flavín-mononucleótido del complejo
I al complejo III, se produce acumulación del
complejo flavín-mononucleótido-semiquinona y una generación incrementada de radicales superóxido dentro del complejo I. Estas
38
lesiones, inducidas por el consumo crónico
de etanol incrementan la producción de superóxido y peróxido de hidrógeno dentro de
la mitocondria afectando a la integridad mitocondrial y con ello la función de la enzima
ALDH2 (25).
El consumo de etanol es un detonante muy
importante para la generación de estrés oxidativo- nitrosativo en el hepatocito y ya sea a
corto o a largo plazo, puede desencadenar la
muerte de la célula, el tejido y el organismo
en general. Algunos factores que están involucrados en la generación de estrés oxidativo-nitrosativo por etanol son: cambios en el
estado redox intracelular como resultado del
metabolismo oxidativo del etanol, hipoxia celular, porque las alteraciones en el metabolismo redox afectan el consumo de oxígeno y la
producción de ATP en la mitocondria.
El metabolismo del etanol y el estrés oxidativo asociado, producen una serie de compuestos de alta reactividad, capaces de unirse
a un gran número de residuos proteicos. El
resultado de estas uniones es la generación de
nuevas entidades bioquímicas que, de forma
genérica, reciben el nombre de aductos.
Estos aductos, según su composición, pueden
clasificarse en cuatro grandes grupos: aductos
derivados del acetaldehído, que provienen de
la formación de enlaces entre el acetaldehído
y residuos lisina o grupos amino de distintas
proteínas. Estos enlaces pueden ser covalentes o no covalentes, lo que determina la estabilidad del aducto formado, algunos productos aldehídicos derivados de la peroxidación
lipídica, como el MDA o el 4-hidroxinonenal,
también son capaces de formar aductos con
proteínas, también se ha documentado la formación de aductos mixtos o híbridos, que son
aquellos formados por la unión de acetaldehído y MDA a un mismo sustrato, finalmente,
en presencia de átomos de hierro, los radicales hidroxietilos formados en la oxidación del
etanol, pueden formar aductos con proteínas.
La unión de grupos aldehído a una biomolécula resulta en una alteración estructural y
consecuentemente, funcional de dichos sustratos. Así, se ha descrito que tras su unión
con moléculas de acetaldehído (formación
de aductos), la actividad de algunas enzimas
se reduce o incluso se suprime. El metabolismo del etanol y la consecuente formación
de aductos en el SNC, están implicados en los
efectos tóxicos del etanol en otros órganos y
tejidos y en la generación de alteraciones en
el cerebro.
Los aductos de acetaldehído pueden formar
complejos con serotonina llamados tetrahidrocarbolinas, ambos complejos aumentan la
preferencia por etanol y pueden inhibir competitivamente la MAO y la COMT.
En el presente estudio existió un incremento
en las concentraciones de MDA en los grupos
no tratados con ozono lo cual indica peroxidación lipídica. Este aldehído tiene una especial
importancia en el alcoholismo y en la abstinencia alcohólica ya que el MDA es un sustrato de la Aldehído Deshidrogenasa (ALDH2).
Esta enzima es responsable del metabolismo
del acetaldehído (inductor del daño cerebral
por consumo crónico de etanol) y otros aldehídos lipídicos producidos por el metabolismo del etanol. Por lo tanto el incremento del
MDA sugiere la inhibición de ALDH2, la cual
puede ser consecuencia de las especies reactivas de oxígeno y nitrógeno (26). La administración de ozono preservó la actividad de la
enzima ALDH2 cerebral y disminuyó las concentraciones de MDA, séricas y cerebrales.
Estos resultados indican el efecto protector
del ozono sobre el daño que provocó el etanol y por tanto la capacidad de estimular la
recuperación funcional de una de las enzimas
que de forma más significativa participa en su
metabolismo. Al tener en cuenta que la propia ALDH2 participa en el metabolismo del
MDA, la disminución de las concentraciones
del mismo indican que también el ozono fue
capaz de disminuir el proceso de POL. Estos
resultados se corresponden con estudios anteriores (27) donde se evidencia, tanto preclínica como clínicamente, el efecto antioxidante del ozono y sus consecuencias favorables
sobre trastornos conductuales durante la abstinencia alcohólica.
El mecanismo por el cual el ozono médico
provoca efectos antioxidantes se resumen en
dos moléculas intermediarias: Peróxido de
Hidrógeno y los productos de POL (MDA).
El O3/O2 induce la formación de H2O2 e
hidroperóxidos, en cantidades pequeñas, los
cuales son capaces de actuar sobre los eritrocitos, plaquetas, monocitos y células endoteliales estimulando la acción de la Glutatión
Peroxidasa (GSH-Px) para formar H2O. Esta
actividad prooxidante estimula no sólo la
GSH-Px sino también la Glutatión Reductasa
(GSH-Rx) y aumenta los niveles de Glucosa
6-Fosfato (G-6P). Como consecuencia se produce un aumento del intercambio iónico, de
los niveles de 2,3 difosfoglicerato (2,3-DPG),
de los niveles de ATP y, por ende, de la glicólisis. El O3/O2 también estimula un aumento
de la relación NAD+/NADH activando la vía
del monofosfato de hexosa dependiente induciendo la liberación de ATP de los eritrocitos
y vasodilatación (28).
Se ha demostrado que el ozono es capaz de
proteger a las células hepáticas frente al daño
producido por los radicales libres del oxígeno;
así como la preservación de las concentraciones de glucógeno hepático, evitando la sobreproducción de lactato, que en el homogenado
de hígado está asociado al daño hepatotóxico
(29). El restablecimiento en las concentraciones de MDA, sugiere la preservación de la
enzima ALDH2, evitando la acumulación de
acetaldehído, a nivel sistémico y del SNC, con
la consiguiente disminución en la formación
de aductos.
La disminución de las concentraciones de
MDA y el aumento de la actividad de la enzima ALDH2 sugieren que el tratamiento con
ozono protegió la integridad mitocondrial
manifestada con la disminución de la peroxidación lipídica lo cual puede ser con secuencia del incremento en el metabolismo tanto
del acetaldehído como del malonildialdehido
sustratos de la enzima y relacionados con el
daño cerebral presente en la Abstinencia Alcohólica.
La aplicación de nuevos procedimientos experimentales, así como la identificación de
posibles biomarcadores que sean confiables y
sensibles, es un gran avance que puede contribuir a implantar un adecuado sistema de
prevención y diagnóstico para detectar, en lo
posible, el daño ocasionado por la ingestión
de etanol.
“Estos resultados se
corresponden con
estudios anteriores
(27) donde se
evidencia, tanto
preclínica como
clínicamente, el
efecto antioxidante
del ozono y sus
consecuencias
favorables
sobre trastornos
conductuales
durante la
abstinencia
alcohólica”.
Agradecimientos:
Los autores queremos expresar nuestra gratitud al Instituto de Farmacia y Alimento de la
Universidad de la Habana por todo el soporte
profesional en el desarrollo de este estudio
39
REFERENCIAS BIBLIOGRÁFICAS
1-Assanangkornchai S, Srisurapanont M. The
treatment of alcohol dependence. Curr Opin
Psychiatry.; 2007,20:222-227.
2-Gonzáles R. Clínica psiquiátrica básica actual.
Ciencias Médicas 1998,7:74.
3-Janicak P., Piszczor J., Scott M. Alcohol related
disorders. Psychiatry diagnosis and therapy. 2 Ed.
Edited by Joseph Flaherty. 2000 193: 247 – 265.
4- Adan A. Bases biológicas del alcoholismo. Psicología Conductual. 2015 Vol. 6, Nº 1
5- Kumar S, Sandell LL, Trainor PA, Koentgen
F, Duester G. Alcohol and Aldehyde Dehydrogenases: Retinoid Metabolic Effects in Mouse
Knockout Models. . Biochim Biophys Acta. 2012
January; 1821(1): 198–205.
6-. Ambrosio, E. y Fernández, E. Fundamentos
Neurobiológicos de las Adicciones. Neurociencia
y Adicción.2011. 19-46.
7-Koob GF .Theoretical Frameworks and Mechanistic Aspects of Alcohol
Addiction: Alcohol Addiction as a Reward Deficit Disorder .Curr Top Behav Neurosci. 2013,
13: 3–30. 109
8-Laniewska-Dunaj M, Jelski W, Orywal K, Kochanowicz J, Rutkowski R, Szmitkowski M. The
Activity of Class I, II, III and IV of Alcohol Dehydrogenase (ADH) Isoenzymes and Aldehyde
Dehydrogenase (ALDH) in Brain Cancer. Neurochem Res 2013, 38:1517–1521.
9- Forn Frías C., Sanchis Segura C. Posible papel del acetaldehído en el daño cerebral derivado del consumo crónico de alcohol. Rev Neurol
2003; 37: 485-93
10- Samson, H.H., and Hodge, C.W. Neurobehavior al regulation of ethanol intake. In: Deitrich,
R.A., and Erwin, V.G., eds. Pharmacological
Effects of Ethanol on the Nervous System. Boca
Raton, FL: CRC Press, 1996. pp. 203–226
11- Smolen, A., M.J. Marks, T.N. Smolen, and
A.C. Collins. “Dose and Route of Administration Alter the Relative Elimination of Ethanol by
Long-Sleep and Short-Sleep Mice.” Alcohol Clin.
Exp. Res. 1986, 10: 198–204.
12- Pukka K, Hietala J, Koivisto H, Anttila P, Bloigu R, Niemelä O. Obesity and the clinical use of
serum GGT activity as a marker of heavy drinking.
Scand J Clin Lab Invest. 2007; 67 (5):480-8.)
13- Hiroshi Nakamura, Robert L.J. Jilka y Ricardo Boland Mecanism of ATP hydrolisis by Sarcoplasmic Reticulum and the role of phospholipids. Nota: Técnica modificada para el uso del
Homogenizador, Torres y col, 1992.
14- Esterbauer, H., Cheeseman, K.H. Determination of aldehydic lipid peroxidation product:
malonaldehyde and 4-hydroxynonenal. Methods Enzymol. 1990; 186: 407–421.
15- Sedlak J et al. ALDH rapid and sensitive method for the quantitation of microgram quantities
of protein utilising the principle of protein-dye
binding. Anal Biochem 1976; 72: 248–54
16- Sedlak J, Lidsay RH. Estimation of total protein bound and non-sulfhydryl groups in tissue
40
with Ellman’s reagent. Anal Biochem 1969; 25:
192-205
17-Mirandola S.R., Melo, D.R., Saito, ^A., Castilho, R.F. 3-nitropropionic acidinduced mitochondrial permeability transition: comparative study
of mitochondria from different tissues and brain
regions. J. Neurosci. Res. 2010; 88, 630e639
18- Nicholls, D.G., Johnson-Cadwell, L., Vesce,
S., Jekabsons, M., Yadava, N. Bioenergetics of
mitochondria in cultured neurons and their role
in glutamate excitotoxicity. J. Neurosci. , 2007,
Res. 85, 3206e3212
19- Roerecke M, Rehm J. Alcohol intake revisited: risks and benefits. Curr Atherosclerosis
Rep. 2012; 14: 556-562 42
20- Lüscher C, Malenka RC. Drug-evoked synaptic plasticity in addiction: from molecular
changes tocircuit remodeling. Neuron. 2011;
69(4):650-63.
21-Rickert WS, Robinson JC, Young JC. Estimating
the hazards of “less hazardous” cigarettes. i. tar, nicotine, carbon monoxide, acrolein, hydrogen cyanide, and total aldehyde deliveries of Canadian cigarettes. J Toxicol Environ Health. 1980; 6:351–365.
22-National Institute on Drug abuses. Las drogas, el cerebro y el comportamiento. NIDA La
ciencia de la adicción, Departamento de Salud y
Servicios Humanos de los Estados Unidos. Publicación NIH. 2014 No. 08-5605.
23- Al-Dalain, S.M., G. Martínez, E.C. Jalil, and
O.S. León Fernández..“Ozone Treatment Reduces Markers of Oxidative and Endothelial Damage in an Experimental Diabetes Model in Rats.”
Pharmacol. Res.2001, 44: 391–396.
24-.Rokitanski O. Klinik und Biochemie der
Ozotherapie. Hospitalis 1982; 52:643-7.
25- Medici V, Halsted CH. Folate, alcohol, and liver disease. MolNutr Food Res. 2013; 57: 596-606
26- Díaz-Soto M. T., Fraga Pérez A, Dranguet
Vaillant J, A. Mallok, Renate Viebahn-Hänsler, Menéndez Cepero S, and León Fernández
O S. Ozone Oxidative Postconditioning Protects
Against the Injury Associated with Alcohol Withdrawal Syndrome in Rats. Ozone: Science &
Engineering, 2012; 34: 425–431.
27- M D. Escarabajal Arrieta. ¿Alcoholismo o
acetaldehismo? El papel de la catalasa y la aldehido deshidrogenasa cerebrales. Área de Psicobiología. Universidad de Jaén. 2016:32-39
28- Díaz-Soto M. T., Fraga Pérez A, Dranguet
Vaillant J, A. Mallok, Renate Viebahn-Hänsler,
Menéndez Cepero S, and León Fernández O S.
Ozone Therapy Ameliorates Nervous System
Disorders and Oxidative Stress in Patients During Ethanol Withdrawal—A Pilot Study Ozone:
Science & Engineering, 2012; 34: 432–437
29- Mallok A, Dranguet Vaillant J, Díaz Soto
M T, Viebahn-Ha¨nsler R, Bécquer Viart M A,
Fraga Pérez A, Donoso Cedeño R I, León Fernández O S. Ozone protective effects against
PTZ-induced generalized seizures are mediated
by reestablishment of cellular redox balance and
A1 adenosine receptors Neurological Research,
2015 Vol. 37 NO. 3
41
42
Función fisiológica y fisiopatológica de las membranas
asociadas a mitocondrias (MAMs): Posible rol en el
procesamiento de APP y en la disfunción mitocondrial en la
enfermedad de Alzheimer.
Panes-Fernandez J 1, Flores-Nuñez O 1, Fuentealba J1*
1 Laboratory of Screening of Neuroactive Compound, Physiology Department, Faculty of Biological Sciences, Universidad de
Concepción, Concepción, Chile
Resumen
La Enfermedad de Alzheimer (EA) es la principal forma de demencia que afecta a los adultos
mayores. Se caracteriza por una muerte neuronal progresiva, especialmente en hipocampo y corteza, y uno de los principales eventos
celulares con los que cursa esta patología es la
disfunción mitocondrial. Se postula que esta
enfermedad neurodegenerativa se produce
principalmente por el aumento en los niveles del péptido beta-amiloide (Aβ), que se
genera a partir del procesamiento proteolítico de la proteína precursora amiloide (APP).
Se ha descrito que tanto APP como la maquinaría enzimática de la vía amiloidogénica
y no amiloidogénica presentaría diferente
localización espacial; mientras que los componentes de la via no amiloidogénica se encontrarían en la membrana plasmática y
compartimentos secretores tardíos, los compartimentos endosómales, Golgi y estructuras
tipo balsas lipídicas denominadas MAMs (del
inglés Mitochondria-Associated RE Membranes) serían los encargados de procesar
APP por la vía amiloidogénica, y producir Aβ.
Las MAMs son dominios especializados entre
Retículo endoplásmico (RE) y mitocondrial,
donde existe una alta aposición entre ambos
organelos de entre 10-20 nm, que les permite comunicarse entre sí tanto física como
bioquímicamente, lo que facilita las funciones de esta región tales como la homeostasis del calcio, colesterol, fosfolípidos, y el
metabolismo mitocondrial. Existe evidencia
reciente que ha encontrado que las funciones localizadas en las MAMs estarían aumentadas significativamente en los modelos
animales y muestras de pacientes con la EA.
En esta revisión bibliográfica estudiaremos
los principales roles fisiológicos y fisiopatológicos de las MAMs, y su impacto en la funcionalidad mitocondrial y en el procesamiento
de APP.
Introducción
La Enfermedad de Alzheimer (EA) es una
enfermedad neurodegenerativa, caracterizada por la pérdida progresiva de la memoria y
aprendizaje (Castellani et al., 2010). Si bien se
desconoce su causa, histológicamente se han
identificado dos marcadores; las placas seniles y los ovillos neurofibrilares (Ballard et al.,
2011). El péptido Aβ se postula como principal agente responsable de la EA (Hardy and
Higgins, 1992), por su capacidad de generar
dishomeostasis del Ca+2, ATP, y lípidos (Blass
et al., 2000; Lal et al., 2007)electrophysiological, and neuroimaging examinations. Therefore, impairment in energy metabolism in
AD can not be attributed to loss of brain substance or to electrophysiological abnormalities. Among the characteristic abnormalities
in the AD brain are deficiencies in several
enzyme complexes which participate in the
mitochondrial oxidation of substrates to yield
energy. There include the pyruvate dehydrogenase complex (PDHC. En este contexto, la
mitocondria es un factor clave, ya que funciona como tampón de Ca+2, productor de ATP, y
participa en el metabolismo de los lípidos, por
ende, se ha asociado la disfunción mitocondrial como evento temprano de la pérdida de
la conexión de la red neuronal, falla sináptica,
y finalmente de la muerte neuronal (Onyango
et al., 2016).
La producción del péptido Aβ comienza por
el procesamiento proteolítico de la proteína
precursora amiloide (APP), proteína integral de membrana que se puede procesar por
dos vías. La vía no amiloidogénica involucra
la participación de α-secretasa, una enzima
que pertenece a la familia de metaloproteasas
ADAM (del inglés A Disintegrin and Metalloprotease), y γ-secretasa, complejo compuesto
por 4 proteínas: Presenilina 1 o 2 (PS1 o PS2),
Nicastrina (Nct), Aph-1 (del iglés Anterior
Pharinx-Defective-1) y Pen-2 ( del inglés presenilin enhancer 2); de estas proteínas, PS1
y PS2 tienen la actividad catalítica y el resto
tiene funciones regulatorias. Estas enzimas
forman los productos: APPsα (APP soluble
α), C83(APP-C83), AICD(Dominio intracelular carboxi terminal de la proteína APP) , y
p3. Por otra parte, en la vía amiloidogénica,
actúan β y γ-secretasa, que forman APPsβ,
C99 (APP-C83), AICD y el péptido Aβ (Muller
et al., 2017; Suh and Checler, 2002)the cause
and progression of both familial and sporadic
AD have not been fully elucidated. The autosomal-dominant inherited forms of early-onset Alzheimer’s disease are caused by muta-
43
tions in the genes encoding APP, presenilin-1
(chromosome 14.
Se ha descrito que tanto APP como la maquinaría enzimática de ambas vías presentarían
diferente distribución subcelular; la vía no
amiloidogénica se produciría principalmente
en la membrana plasmática y compartimentos
secretorios tardíos, mientras que la vía amiloidogénica se produciría principalmente en
compartimentos endosomales, balsas lipídicas
(Zhang and Song, 2013) y estructuras denominadas MAMs (Membranas de Retículo Endoplásmico asociadas a mitocondrias) (Area-Gomez et al., 2012). Por lo tanto, sería en estos
últimos compartimentos intracelulares donde
se produciría el péptido Aβ, y no en la superficie de la membrana plasmática, como se ha
sugerido clásicamente.
Las MAMs son dominios especializados entre
retículo endoplásmico (RE) y mitocondrial,
de una naturaleza semejante a las balsas lipídicas ya que son microdominios ricos en
esfingomielina y colesterol resistentes a los
detergentes (Aria Gómez et al., 2012), donde
existe una asociación estrecha entre ambos
organelos (10-20 nm), que les permite comunicarse entre sí física y bioquímicamente,
facilitando las funciones de esta región tales
como metabolismo del Ca+2, lípidos, y regular funciones mitocondriales (Bernard-Marissal et al., 2018). Existe evidencia reciente
de que las funciones asociadas a las MAMs
aumentan significativamente en los modelos
animales de la EA y en las células de pacientes con EA (Area-Gomez et al., 2012). Es por
ello por lo que a continuación describiremos
los principales roles que cumplen los MAMs
y sus implicancias en la EA cuando estos se
incrementan de forma patológica.
Rol de los MAMs en la transferencia
de Ca+2
meses). Además, en tejido cerebral de pacientes con EA (etapa Braak V-VI) los niveles de
VDAC1 aumentaron significativamente (55%)
comparados con individuos control (Cuadrado-Tejedor et al., 2011).
En relación con el IP3R, existen trabajos que
muestran resultados distintos dependiendo
de los modelos de estudio. En células CHO7PA2 que expresan APPV717F, se encontró un
incremento en los niveles del IP3R en comparación a células control, mismo resultado
que se observó en modelos celulares de sobreexpresión de APP swedish (APPswe); sin
embargo, en muestras de corteza de ratones
transgénicos Tg2576 la expresión de IP3R no
se vio afectada significativamente (Oules et
al., 2012).
Por otra parte, en ratones P301L tau, que fueron inyectados en la amígdala con el péptido
Aβ1-42, no se observaron cambios significativos en muestras de corteza para la proteína
GRP75 en comparación a muestras de corteza
control. Esto también fue observado por análisis de western blot de la corteza temporal
de individuos con EA, donde no se observaron cambios significativos en los niveles de
GRP75 en comparación con individuos control (David et al., 2006).
Además, Dyzma describió una evidencia funcional de que el incremento en las asociaciones MAMs estaría relacionada con una mayor
transferencia de Ca+2 desde el retículo hacia la
mitocondria mediado por la vía IP3R-VDAC1
en un modelo de la EA. En células SHSY5Y al
co-incubar con una agonista de la chaperona
del IP3R Sigma R1 (Receptor Receptor intracelular no-opioide sigma 1), localizada en la
interfaz RE-mitocondria, y el péptido Aβ, se
producía una potenciación del flujo de Ca+2
desde el RE hacia la mitocondria (Dyzma et
al., 2012).
Rol de los MAMs en mitofagia
Los MAMs tiene un rol en la transferencia de
Ca+2 desde RE a la mitocondria, donde participarían los proteínas IP3R (receptor de IP3),
Sigma R1 (Receptor intracelular no opioide
sigma 1) a nivel de RE, y VDAC1(canal selectivo de aniones dependiente de voltaje 1) a nivel
de la membrana mitocondrial externa (MME).
Esta comunicación inter-organelar coordina
tanto el metabolismo de Ca+2 como preservación de la integridad de la MME (Vance, 2014).
Los estudios de proteómica para evaluar los
niveles las proteínas del complejo IP3RVDAC1, mostraron un aumento dependiente
de la edad de los niveles de proteína VDAC1
en el hipocampo de ratones Tg2576, un modelo de la EA (Hsiao et al., 1996), comparadas
con ratones control de la misma edad (6-12
44
El proceso de mitofagia es un proceso degradativo que regula el control de calidad mitocondrial, en el cual las mitocondrias depolarizadas asociadas un fenotipo hiperfisionado
activan su reciclaje selectivo, y por lo tanto la
célula es capaz de adaptar la cantidad y calidad de las mitocondrias dependiendo de sus
necesidades energéticas. Este proceso estaría
mediado por receptores Atg8 y Atg32 (Proteína relacionada con la autofagia 8 y 32), que
reclutan la maquinaria autofágica en la mitocondria; evento que promueve su envoltura
por un fagóforo que posteriormente se fusiona con un lisosoma (Bockler and Westermann, 2014).
De acuerdo al trabajo de Gelmetti (Gelmetti
et al., 2017), se logró describir que la formación de autofagosomas (mitofagosomas) ocurriría en las MAMs; y que este proceso estaba
mediado por la relocalización de las proteínas
mitofágicas PINK1, BECN1 en las MAMs. En
otro trabajo, Böckler en un modelo de levaduras realizó mutantes de proteínas MAMs
Mmm1, Mdm10, Mdm34, Mdm12 encargadas de estabilizar el acoplamiento RE-mitocondria, observado que estas mutantes eran
defectuosas específicamente para el proceso
mitofágico. Además, encontró una asociación
entre atg8 y Mmm1, resultado que indicaría
que la asociación MAM es necesaria para iniciar el proceso de la mitofagia de forma selectiva (Bockler and Westermann, 2014).
Rol de los MAMs en transporte
mitocondrial
La comunicación entre el cuerpo celular y
los terminales sinápticos en las neuronas se
basan en el transporte axonal, el cual es altamente eficiente para las mitocondrias, ya que
deben alcanzar sitios especializados con alta
energía de en un breve tiempo. Se ha descrito
una correlación positiva entre el transporte
de mitocondrias axonal con proteínas MAMs,
en donde sería necesaria la interacción de
Mfn2 (mitofusina 2), MIRO1/2 (Rho GTPase 1 y 2 mitocondrial) y las proteínas motoras quinesinas y dineinas para promover el
transporte de las mitocondrias retrogrado y
anterógrado, entre el soma neuronal y el axón
(Bernard-Marissal et al., 2018).
Sin embargo, existe escasa información del
papel de las MAMs en el compartimento axonal, y si la disfunción de MAM puede afectar
específicamente el transporte axonal, ya que
la mayoría de los estudios están hechos en líneas celulares. En modelos de esclerosis lateral amiotrófica (ELA) y de neuropatías hereditarias motoras y sensoriales Villegas en un
explante de nervio usando mutantes de proteínas MAMs SigmaR1 y Mfn2, logró visualizar que había una degradación axonal asociada a una falla en el transporte mitocondrial,
lo que estaría sugiriendo que este transporte
es dependiente de la formación de las MAMs
(Villegas et al., 2014).
En motoneuronas, al suprimir la expresión de
la proteína SigmaR1 disminuyó el transporte anterógrado mitocondrial, conduciendo
a una acumulación mitocondrial en la zona
distal del axón, interesantemente esta acumulación también condujo a un cambio en la
morfología mitocondrial (Bernard-Marissal
et al., 2018).
Rol de los MAMs en dinámica
mitocondrial
La dinámica mitocondrial es el proceso que
regula la fisión y fusión de mitocondrias, dependiendo de los requerimientos energéticos
de la célula, suceso donde participan pro-
45
teínas para fisión como DRP1 y Fis1, y para
fusión, como Mfn1,2 y OPA1. Se ha descrito
que estos complejos MAMs se encontrarían
directamente asociados con un rol directo en
la dinámica mitocondrial, ya que se ha encontrado que tanto DRP1 como mitofusina 1 y 2
se encontrarían reclutados en estos complejos, sin embargo, se le han adjudicado a los
MAMs un rol en la desarrollo un fenotipo más
fisionado. De hecho, cuando hay una disrupción de los complejos MAMs por la deleción
de Sigmar1, se observó una alteración en la
dinámica mitocondrial, que condujo a que las
mitocondrias presentasen un fenotipo mitocondrial más fusionado (Bernard-Marissal et
al., 2018).
Otra proteína altamente estudiada que coordina la fusión mitocondrial es Mfn2, proteína que forma homodiméros entre RE y mitocondria (ya que se encuentra expresada en
ambos organelos), o heterodímeros con Mfn1
(se encuentra sólo en mitocondria). En efecto,
en células MEFs ( del inglés Mouse Embryonic Fibroblasts) de un ratón MFN2-KO, hubo
una menor obtención de MAMs por fraccionamiento subcelular, lo que estaba directamente correlacionado con la evidencia funcional
que mostró que había una menor liberación
de Ca+2 del RE a la mitocodnria, comparados
con MEFs WT (Filadi et al., 2018). Sin embargo, en el año 2012 el grupo de Orci (Cosson
et al., 2012), en el mismo modelo, se observó
un efecto opuesto; por microscopía de transmisión electrónica e inmunofluorescenca en
células MEFs del ratón MFN2-KO hubo un
incremento en la yuxtaposición de los MAMs
(~4.9%), comparadas con MEFs WT (~2,2%),
y desde un punto de vista funcional hubo una
directa correlación con el aumento de MAMs,
ya que había una mayor liberación de Ca+2 del
RE a la mitocondria en MEFs MFN2-KO que
en MEFs WT (Filadi et al., 2018). Interesantemente, estos efectos estuvieron asociados
a una disminución de la expresión del MCU
(uniportador de Ca2+ mitocondrial), el cual
media el importe de Ca+2 por la membrana mitocondrial interna. Por lo tanto, y a pesar de
que aún es controversial el rol de Mfn2 en los
MAMs, si podemos establecer que participa
activamente como espaciador de la conformación de las asociaciones RE-mitocondria, aunque serán necesarias más investigaciones para
abordar este problema.
Rol de los MAMs en la EA
En fibroblastos de pacientes humanos el grupo de Aria Gómez describió que existían distintas conformaciones entre retículo y mitocondria en fibroblastos de pacientes control,
46
y observó que los MAMs principalmente presentaban áreas de superficie de contacto que
alcanzaban asociaciones cortas (menores a
los 50nm) (superiores al 50%), en tanto que
áreas de contacto larga (entre 50 a 200 nm)
se encontraban en torno al 30%, y las áreas de
contacto muy largas (mayores a los 200nm)
sólo alcanzaban entorno al 4%. En contraparte, se observó que en fibroblastos de pacientes con FAD y SAD, tenían áreas de contacto
principalmente largas y muy largas, alcanzando porcentajes del 40% y del 35%, respectivamente (Area-Gomez et al., 2012).
Para lograr comprender por qué estas conformaciones altamente especializadas entre
retículo y mitocondrias presentan una sobreexpresión, el grupo de Aria Gómez comenzó
a estudiar si componentes de la vía amiloidogénica estaban regulando este aumento.
Describió que las presinilinas en fibroblastos
humanos se encontraban enriquecidas en los
MAMs, utilizando marcadores contra proteínas como es FACL-4, proteína descrita clásicamente como proteína MAM. Además, por
fraccionamiento subcelular (para separar los
MAMs del retículo y mitocondria), se observó
que las presenilinas 1 y 2 se encontraban altamente enriquecidas en los compartimentos
MAM, respecto de la membrana plasmática,
la mitocondria y el retículo endoplasmático.
Además, al analizar la actividad de esta proteína por un ensayo enzimático se pudo concluir que son los compartimiento MAMs los
que tienen una mayor actividad de la γ-secretasa con respecto a los otros compartimientos subcelulares. En otro trabajo similar, el
grupo de Schreiner describió que además este
compartimiento MAM era el que presentaba
una mayor producción del péptido β-amiloide, respecto de RE, mitocondria y membrana
plasmática (Schreiner et al., 2015).
Respecto de la localización de los otros componentes de la vía amilodogénica, se analizó
la localización de β-secretasa y APP en compartimentos intracelulares. Primero, por fraccionamiento subcelular con separación de
gradiente de sucrosa en fracciones mitocondriales y fracciones endosomales lisosomales,
se pudo observar con distintos marcadores
para endosomas, Golgi, retículo y MAMs, que
APP y BACE1, el componente catalítico de la
β-secretasa, cofraccionan parcialmente con
marcadores de endosomas tempranos y tardíos, y con marcadores MAM. Sin embargo,
faltan evidencias funcionales de la actividad
de β-secretasa en estos compartimentos (Pera
et al., 2017). Otra observación importante de
estos autores fue que en cerebros de ratones
WT no había un inmunomarcaje positivo
para los fragmentos derivados de la escisión
en el fragmento C-terminal de APP por la
β-secretasa (APP-CTF), que corresponden al
fragmento C83 y al C99. Esto podría evidenciar 2 fenómenos; el primero es que no estarían presentes en estos complejos MAMs y la
otra posibilidad es que es la alta la actividad
de la γ-secretasa estaría rápidamente escindido el fragmento APP-CTF de la vía amiloidogéncia (C99) para producir el péptido β-amiloide y el dominio intracelular AICD.
Por ende, utilizando un doble KO para las presinilinas 1 y 2, observaron que los APP-CTF se
encuentran cofraccionando con endosomas y
con los MAMs. En células COS-7, línea celular de riñón humano, tratadas con DAPT, un
inhibidor de la γ-secretasa, se observó una
triple colocalización positiva entre C99, SEC61β, una proteína de retículo, y una sonda
mitocondrial, indicando la presencia de este
fragmento en los MAMs (Pera et al., 2017).
Además, en células MEFs del doble KO de las
presenilinas utilizando anticuerpos APP-CTF,
se observó la presencia de C99 en los MAMs
(Pera et al., 2017).
Sin embargo, falta evidencia que indique que
efectivamente sería C99 el fragmento que se
encuentra en los MAMs, debido a que estos
autores utilizaron un anticuerpo que también
sería positivo para C83; por lo tanto, faltan
más trabajos que indiquen efectivamente es
este fragmento de la vía amiloidogénica que
estaría localizado en estos compartimientos.
Se observó que los MAMs se encuentran regulados negativamente por las presinilinas. En
células MEFs de ratones DKO para las presinilinas, se observó que había un incremento
en la colocalización entre marcadores de RE y
mitocondria, respecto de células MEFs de ratones WT. Además, al utilizar BI, un inhibidor
de β-secretasa, se observó una disminución
significativa de la colocalización entre ambas
marcas. Por lo tanto, C99 estaría regulando directamente la sobrerregulación MAM. Este resultado también fue confirmado por ensayo de
microscopía electrónica, en donde observó que
había un incremento en las áreas de contacto
largas y muy largas de los MAMs (>200nm)
en células DKO, que además presentaban una
conformación punteada no superior al 20%,
respecto de las células MEFs-WT, que principalmente tenían una conformación punteada
(sobre el 80%), en tanto que alcanzaban áreas
de contacto en bajo porcentaje (menores del
16%), y las muy largas no más de un 4%. Además, se observó un aumento de la funcionalidad de los MAMs en células MEFs de ratones
DKO, respecto de células MEFs de ratones WT
(Area-Gomez et al., 2012).
Desde un punto de vista funcional, por ensayos
de radioactividad donde se midió la activad de
ACAT1, enzima que transforma el colesterol al
colesterol éster, se observó que en células WT
MEFs tratadas con un inhibidor de la γ-secretasa (DAPT) había un aumento en la actividad
de ACAT1 en comparación al vehículo con
DMSO y este efecto se revirtió al añadir un inhibidor de la β-secretasa (BI). Cabe destacar
que este efecto no se vio revertido en este caso
por in inhibidor de la α-secretasa (TAPI); confirmando entonces que sería C99 y no C83 el
que estaría regulando las MAMs.
Interesantemente, se observó que había una
comunicación entre presinilinas y la proteína
Mfn2, ya que ambas participaban en la misma
ruta de actividad de las MAMs (Area-Gomez
et al., 2012). Para ello, en células WT MEFs
comparadas con doble KO (DKO) de las presinilinas 1 y 2 se observó que el escisión de C99
se vio inhibida en células MEFs DKO. Por otra
parte, al utilizar un ratón KO para mitofusina
2 en células MEFs y al compararlos con ratones WT MEFs, se observó un efecto similar en
células MEFs DKO; ambas presentaban una
disminución de escisión de C99. Estos datos
establecen entonces que Mfn 2 regularía positivamente el clivaje de C99 y la actividad de
γ-secretasa, y, por ende, estaría potenciando
la vía amiloidogenica.
Rol de los MAMs en la disfunción
mitocondrial
Ha sido ampliamente estudiado que en la
EA ocurre una disfunción mitocondrial, pero
aún se desconoce la razón de esta alteración.
Una de las hipótesis que ha sido ampliamente estudiada por el grupo de Área Gómez
(Area-Gomez et al., 2009, 2012; Pera et al.,
2017), es que el aumento de la comunicación
mitocondrial ER y la función MAM en la EA
sería la responsable de esta alteración, y esta
alteración esta mediada por una potenciación
de la vía amiloidogénica. En efecto, el rol que
tendría C99 de la vía amiloidogénica tendría
un alto impacto en la funcionalidad mitocondrial. Esta alteración en funcionalidad mitocondrial fue observada mediante ensayos de
Seahorse de fracciones enriquecidas en mitocondrias, que permiten medir en tiempo real
medir el consumo de oxígeno (ORC), fenómeno asociado directamente a la funcionalidad
de los complejos respiratorios de la cadena
transportadora de electrones, y por ende, a la
capacidad de producir ATP.
Se observó que células MEFs APP-DKO (doble KO de APP) presentaban un mayor consumo de oxígeno en comparación a MEFs WT,
indicando que C99 estaría regulando negativamente la función mitocondrial. Este resultado fue confirmado al utilizar un fragmento
47
En la siguiente esquema se encuentra un modelo propuesto que resume lo
expuesto anteriormente en esta revisión:
Figura 1. Rol de las MAMs en la EA. Esquema representativo que muestra las vías que estarían involucradas en la falla sináptica. Se puede observar que el incremento en la asociación
RE-mitocondria mediaría la disfunción mitocondrial mediante alteración de los componentes encargados de regular la homeostasis de calcio (I), mitofagia (II), transporte y dinámica
mitocondria (III), y la producción del péptido Aβ (IV).
de C99 en el ratón APP-DKO donde hubo
una disminución significativa de la función
mitocondrial en ratones APP-DKO C99, comparadas con ratones APP-DKO. Es más, esta
disminución se vio potenciada al utilizar este
fragmento C99 en conjunto con un inhibidor
de la γ-secretasa (DAPT), indicando que C99
estaría promoviendo la pérdida de la correcta
función mitocondrial (Pera et al., 2017).
Por otra parte, en células MEFs DKO (para
presinilina 1 y 2), hubo una disminución significativa del consumo de oxígeno respecto
a células MEFs WT. Sin embargo, cuando se
utilizó un inhibidor de la síntesis de Novo de
lípidos (Miriocina), y por ende, bloqueando
el flujo de lípidos hacia la mitocondria, hubo
una reversión de este fenómeno de disfunción
mitocondrial, reflejando en la disminución en
la capacidad respiratoria mitocondrial estaría
mediada por el aumento del metabolismo lipídico en los MAMs (Pera et al., 2017). Cabe
destacar que falta evidencia de posibles efectos de AICD y de los fragmentos de APP solu-
48
ble β que no se ha descrito hasta el momento,
que y pudiesen también estar afectando la
funcionalidad mitocondrial. Por lo tanto, podemos concluir que la alteración o mutación
las presinilinas impiden el correcto corte de
C99, lo que estaría induciendo disfunción
mitocondrial, evento que se ha asociado directamente con la falla sináptica y la muerte
neuronal en pacientes con EA.
En suma, como hemos visto, es posible que la
intercomunicación entre RE y Mitocondrial va
más allá de los roles clásicamente atribuidos
a estas dos importantes organelas; y permiten
pensar que frente a la carencia de selectividad o especificidad de los actuales fármacos
indicados en las etapas leves a moderadas de
la EA, la identificación de nuevas dianas terapéuticas, que permitan abrir nuevos espacios
de desarrollo de moléculas de síntesis, o moléculas biológicas que puedan modular la función de las MAMs, representa una interesante
oportunidad para el desarrollo farmacológico
de medicamentos neuroprotectores.
Referencias
Area-Gomez, E., de Groof, A. J. C., Boldogh,
I., Bird, T. D., Gibson, G. E., Koehler, C. M., et
al. (2009). Presenilins are enriched in endoplasmic reticulum membranes associated with
mitochondria. Am. J. Pathol. 175, 1810–1816.
doi:10.2353/ajpath.2009.090219.
Area-Gomez, E., Del Carmen Lara Castillo, M.,
Tambini, M. D., Guardia-Laguarta, C., de Groof,
A. J. C., Madra, M., et al. (2012). Upregulated
function of mitochondria-associated ER membranes in Alzheimer disease. EMBO J. 31, 4106–
4123. doi:10.1038/emboj.2012.202.
Ballard, C., Gauthier, S., Corbett, A., Brayne, C.,
Aarsland, D., and Jones, E. (2011). Alzheimer’s
disease. Lancet (London, England) 377, 1019–
1031. doi:10.1016/S0140-6736(10)61349-9.
Bernard-Marissal, N., Chrast, R., and Schneider,
B. L. (2018). Endoplasmic reticulum and mitochondria in diseases of motor and sensory neurons: a broken relationship? Cell Death Dis. 9,
333. doi:10.1038/s41419-017-0125-1.
Blass, J. P., Sheu, R. K., and Gibson, G. E.
(2000). Inherent abnormalities in energy metabolism in Alzheimer disease. Interaction with
cerebrovascular compromise. Ann. N. Y. Acad.
Sci. 903, 204–221.
Bockler, S., and Westermann, B. (2014). Mitochondrial ER contacts are crucial for mitophagy
in yeast. Dev. Cell 28, 450–458. doi:10.1016/j.
devcel.2014.01.012.
Castellani, R. J., Rolston, R. K., and Smith, M. A.
(2010). Alzheimer disease. Dis. Mon. 56, 484–
546. doi:10.1016/j.disamonth.2010.06.001.
Cosson, P., Marchetti, A., Ravazzola, M., and
Orci, L. (2012). Mitofusin-2 independent juxtaposition of endoplasmic reticulum and mitochondria: an ultrastructural study. PLoS
One 7, e46293–e46293. doi:10.1371/journal.
pone.0046293.
Cuadrado-Tejedor, M., Vilariño, M., Cabodevilla,
F., Del Río, J., Frechilla, D., and Pérez-Mediavilla,
A. (2011). Enhanced expression of the voltage-dependent anion channel 1 (VDAC1) in alzheimer’s
disease transgenic mice: An insight into the pathogenic effects of amyloid-β. J. Alzheimer’s Dis.
23, 195–206. doi:10.3233/JAD-2010-100966.
David, D. C., Ittner, L. M., Gehrig, P., Nergenau,
D., Shepherd, C., Halliday, G., et al. (2006).
Beta-amyloid treatment of two complementary
P301L tau-expressing Alzheimer’s disease models reveals similar deregulated cellular processes. Proteomics 6, 6566–6577. doi:10.1002/
pmic.200600634.
Dyzma, M., Szopa, P., and Kaźmierczak, B.
(2012). Membrane associated complexes : new
approach to calcium dynamics modelling. Math.
Model. Nat. Phenom. 7, 167–186. doi:DOI:
10.1051/mmnp/20127608.
Filadi, R., Pendin, D., and Pizzo, P. (2018). Mitofusin 2: from functions to disease. Cell Death
Dis. 9, 330. doi:10.1038/s41419-017-0023-6.
Gelmetti, V., De Rosa, P., Torosantucci, L., Marini, E. S., Romagnoli, A., Di Rienzo, M., et al.
(2017). PINK1 and BECN1 relocalize at mitochondria-associated membranes during mitophagy and promote ER-mitochondria tethering
and autophagosome formation. Autophagy 13,
654–669. doi:10.1080/15548627.2016.1277309.
Hardy, J. A., and Higgins, G. A. (1992). Alzheimer’s disease: the amyloid cascade hypothesis.
Science 256, 184–185.
Hsiao, K., Chapman, P., Nilsen, S., Eckman,
C., Harigaya, Y., Younkin, S., et al. (1996). Correlative memory deficits, Abeta elevation, and
amyloid plaques in transgenic mice. Science
274, 99–102. doi:10.1126/science.274.5284.99.
Lal, R., Lin, H., and Quist, A. P. (2007). Amyloid
beta ion channel: 3D structure and relevance to
amyloid channel paradigm. Biochim. Biophys.
Acta 1768, 1966–1975. doi:10.1016/j.bbamem.2007.04.021.
Muller, U. C., Deller, T., and Korte, M. (2017).
Not just amyloid: physiological functions of the
amyloid precursor protein family. Nat. Rev. Neurosci. 18, 281–298. doi:10.1038/nrn.2017.29.
Onyango, I. G., Dennis, J., and Khan, S. M.
(2016). Mitochondrial Dysfunction in Alzheimer’s Disease and the Rationale for Bioenergetics Based Therapies. Aging Dis. 7, 201–214.
doi:10.14336/AD.2015.1007.
Oules, B., Del Prete, D., Greco, B., Zhang, X.,
Lauritzen, I., Sevalle, J., et al. (2012). Ryanodine receptor blockade reduces amyloid-beta load
and memory impairments in Tg2576 mouse model of Alzheimer disease. J. Neurosci. 32, 11820–
11834. doi:10.1523/JNEUROSCI.0875-12.2012.
Pera, M., Larrea, D., Guardia-Laguarta, C., Montesinos, J., Velasco, K. R., Agrawal, R. R., et al.
(2017). Increased localization of APP-C99 in
mitochondria-associated ER membranes causes mitochondrial dysfunction in Alzheimer
disease. EMBO J. 36, 3356–3371. doi:10.15252/
embj.201796797.
Schreiner, B., Hedskog, L., Wiehager, B., and
Ankarcrona, M. (2015). Amyloid-beta peptides
are generated in mitochondria-associated endoplasmic reticulum membranes. J. Alzheimers.
Dis. 43, 369–374. doi:10.3233/JAD-132543.
Suh, Y.-H., and Checler, F. (2002). Amyloid
precursor protein, presenilins, and alpha-synuclein: molecular pathogenesis and pharmacological applications in Alzheimer’s disease. Pharmacol. Rev. 54, 469–525.
Vance, J. E. (2014). MAM (mitochondria-associated membranes) in mammalian cells: lipids
and beyond. Biochim. Biophys. Acta 1841, 595–
609. doi:10.1016/j.bbalip.2013.11.014.
Villegas, R., Martinez, N. W., Lillo, J., Pihan, P.,
Hernandez, D., Twiss, J. L., et al. (2014). Calcium
release from intra-axonal endoplasmic reticulum leads to axon degeneration through mitochondrial dysfunction. J. Neurosci. 34, 7179–
7189. doi:10.1523/JNEUROSCI.4784-13.2014.
Zhang, X., and Song, W. (2013). The role of APP
and BACE1 trafficking in APP processing and
amyloid-beta generation. Alzheimers. Res. Ther.
5, 46. doi:10.1186/alzrt211.
49
50
Especial Congreso
51
Estimadas socias y socios
S
ean todas y todos muy bienvenidos a
Concepción, la ciudad Universitaria
de Chile, que les recibe en un año especial donde la comunidad penquista
celebra a la Universidad de Concepción, su
emblema más reconocido, en el aniversario
de sus primeros 100 años, periodo en el que
ha aportado a la generación de conocimiento
en las ciencias, las artes y las humanidades,
a la política social del país y a su desarrollo.
Hemos trabajado arduamente como directiva
para lograr que el presente Congreso cuente
con toda la excelencia, experiencia y juventud que caracteriza a nuestra organización.
Recibiremos en Concepción a una veintena
de investigadores internacionales provenientes desde México, Cuba, Venezuela, Uruguay,
Ecuador, Brasil y Argentina, quienes se han
interesado en compartir con nosotros sus investigaciones, exposiciones que se sumarán
a las de destacados especialistas en farmacología del país y a las de nuestros invitados
especiales.
Es así como hemos construido un programa
académico y científico de alto nivel que contará con 9 simposios, 2 mini simposios, más de
70 expositores nacionales e internacionales,
entre quienes destacan 5 reconocidos conferencistas de prestigio mundial. Las jóvenes
huestes científicas también estarán presentes, representados por cerca de 190 investigadores que nos mostrarán sus avances en el
desarrollo de sus trabajos de investigación.
Especial mención y agradecimientos debemos por la generosa participación del Instituto-Fundación Teófilo Hernando de la
Universidad Autónoma de Madrid, cuyos
representantes participarán impartiendo un
simposio de sus actividades de I+D del medicamento. También extendemos este reconocimiento a los representantes de La Sociedad
Española de Farmacología (SEF), a quienes
agradecemos también por haber acogido a
una delegación de la Sofarchi en su reciente
congreso anual en julio de 2019.
Queridas amigas y amigos, en sus 41 años de
existencia la Sofarchi se encuentra en un momento clave de su desarrollo. A consecuencia
de las movilizaciones de millones de personas y familias que han ocurrido a lo largo y
ancho de nuestro Chile, tenemos el enorme
desafío y la responsabilidad de poner nues-
52
tra experiencia a disposición de la sociedad,
para convertirnos en referentes y líderes de
opinión en el área de nuestra experiencia.
Tenemos el deber ético de estar estrechamente vinculados a las necesidades de nuestros
ciudadanos. Ya hemos visibilizado este compromiso a través de actividades de extensión,
como lo hicimos con las charlas de difusión
realizadas en diversos colegios de la comuna
de Santa Cruz en el año 2018, las presentaciones en colegios de La Serena a comienzos
de este año y nuestra participación en el Brain Awarennes Week Concepción 2019, donde
ocho de nuestros socios realizaron exposiciones científicas para más de 2000 alumnos de
colegios municipales, un hecho inédito para
nuestros registros.
También hemos contribuido a cursos de actualización en temas de Cáncer, Drogas de
Abuso y Adicciones, Neurofarmacología y
Fármaco-endocrinología, entre otros. Nuestra excelencia científica una vez más se ve
reconocida por la revista Frontiers in Pharmacology, que nos ha dedicado una sección
especial (Research Topic) donde tenemos
cerca de 70 trabajos científicos comprometidos por los socios, de los cuales cerca de 40
se encuentran ya evaluados o en proceso de
revisión.
Finalmente quiero agradecer a los todos los
miembros de la Directiva, al Editor de la Revista de Farmacología de Chile, Dr. Ramón
Sotomayor, al Editor Asociado de Frontiers
in Pharmacology, Dr. Gonzalo Yévenes y a
todos quienes han dedicado gran trabajo y
esfuerzo para lograr los hitos mencionados y
permitir el continuo crecimiento de nuestra
organización.
Con todos estos elementos, esperamos que
este XLI Congreso Anual de la Sociedad de
Farmacología de Chile supere con creces sus
expectativas científicas y, además, nos permita compartir agradables jornadas de camaradería entre nosotros, disfrutando de las hermosas postales de nuestra histórica ciudad
de Concepción.
Un fraterno abrazo
Dr. Jorge Fuentealba Arcos, PhD.
Presidente
PROGRAMA XLI CONGRESO ANUAL SOCIEDAD DE FARMACOLOGIA DE CHILE 2019
UNIVERSIDAD DE CONCEPCION, CONCEPCION, CHILE
LUNES 04 DE NOVIEMBRE
lunes 04 de noviembre
11:00 – 15:00
Inscripciones
15:00 – 17:00
Inauguración (Dr. J. Fuentealba)
Conferencia 1: Dr. A. García, España
17:00 – 17:30
Coffee break
17:30 – 19:30
Simposio 1: “Purinergic signalling: from structure-activity to application in pathologies”
19:30 – 20:30
Conferencia 2: Dr. S. Moncada, UK
20:30 – 22:00
Cóctel bienvenida
MARTES 05 DE NOVIEMBRE
09:00 – 11:00
Simposio 2: “Toxicología como una ciencia multidisciplinaria”
11:00 – 11:30
Coffee break
11:30 – 12:30
Conferencia 3: Dr. S. Stojilkovic, USA
12:30 – 13:30
Premio Mardones (3)
13:30 – 15:00
Almuerzo libre
15:00 – 17:00
Simposio 3: “Translational options for the treatment of drug-abuse disorders: opportunities, successes and pitfalls”
17:00 – 17:30
Coffee break
17:30 – 19:30
Minisimposios: “Desregulación del metabolismo proteico en esquizofrenia”, “Envejecimiento cardiovascular”
19:30 – 22:00
Sesión de Posters
MIERCOLES 06 DE NOVIEMBRE
09:00 – 11:00
Simposio 4: “Estrategias para el diseño y desarrollo de nuevo fármacos”
11:00 – 11:30
Coffee break
11:30 – 12:30
Conferencia 4: Dr. T. Gallagher, UK
12:30 – 13:30
Incorporaciones (4)
13:30 – 15:00
Almuerzo libre
15:00 – 17:00
Simposio 5: “Enfermedades Neurodegenerativas y Neuroprotección”
17:00 – 17:30
Coffee break
17:30 – 19:30
Simposio 6: “Química Medicinal: Diseño Racional y Relaciones Estructura Actividad, la aproximación sintética de
nuevas sustancias biológicamente activas”
19:30 – 22:00
Sesión de Posters
JUEVES 07 DE NOVIEMBRE
09:00 – 11:00
Simposio 7: “Antimicrobial activity of herbal extracts against clinically relevant pathogens”
11:00 – 11:30
Coffee break
11:30 – 12:30
Conferencia 5: Dr. C. Bauzat, Argentina
12:30 – 13:30
Incorporaciones (2), Comunicaciones orales (4) (2 Chile, 2 México)
13:30 – 15:00
Almuerzo libre
15:00 – 22:00
Tarde libre
VIERNES 08 DE NOVIEMBRE
09:00 – 11:00
Simposio 8: “Biotechonology Aspects of Asparaginase clinical and industrial development”
11:00 – 11:30
Coffee break
11:30 – 12:30
Conferencia 6: Dr. A. Bush, Australia
12:30 – 13:30
Comunicaciones orales (6)
13:30 – 15:00
Almuerzo libre
15:00 – 17:00
Simposio 9: “Neurobiological topics in chronic pain management: from molecules to behavior”
20:30
Cena clausura
53
Conferencistas
Dr. Salvador Moncada, School of Medical Sciences, Manchester
Cancer Research Centre, University of Manchester, Inglaterra. Médico, cirujano
y farmacólogo hondureño. Realizó labores docentes en las Universidades de El
Salvador y Honduras y distintos estudios en los laboratorios Wellcome Research
entre 1975 y 1985, cuando estuvo a cargo del equipo de investigación que trabajaba
en las prostaglandinas. Su trabajo se ha centrado en efectos farmacológicos de las
sustancias vasoactivas, inflamación e interacción entre plaquetas y pared vascular;
además de realizar importantes trabajos sobre la trombosis y la arterioesclerosis.
Respecto a este último punto, destacó en los años 70 por ser el descubridor de
la prostaciclina, un vasodilatador muy potente que actúa como inhibidor de los
trombos que obstruyen las arterias.
Fue profesor visitante en diferentes universidades de Europa, Estados Unidos,
Hispanoamérica y Japón y desde 1972 ha sido asesor de la Organización Panamericana
de la Salud. Sus muchos méritos profesionales le han valido el reconocimiento de todo
el mundo; es miembro de la Royal Society, de la Sociedad Británica de Farmacología,
de la Sociedad Colombiana de Medicina Interna, de la Sociedad Farmacológica
Peruana y académico de Honor de la Real de Medicina de Valencia; es también
doctor Honoris causa por las Universidades de Honduras, Cantabria y Complutense
de Madrid. Está en posesión de cinco patentes correspondientes a distintos fármacos,
y es autor, colaborador o director de unas cuatrocientas publicaciones científicas.
54
internacionales
Dr. Antonio G. García, Instituto Teófilo Hernando and Departamento
de Farmacología, Facultad de Medicina, Universidad Autónoma de Madrid. MD,
PhD Presidente de la Fundación Teófilo Hernando (FTH) y Profesor Emérito de
Farmacología Universidad Autónoma de Madrid.
Desde 1975 sus estudios se han enfocado en la regulación farmacológica y
neuroquímica de los canales iónicos, junto con la señalización del calcio; ambas
líneas de investigación proyectadas hacia la comprensión de los principios básicos
que gobiernan la liberación exocítica de catecolaminas, la supervivencia neuronal y la
muerte neuronal. Más recientemente, se ha orientado al estudio de las enfermedades
de Alzheimer (EA) y de Huntington (EH), además de la esclerosis lateral amiotrófica
(ELA), a través de la búsqueda de un medicamento neuroprotector capaz de retrasar
la progresión de la EA y / o la ELA y mejorar la calidad de vida de los pacientes
afectados por estas patologías.
55
Dr. Ashely Bush, Melbourne Dementia Research Centre, Florey Institute
of Neuroscience and Mental Health, The University of Melbourne, Australia.
Psiquiatra y neurocientífico translacional, Director del Centro de Investigación
de la Demencia de Melbourne y Jefe de la Unidad de Biología de la Oxidación del
Florey Institute of Neuroscience and Mental Health. Es considerado una de las
mentes científicas más influentes del mundo, tras descubrir la interacción del betaamiloide con el zinc como un factor importante en la patogénesis de la enfermedad
de Alzheimer y centrarse en la neurobiología de los iones metálicos y del estrés
oxidativo en los trastornos neurodegenerativos y psiquiátricos.
Gracias a sus resultados, el estudio de la neurodegeneración se orientó hacia
una apreciación de una perturbación subyacente en la homeostasis del metal del
cerebro, proporcionando información sobre las enfermedades de Alzheimer,
Parkinson y Esclerosis Lateral Amiotrófica. Además, ha desarrollado nuevas pruebas
predictivas para la EA y estrategias innovadoras, potencialmente modificadoras de
la enfermedad.
Su exitosa carrera ha sido merecedora de diversos galardones, entre ellos el Premio
Beeson de la Federación Estadounidense de Investigación sobre el Envejecimiento,
el Premio Senator Hatfield de la Asociación de Alzheimer de EE. UU., El Premio
Potamkin para la investigación de la enfermedad de Alzheimer, el Premio de
Investigación Superior Schering-Plough del Colegio Australiano de Psiquiatras.
, una beca de la Federación ARC, el Premio Victoria de Ciencia e Innovación, y
actualmente una beca principal de investigación del NHMRC; entre otros.
Es autor de más de 447 publicaciones (más de 41 mil citas), 28 patentes y fundador
de 4 empresas de biotecnología.
56
Dra. Cecilia Bouzat, Instituto de Investigaciones Bioquímicas (INIBIBB),
Consejo Nacional de InvestigacioneS Científicas y Técnicas (Conicet), Universidad
Nacional del Sur, Argentina.
Doctora en Bioquímica de la Universidad Nacional del Sur (UNS), posteriormente
realizó estudios postdoctorales en el Departamento de Fisiología y Biofísica, en la
Clínica y Fundación Mayo (Rochester, USA). A partir de su experiencia, la Dra.
Bouzat comenzó su carrera como investigadora independiente en el Instituto de
Investigaciones Bioquímicas Bahía Blanca (INIBIBB), dependiente de la UNS, a
fines de los años 90. En el área de la investigación ha contribuido significativamente
al conocimiento a nivel funcional, farmacológico y bioquímico de los canales iónicos
activados por acetilcolina y por serotonina del sistema nervioso central, a través de
87 publicaciones científicas, lo que incluye artículos publicados en Nature, Journal
of Biological Chemistry y Molecular Pharmacology, entre otras, además de 30
proyectos de investigación financiados por entidades nacionales e internacionales.
Producto de su trabajo y esfuerzo, la Dra. Bouzat ha sido distinguida con numerosos
premios y distinciones, dentro cuales se destaca el premio L´Oréal-UNESCO for
Women in Science, Latin America International Laureate, obtenido el año 2014. El
liderazgo de la Dra. Bouzat también se ha reflejado en numerosas participaciones en
comités nacionales e internacionales, entre las cuales se destacan la presidencia y
vicepresidencia de la Sociedad Argentina de Investigación en Neurociencias (SAN),
la membresía titular de la comisión de Ciencias Médicas del CONICET, la membresía
del Comité Regional de América Latina de International Brain Research Organization
(IBRO-LARC) y la membresía permanente del comité editorial de la revista
Molecular Pharmacology. Actualmente, dirige el Laboratorio de Neurofisiología y
Farmacología Molecular del INIBIBB y es la directora del INIBIBB, en Bahía Blanca.
Además, preside el Comité Regional de América Latina de International Brain
Research Organization (IBRO-LARC) y es la directora del programa de Doctorado
en Ciencias Farmacéuticas de la UNS.
57
Dr. Tim Gallagher, PhD Universidad de Liverpool, profesor de Quimica
Orgánica de la Escuela de Química de la Universidad de Bristol (Reino Unido ),
Sus intereses de investigación abordan la química heterocíclica y abarcan una
variedad de temas, los que incluyen el desarrollo de metodología sintética, el estudio
del mecanismo de reacción, la síntesis de productos naturales y la aplicación de
síntesis a áreas de interés biológico. Tiene intereses particulares en el diseño y
síntesis de ligandos nicotínicos selectivos (basados en la citisina) y en la comprensión
del mecanismo de acción, así como la química de los sulfamidatos cíclicos como
vehículos para la síntesis asimétrica. Tiene colaboraciones dentro del área de
carbohidratos basadas en un interés en comprender y explotar las interacciones
basadas en carbohidratos.
Su trabajo ha recibido amplios galardones, tales como el Premio Katritzky de la
Sociedad Internacional de Química Heterocíclica (1999), el Tilden Lectureship de
la Royal Society of Chemistry (RSC) (2004) y el Premio de Química Heterocíclica
(2005). Además, es supervisor en el Centro EPSRC de Formación Doctoral en
Síntesis Química.
Por otra parte, ha sido consultor y presentador de tres programas de televisión
relacionados con la ciencia en el mundo antiguo, entre ellos “Antiguo Descubrimientos
III “, serie encargada por History Channel.
58
59
Abstracts for XLI of the Chilean Society of Pharmacology
CONFERENCES
1. THE EMOTION OF SCIENTIFIC
DISCOVERY.
Antonio G. García
Instituto Teófilo Hernando, Departamento
de Farmacología y Terapéutica, Facultad de
Medicina, Universidad Autónoma de Madrid,
Spain
Stephen Hawking said that “science is not
only a matter of reason; it is also a matter
of romance and passion”. This was already
written by Santiago Ramón y Cajal in his
famous book “Reglas y Consejos sobre
Investigación Científica”, a hundred years
ago. More recently, the National Academy
of Sciences of the USA wrote a short
guide entitled “ON being a scientist”, that
summarizes the emotions that a scientist
may feel along his carrier when pursuing
a problem and finding the response
sometimes after years of hard work. I will
illustrate the way science is practiced with
some experimental findings on the topic of
calcium signaling and exocytosis in adrenal
chromaffin cells. In doing so, I will focus first
on basic science, describing how we arrived
to the concept of a functional triad that
includes the voltage-gated calcium channels
(VGCCs), the endoplasmic reticulum (ER)
and mitochondria (MIT). Such triad shapes
the cytosolic calcium signals that control both
pre-exocytotic and exocytotic responses, the
basis of the fight-or-flight stress response
of W. Cannon. Then, I will focus on more
recent translational research done in
chromaffin cells from mouse models of
neurodegenerative diseases. I will comment
on dysfunctions of Ca2+ and exocytosis
occurring even at pre-symptomatic disease
stages. I will next make some comments on
the failure of clinical trials in AD, to end with
some hints on the pressure to “publish-orperish” and how science is becoming just a
mere business for editorials and else.
Recent and ongoing work from AGG’S
laboratory is being founded by
1. European Union Horizon 2020 Research
and Innovation Programme under the Marie
Skoldowska-Curie Grant Agreement 766124);
2. Grant SAF-2016-48892-R, from Ministerio
de Ciencia, Innovación y Universidades,
Spain; and
3. Fundación Teófilo Hernando, Madrid,
Spainen un contexto general y discutiré su
60
importancia con relación a la salud y a la
enfermedad.
2. THE PLEASURE OF SCIENCE: MY
LIFE IN PHARMACOLOGY.
Salvador Moncada
Manchester Cancer Research Centre, The
University of Manchester, U.K.
I will describe the work that opened several
fields of investigation. From the mechanism
of action of non-steroidal anti-inflammatory
drugs, to the discovery of thromboxane
synthase and prostacyclin, to the identification
of nitride oxide and its metabolic pathway of
synthesis. I will finish with a reference of the
role of mitochondria in oxidative stress. I
will put all this work in a general context and
its importance in health and disease will be
discussed.
3. CELL TYPE- AND SEX-DEPENDENT
TRANSCRIPTOME PROFILES OF RAT
ANTERIOR PITUITARY CELLS.
Stanko S. Stojilkovic
Section on Cellular Signaling, Eunice Kennedy
Shriver National Institute of Child Health and
Human Development, NIH, Bethesda, MD,
USA
We are investigating receptors and channels
expressed in neuronal and endocrine
cells and their roles in signaling, gene
transcription, and hormone secretion. To
gain better understanding of the cell typespecific expression and role of these and other
proteins in anterior pituitary cell functions
and related disorders, we performed single
cell RNA sequencing on freshly dispersed
cells from adult male and female rats. Our
analysis based on over 7000 cells confirmed
the expression of six pituitary-specific
cell: folliculostellate cells and hormone
producing corticotrophs, gonadotrophs,
thyrotrophs, somatotrophs, and lactotrophs.
Also identified were endothelial and blood
cells from the pituitary capillary network.
The expression of numerous developmental
and neuroendocrine marker genes in both
folliculostellate and hormone producing cells
supports that they have a common origin. For
several genes, the validity of transcriptome
analysis was confirmed by qRT-PCR and single
cell immunocytochemistry. Folliculostellate
cells exhibit impressive transcriptome
diversity, indicating their major roles in
production of endogenous ligands and
detoxification enzymes, and organization of
extracellular matrix. Transcriptome profiles
of hormone producing cells also indicate
contributions toward those functions, while
also clearly demonstrating their endocrine
function. This include the expression of
genes encoding numerous voltage-gated,
ligand-gated and other channels in hormoneproducing but not follicolostellate cell. This
survey highlights many novel genetic markers
contributing to pituitary cell type identity,
sexual dimorphism, and function and points
to relationships between hormone producing
and folliculostellate cells.
4. DIRECT C-H FUNCTIONALIZATION
OF CYTISINE. NICOTINIC RECEPTOR
SELECTIVITY AND MECHANISM OF
ACTIVATION.
Tim Gallagher
University of Bristol, UK
The talk will cover the application of
subtype-selective nicotinic partial agonists
to manage nicotine addiction, with a focus
on the chemistry of cytisine. Already
used for smoking cessation, cytisine (and
varenicline) target subsets of nicotinic
receptors, and the opportunity to generate
novel structural variants of cytisine raises
the question of whether more subtype
selective ligands are available and of value
or indeed are even desirable. The talk will
cover recent work in this area, much of
which is underpinned by development of C-H
functionalisation chemistry that provides
very direct and efficient access to new C-10
cytisine derivatives, which in turn, offer more
selective subtype profiles. Recent studies (in
collaboration with Henry Lester and Dennis
Dougherty) have probed the influence of
steric vs. electronic factors in determining
the binding mode of cytisine. We have also
pursued extensive molecular dynamics
simulations to probe the mechanism (timing)
of signal propagation through the protein
scaffold that occurs on ligand association
to the receptor, and addressed the question
of the applicability and generality of that
mechanism across other receptors.
5. GENETIC, PROTEIN AND
PHARMACOLOGICAL MODULATION
OF HUMAN α7 NICOTINIC
RECEPTORS.
Cecilia Bouzat
Instituto de Investigaciones Bioquímicas de
Bahía Blanca, INIBIBB (CONICET-UNS),
Departamento de Biología, Bioquímica y
Farmacia, Universidad Nacional del Sur,
Bahía Blanca, Argentina.
The α7 nicotinic acetylcholine receptor is
a pentameric ligand-gated ion channel. It
is widely expressed in the central nervous
system where it is involved in cognition,
attention, and memory. It is also expressed
in many non-neuronal cells and its activation
has anti-inflammatory and neuroprotective
roles. Enhancement of α7 activity is emerging
as a therapeutic strategy for cognitive,
neurodegenerative
and
inflammatory
disorders. We have focused on understanding
α7 function and its different mechanisms
of modulation associated to physiological,
pathological and therapeutic situations. By
single-channel recordings we determined
that positive allosteric modulators (PAMs)
enhance α7 activation by increasing openchannel lifetime and inducing prolonged
activation episodes, and we also identified
novel PAMs. Although α7 has been
considered the homomeric member of the
family, heteromeric α7β2 receptors have been
detected in human brain. We generated α7β2
receptors with different stoichiometries and
determined how the β2 subunit modifies α7
kinetics and its allosteric modulation. This
information is required to decipher the role
of α7β2 receptors in native cells. In humans,
there is a truncated α7 subunit (dupα7)
that lacks part of the ACh-binding site and
results from partial duplication of the α7
gene. We demonstrated that dupα7 acts as a
negative modulator and can assemble with
α7 into functional heteromeric receptors.
Deciphering the molecular basis underlying
α7 function has implications for the design of
novel therapeutic compounds as well as for
clarifying its pleiotropic actions.
61
6. IRON AND FERROPTOSIS
IN AGING AND AGE-RELATED
NEUROLOGICAL DISEASES.
Ashley Bush
The Melbourne Dementia Research Centre,
The Florey Institute of Neuroscience
and Mental Health and the University of
Melbourne, Australia.
Recent research has implicated increased
brain iron as a trait that can propel various
neurodegenerative
diseases,
including
Alzheimer’s disease, Parkinson’s disease,
Motor Neuron disease and the complications
of stroke. Ageing itself causes iron to increase
in the brain to a point where it is “too much of
a good thing” and can set up conditions that
lead to neurodegeneration. During childhood
and reproductive life, iron recruitment is
geared towards avoiding iron deficiency,
but there is no natural mechanism for offloading excess iron. After reproductive life
the systems that harvest iron so efficiently
do not turn off, and lead to accumulation
in tissues that are not normally shed, like
brain. In the C. elegans model of ageing, we
find that such iron elevation limits lifespan.
In Alzheimer’s disease brain iron elevation is
associated with the rate of cognitive loss, lipid
peroxidation products and features of the
regulated cell death mechanism, ferroptosis.
Anti-ferroptosis agents have been effective in
animal models of neurodegenerative disease,
and a recent phase 2 clinical trial of the antiferroptotic chelator deferiprone in Parkinson’s
disease lowered nigral iron and improved
clinical readouts. We are currently testing
this drug in a phase 2 RCT in Alzheimer’s
disease. CuATSM, has recently reported
benefits in phase 1 studies of Parkinson’s
disease and Motor Neuron Disease, and we
have identified that it possesses potent antiferroptotic properties.
References:
Stockwell et al. Ferroptosis: A Regulated Cell
Death Nexus Linking Metabolism, Redox
Biology, and Disease. Cell, 171, 273–285
(2017).
Ayton et al. Brain iron is associated with
accelerated cognitive decline in people with
Alzheimer pathology. Molecular Psychiatry,
in press doi: 10.1038/s41380-019-0375-7.
Southon
et
al.
CuII(atsm)
inhibits
ferroptosis: implications for treatment of
neurodegenerative disease. British Journal of
Pharmacology, in press.
62
SYMPOSIA
1. PURINERGIC SIGNALLING:
FROM STRUCTURE-ACTIVITY TO
APPLICATION IN PATHOLOGIES
ALLOSTERIC REGULATION OF THE
P2X4 RECEPTOR CHANNEL GATING.
Stojilkovic, S.S.
Section on Cellular Signaling, Eunice Kennedy
Shriver National Institute of Child Health and
Human Development, NIH, Bethesda, MD,
USA.
In general, allosteric modulators of
ligand-gated
receptor-channels
induce
conformational changes of the entire
protein that alter potencies and efficacies for
orthosteric ligands, expressed as the EC50 and
maximum current amplitude, respectively.
Recently, we studied the influence of allostery
on channel gating using the rat P2X4 receptor
expressed in HEK-293T cells and gated by
ATP in the presence and absence of ivermectin
(IVM), an established positive allosteric
regulator of this channel. In the absence of
IVM, this channel activates and deactivates
rapidly, does not conduct NMDG+, a large
organic cation, desensitizes completely with a
moderate rate, and recovers only fractionally
during washout. IVM treatment increases the
efficacy of ATP to activate the channel, slows
receptor desensitization during sustained
ATP application and receptor deactivation
after ATP washout, and makes channel
permeable to NMDG+. Experiments with
vestibular and transmembrane domain
receptor mutants further established that
IVM has distinct effects on the channel
pore opening, the first accounting for
increased peak current amplitude and the
latter correlating with changes in the EC50
and kinetics of receptor deactivation. The
corresponding kinetic (Markov state) models
can reproduce many of the observed time
series of evoked currents, as well as the
transient changes in desensitization observed
upon IVM application, the significant increase
in ATP-induced current amplitudes at low
IVM concentrations, and the modest increase
in the unitary conductance. In summary, this
study provides a detailed analysis of P2X4R
kinetics and elucidates the mechanism
regulating its channel gating.
CONTRIBUTIONS OF MOLECULAR
DYNAMIC CALCULATIONS
TO STRUCTURAL BIOLOGY
UNDERSTANDING, THE CASE OF
IVERMECTIN AND ALFAXOLONE AS
P2X4R MODULATORS.
Huidobro-Toro J.P.; Latapiat V.; Alveal N.;
Montenegro F.; Barrera N.
Laboratorio de Farmacología de Nucleótidos,
Departamento
de
Biología,
Facultad
de Química y Biología, Universidad de
Santiago de Chile y Centro de Nanociencia y
Nanotecnología, CEDENNA.
Multiple
molecules
modulate
the
electrophysiological activity of P2X receptors
(P2XR). In particular, trace metals such
as Cu(II) or Zn(II), modulate P2X4R
concentrated in neurons by selective and
specific cationic transporters; the putative site
of action of these trace metals is apparently
restricted to the extracellular receptor domain.
Drugs such as the antiparasitic antibiotic
ivermectin (IVN) or alfaxolone (A) a synthetic
neuro-steroid, with hypnotic properties, also
modulate the P2X4R but at intracellular sites,
near the transmembrane P2X4R domain. In
order to reveal and understand the site and
mechanism of the modulator effect, as well as
the allosteric agonism of A, we strategically
used molecular dynamic simulations/
calculations to visualize the docking and
mode of action of these modulators. A
rP2X4R homology model was built; docking
of either IVM or A revealed selective binding
sites confined to the transmembrane domain
as anticipated based on drug lipophilicity. In
addition, molecular dynamic simulations in
the APO and HOLO P2X4R states revealed
allosteric-induced stability. Pore and lateral
P2X4R fenestrations measurements of the
different receptor states, in the absence
and next in the presence of either IVM or
A, showed that both IVM and A can elicit a
larger pore opening that proved larger in the
presence of ATP, as expected for allosteric
modulators. Interestingly, in the case of A,
consonant and consistent with an allosteric
agonist, the hole analysis demonstrated an
even larger opening. The present findings
reveal the power and strategy of using
simulation studies to understand molecular
aspects of the binding and intricate molecular
mechanism of allosteric agonist studies.
Based on these findings, dynamic simulation
calculations offer opportunities to design
novel, P2X4R ligands not based on purines.
P2X2 RECEPTOR: NEW
PHARMACOLOGICAL TARGET TO
AD.
Fuentealba, J.
Depatamento de Fisiología, F. de Ciencias
Biológicas, Centro de Investigaciones
Avanzadas en Biomedicina (CIAB-UdeC),
Universidad de Concepción, Chile.
Soluble oligomers of amyloid beta peptide
(SOAβ) have been considered as central
factors in Alzheimer’s disease (AD). Aβ
peptide is generated through the sequential
cleavage of the amyloid precursor protein
(APP), a process that requires the previous
endocytosis of APP and that can be
modulated by the multidomain adaptor
protein Fe65. This protein is able to regulate
the transcription of key genes directly related
to AD pathogenesis, encoding proteins like
APP and BACE 1. On the other hand, we
have described that chronic SOAβ treatment
induces an increase in the expression of the
P2X2 purinergic receptor in PC12 cells and
hippocampal neurons. Additionally, it has
been described that the P2X2a isoform has
an intracellular domain that can interact
with Fe65, a segment which is absent on the
P2X2b isoform. We found that SOAβ treated
cells displayed an increase in evoked ATP
currents (C: 100 ± 50%; SOAβ: 231 ± 70%;
n=9). Additionally, immunocytochemistry
(ICC) experiments demonstrated that these
cells exhibited an increase in their P2X2R
immunoreactivity (C: 100 ± 1 %; SOAβ:
149 ± 15%; n=5). Moreover, cells treated
chronically with SOAβ showed a reduction
in the Fe65 nuclear-cytoplasmic (N-C) ratio
(C: 100 ± 6%; SOAβ: 80 ± 4%; n=5). A
similar behavior was observed in PC12 cells
transfected to express the P2X2a isoform, but
not in those transfected with P2X2b (C: 100 ±
5%; P2X2a: 70 ± 6%; P2X2b: 95 ± 6%; n=3).
Colocalization analyses demonstrated that
SOAβ decreased the colocalization of Fe65
with APP (C: 100 ± 17%; SOAβ: 47 ± 12%;
n=5); results that correlate with the increase
observed in the colocalization of APP with
clathrin (C: 100 ± 8%; SOAβ: 127 ± 8%; n=4)
and Rab5 (C: 100 ± 6%; SOAβ: 132 ± 16%;
n=5). In conclusion, these results suggest
that chronic SOAβ treatment promotes
the endocytosis of APP, potentiating its
amyloidogenic processing. Additionally, the
calcium dyshomeostasis/overload induced
by P2X2R overexpression, alter the activation
and localization of CAMKIIα, in the context
of AD. Using molecular biology techniques,
we observed that after chronic SO-Aβ
treatments, mice hippocampal neurons
63
showed an increase on the levels of P2X2R
compared to the control cells (C: 100.0 ±
6.4%; SOAβ: 130.1 ± 10.7%, n=5). This was
correlated with increased Ca2+ signal evoked
by ATP (C: 100.0 ± 12%, SOAβ: 194 ± 24%,
n=4). Immunocytochemistry approaches on
mice hippocampal neurons, showed that the
overexpression of P2X2R induced changes on
the immunorreactivity pattern of pCAMKIIα
(in soma and neurites), which induced
alterations on the cells morphology, and
electrophysiological recordings assessed by
Sholl Analysis and Patch Clamp, respectively.
These
results
suggest
that
P2X2R
overexpression can potentiate the toxicity of
SO-Aβ, due to the chronic Ca2+ overload and
inactivation of CAMKIIα, and thus, altering
the mechanisms of neuronal plasticity, the
basis of the pathophysiological mechanism of
AD.
ROLE OF PURINERGIC SIGNALING
AND IN GASTRIC CANCER.
Coddou C.1; Castro P.2; Cerda D.1; ReynaJeldes M.1; De la Fuente E.1.
1, Laboratorio de Señalización Purinérgica,
Departamento de Ciencias Biomédicas,
Facultad de Medicina, Universidad Católica
del Norte, Coquimbo, Chile. 2, Departamento
de Fisiología, Facultad de Ciencias Biológicas,
Universidad de Concepción, Concepción,
Chile.
Gastric cancer (GC) is the one of the most
prevalent cancers worldwide. Here, we studied
in detail purinergic signaling in several gastric
adenocarcinoma-derived cell lines: AGS,
MKN-45 and MKN-74, and compared them
to a non-tumoral epithelial cell line: GES-1. In
GC-derived cells, we detected the expression
of several purinergic receptors, and found
important differences as compared to GES1 cells. Functional pharmacological studies
with calcium imaging and proliferation
assays revealed a strong contribution of
P2YRs, especially P2Y2Rs to increases in cell
proliferation and an antiproliferative effect
induced by the activation of P2X4Rs. Also,
we detected a tonic purinergic response that
is probably a reflect of the paracrine and
autocrine nucleotide signaling, because to
sole application of purinergic antagonists
changed the basal proliferation of GC-derived
cells. In tumor-derived biopsies, we found
an increase of P2Y2R and a decrease in
P2X4R expression; however, we found high
variability between the biopsies and their
respective adjacent healthy gastric mucosa.
Even so, we found a correlation between the
expression levels of P2Y2R and P2X4R and
64
survival rates of gastric cancer patients. Our
latest experiments suggest that purinergic
signaling also can contributes to epithelial
to mesenchymal transition in CG-derived
cells and that transactivation of P2Y2/
HER2 can also contribute to these effects.
Taken together, these results demonstrate
the involvement of purinergic signaling
in GC, and that the changes in expression
and nucleotides release observed in GC
could direct nucleotide signaling from antiproliferative effects in healthy tissues to
proliferative effects in cancer.
2. TOXICOLOGY AS A
MULTIDISCIPLINARY SCIENCE
TOXICOLOGY AS A
MULTIDISCIPLINARY SCIENCE.
Schulz B.
Toxicología, Departamento de Farmacia,
Facultad de Farmacia, Universidad de
Concepción.
The development of the Toxicology is
strong related to the human history and its
relationship with the environment, as well as
geographic, social or politic environment. In
the primitive phase of the toxicology as health
topic, the principal focus of the knowledge
was the description of the toxic events on
humans, and its accidental or intentional
character. Many of the early register of
intoxications were based on the contact of
plants, minerals or animals with the human
bodies and it deleterious results. This
description of events summarized the botanic,
chemical, zoological and medical sciences.
With the human enrichment on scientific
information, the description of intoxications
are more specific and included clinical
symptoms, treatment of the intoxicated
patients or forensic evidences in the body.
For that are applied the physiopathology,
forensic medicine, clinical medicine and
pharmacology. The actual toxicology is
related to many complex and heterogeneous
knowledge, as well as analytical, molecular,
computational, clinical, legal, epidemiological
and pharmacological methods. In a more
general aspect, toxicology is implicated in
diverse applied areas, as clinical toxicology,
forensic and legal toxicology, occupational
toxicology, ecotoxicology and environmental
toxicology. Therefore, toxicology is clearly a
multidisciplinary science, it may be used in
other areas, and toxicology needs information
of other different disciplines as pharmacology.
STATE OF THE ART OF TOXICOLOGY
IN CHILE.
Cavieres, F.
Toxicología,
Facultad
de
Farmacia,
Universidad de Valparaíso, Chile.
The first scientific meeting of Sociedad de
Toxicología de Chile (SoTox) was held in
november 2012. The event brought together
toxicologists from academia, regulatory
agencies and industry that represented the
practice of toxicology in Chile. Only a few
months earlier, SoTox had acquired its legal
personality becoming Chile´s first scientific
society in toxicology, so the meeting was an
unprecedented opportunity to talk about
the state of the art of basic and applied
toxicological sciences in Chile. In general
terms, it was acknowledged that toxicology
played an important yet undefined role in
the Chilean society, mostly due to the lack
of professionals who had received formal
academic training in toxicology. Seven years
later, SoTox continues its efforts to position
toxicology and toxicologists as an important
discipline developed by professionals who can
contribute to better decision making in the
many different areas needed for the growth of
the country. In this talk I will: i) introduce a
modern perspective of what toxicology should
be; ii) describe the state of development of the
science in the country and iii) communicate
SoTox´s goals and achievements.
EPIDEMIOLOGY OF INTOXICATIONS
IN CHILE. 2018 ANNUAL REPORT
OF THE TOXICOLOGICAL
INFORMATION CENTER OF THE
PONTIFICIA CATHOLIC UNIVERSITY
OF CHILE (CITUC).
Silva, L.
Centro de Información Toxicológica, Facultad
de Medicina, Pontificia Universidad Católica
de Chile (CITUC).
The epidemiology of poisonings in countries
is relevant when addressing public health
guidelines, evaluating exposure profiles
and defining treatment and prevention
strategies. This presentation describes the
characterization of exposures to potentially
dangerous substances based on the crosssectional descriptive study of the universe
of calls that entered the CITUC toxicological
emergency center during 2018. The variables
analyzed were: sex, age, circumstance of the
exposure, agent (s), interlocutors of the call,
location of the interlocutor and the incident,
routes of exposure, symptomatology and
severity. This work collects information
regarding calls for toxicological emergencies
that entered CITUC, from the 15 regions, 54
provinces and 346 communes of Chile. During
the telephone call, the emergency center
professionals collect all available information
provided by the caller, required for the
evaluation of the case considering data of the
agent, the circumstances of the exposure and
the patient. After the background evaluation,
the technical recommendations for exposure
management are communicated. The data
is collected in the manual registration form
and subsequently all the data is entered into
the electronic Registration System called
“Call Registry System CITUC SRL”. The
Central of toxicological emergencies provides
free telephone assistance with qualified
professionals 365 days a year in continuous
hours (24/7), answering questions from
health professionals, authorities, emergency
personnel and the general public. The
commitment and dedication of the
professionals, together with the excellence
in the service, guarantee the 27 years of
existence of the center and the 35,000 calls
on average that CITUC receives annually.
CLINICAL MANAGEMENT OF
POISONINGS: SPECIALISTS NEEDED.
Müller C.
Toxicología, Departamento de Farmacia,
Facultad de Farmacia, Universidad de
Concepción.
In Chile, the number of acutely poisoned
patients, admitted into emergency medicine
departments, is increasing every year. Under
this circumstance, medical staff need to
be prepared, in terms of proving poisoned
patients, with adequate clinical treatment.
According to the National Poison Control
Center (CITUC), 57% of phone calls received
at the center are from healthcare settings.
This may be indicative of existing limitations
related to the clinical management of
poisoned patients. Thus, availability of
basic up-to-date knowledge about clinical
management of poisoning events would
optimize treatment regimes, economic
burden, and also improve patients’ prognosis.
A classic example of this is the use of gastric
decontamination techniques, such as gastric
lavage and activated charcoal, when patients
expose to chemicals through the oral route.
However, there are some limitations when
employing these maneuvers. Specifically, the
frame time elapsed between the exposure and
the use of the technique (up to 60 minutes).
After that time, there is a significant
decrease of effectiveness, and the procedure
65
itself turns, not only very traumatic to the
patient, but also in unnecessary expenses to
the healthcare setting. This inappropriate
practice has become common in several
health institutions, which are characterized
by a lack of specialized medical staff with
knowledge in clinical toxicology.
3. TRANSLATIONAL OPTIONS FOR
THE TREATMENT OF DRUG-ABUSE
DISORDERS: OPPORTUNITIES,
SUCCESSES AND PITFALLS
INTRODUCTORY REMARKS.
Herrera-Marschitz, M.
Programme of Mol. & Clin. Pharmacology,
Medical Faculty, University of Chile.
Synthesized molecules and drugs can be used
for rapidly achieving a high pleasant and/or
euphoric mood, bypassing the homeostatic
pathways for pleasure and reward. That can
be escalated by repeating and increasing the
drug experience, abusing of the shortcutting
pathway to pleasure, arriving to dependence
and addiction to the substances providing
that shortcut, changing the physiological
substrate for perpetuating an addicting
behaviour, impacting on the social and
familiar environment for the compulsion of
“experiencing a new trip”. Much has been
investigated about the physiological and
neuronal framework sustaining that condition
in mammals, including humans, arriving
to the pivotal neurocircuitry of pleasure,
identifying a role for dopamine, glutamate
and opioid neuropeptides. The obtained
knowledge is enormous, but that has not led
to a consensus for treating a medical issue,
which is not only menacing the individual,
but is destroying the society. We have hereby
sampled a group of international leaders
and experts, who have devoted a research
life to investigate the issue, both at basic
and clinical levels, to discuss why preclinical
results have not translated into the clinic.
Thus, Gaetano Di Chiara (Cagliari), who first
proposed a role for accumbens dopamine
release as a common substrate for addictive
substances will discuss about translational
approaches for treating cocaine addiction.
Rainer Spanagel (Mannheim), a leader on
neuropeptides and addiction will discuss on
the role of corticotropin-releasing pathways
for sustaining drug abuse and addiction. Yedy
Israel (Santiago), an international referent
on alcohol and alcoholism will discuss on a
neuroinflammatory-oxidative stress cycle
sustaining chronic alcohol intake.
66
TRANSLATIONAL APPROACHES
TO THE TREATMENT OF COCAINE
ADDICTION.
Gaetano Di Chiara
Dept. of Biomedical Sciences, University of
Cagliari, Cagliari, Italia.
Cocaine addiction treatment is probably the
most challenging and paradigmatic example of
the difficulties in translating neurobiological
knowledge into addiction therapy. Thus, to
date, in spite of the advances in the knowledge
of the neural basis of cocaine addiction,
none, among the translationally-based
treatments proposed, has been approved by
national or international agencies. Clearly,
the difficulties with cocaine addiction
might be a case of a general difficulty in
translating experimental results obtained in
animals into human therapy. Although drug
addiction is recognized as a brain disease,
it is an exceedingly complex one and it is
unclear to which extent, as in the case of
schizophrenia and dementia, animal models
are able to model the human condition.
Therefore, critical analysis of the defaillances
with cocaine can provide important clues
as to the models to utilize. As far as the
main lines of research, simple approaches
targeting DA transmission, the indirect site
of cocaine action, with DA receptor agonists
and antagonists or with low abuse liability
DAT blockers have been discouraging,
although the development of allosteric DAT
ligands is currently a major line of research
at NIDA. Acknowledgment of the critical role
of neuroplastic changes at the level of the
glutamatergic/dopaminergic cortico-ventral
striatal circuit is the basis for pharmacological
and physical treatments (DBS and TMS),
aimed at reversing the neural changes induced
by long lasting drug exposure. Preliminary,
small scale observations in humans indicate
that this might be the right way to go.
CORTICOTROPIN-RELEASING
HORMONE RECEPTOR 1 (CRHR1)
AND ADDICTION: WHY THE
PRECLINICAL RESULTS DID NOT
TRANSLATE INTO THE CLINIC?.
Spanagel, R.
Heidelberg-Mannheim, Germany
ALCOHOL-INDUCED
NEUROINFLAMMATION-OXIDATIVE
STRESS CYCLE: INFLAMMATORY
AND ANTIOXIDANT DRUGS INHIBIT
CHRONIC ALCOHOL INTAKE AND
BLUNT RELAPSE BINGE-DRINKING.
Israel Y. 1, Quintanilla ME.1, Ezquer F.3
, Morales P. 1,2 ,Ezquer M. 3, HerreraMarschitz,M.1.
1 Pharmacology Program and 2 Dept
Neuroscience,
Fac.Medicine-ICBM,
University of Chile, and 3 Centro de Med.
Regenerativa, Universidad del Desarrollo,
Santiago CHILE.
Brain of UChB rats chronically consuming
over 10 g ethanol/kg/day show (i) a 200%
increase in hippocampal oxidative stress,
determined as the ratio of oxidized/reduced
glutathione (GSSG/GSH), and (ii) marked
neuroinflammation, shown as 60% increases
in astrocyte glial-fibrillary acidic protein
(GFAP) and increases in microglial density
(Iba-1). Noteworthy, these changes remain
long after ethanol intake is discontinued;
in line with a proposed self-perpetuation
(vicious cycle) of oxidative stress and
neuroinflammation.
Administration
of
a low dose of the antioxidant N-acetyl
cysteine (40 mg/kg) reduces brain oxidative
stress and neuroinflammation and inhibits
chronic alcohol intake by 50-60%. The coadministration of N-acetyl cysteine with
low doses of aspirin (ASA 15 mg/kg) inhibit
alcohol intake by 75%, showing a significant
synergism of both drugs. Following chronic
ethanol intake, co-administration of N-acetyl
cysteine plus the anti-inflammatory drug
during a 2-week alcohol deprivation period
block neuroinflammation and oxidative
stress and inhibit by 85% the relapse bingelike drinking (“ADE”) prompted by the
subsequent ethanol re-access. As will be
shown, studies tie neuroinflammationoxidative-stress and hyper-glutamatergic
conditions as the likely mechanisms that
perpetuate chronic alcohol intake and
promote intoxicating relapse drinking, and
also indicate the pharmacological agents that
block this condition. Studies suggest that
anti-oxidant and anti-inflammatory agents
may add significantly to interventions aimed
at reducing alcohol-use disorders.
4. STRATEGIES FOR THE DESIGN
AND DEVELOPMENT OF NEW
DRUGS
DRUG DESIGN STRATEGIES BASED
ON THE MOLECULE.
Andrades-Lagos, J. 1,2; Vasquez-Velasquez,
D, 2; Campanini-Salinas, J. 3.
1. Facultad de Medicina y ciencia,
Universidad San Sebastian,Campus Los
Leones. 2. Laboratorio de Desarrollo de
Fármacos, Facultad de Ciencias Químicas
y Farmacéuticas, Universidad de Chile. 3.
Escuela de Química y Farmacia, Facultad
de Medicina y ciencia, Universidad San
Sebastian sede Patagonia Puerto Montt.
Resistance to antibacterial agents is a growing
problem of global public health, which affects
the treatment of infectious diseases, reducing
the effectiveness of available antibacterials,
causing an increase in mortality and
morbidity of patients. If innovative initiatives
that seek to solve this problem are not
generated, it is projected that, worldwide, in
the year 2050 there will be around 10 million
deaths caused by resistant microorganisms,
with costs estimated at 100 trillion dollars.
Unfortunately, the pharmaceutical industry
has been leaving research in this area which
is reflected in the approval of only ten drugs
by the FDA, during the last ten years, none
of them with new mechanisms of action.
Due to this is why it is necessary to promote
and encourage the development of a greater
quantity of antibacterial compounds,
different from those already known. For this
reason, the discovery and development of
new molecules is of vital importance. Thus,
different approaches have been used for the
discovery of active compounds, such as the
High-throughput screening, the extraction
of compounds from natural products or the
design based on a biological target. But, what
can be done when an isolated molecule (or
series) is available and its pharmacological
target is unknown? How to know which area
of the molecule can be modified and what
type of modification can be made to obtain
compounds that are more active? How to
know the relationship structure activity of this
family of molecules? In this work, we show the
experience of development of a new family of
antibacterial drugs using different strategies
of traditional medicinal chemistry. References
W. H. Organization, Antimicrobial resistance:
global report on surveillance. World Health
Organization, 2014. J. Campanini-Salinas,
J. Andrades-Lagos, J. Mella-Raipan, and
D. Vasquez-Velasquez, “Novel classes of
antibacterial drugs in clinical development, a
hope in post antibiotic era.,” Curr. Top. Med.
Chem., 2018. J. Campanini-Salinas et al.,
“A New Kind of Quinonic-Antibiotic Useful
Against Multidrug-Resistant S. aureus and E.
faecium Infections,” Molecules, vol. 23, no. 7,
2018.
67
INTEGRATION OF STRUCTURAL
BIOINFORMATICS AND CHEMICAL
BIOLOGY FOR THE DISCOVERY OF
NOVEL DRUGS.
Lagos C. F.
Chemical Biology & Drug Discovery Lab,
Facultad de Medicina y Ciencia, Universidad
San Sebastián.
structural relationship with the antibiotic
drugs predecessors. Under this look, the
present work addresses the development of
the latest antibacterial drugs in clinical phases
II and III, analyzing the design strategies by
which these new molecules were obtained
and the structure-activity relationship of
these new families of antibiotics, in order to
define the state of the vanguard antibacterial
The discovery and design of drugs is drugs in the post-antibiotic era.
increasingly
incorporating
structural
bioinformatics techniques to model and PRECLINICAL DEVELOPMENT OF A
analyze proteins of biological or therapeutic NEW CLASS OF ANTIBACTERIALS, A
interest, perform large-scale virtual screening NATIONAL EXPERIENCE.
programs to identify lead compounds and Campanini-Salinas J. 1, Andrades-Lagos J.
evaluate molecular interactions through 12, Vásquez D. 3.
molecular dynamics simulations. These 1,Facultad de Medicina y Ciencia, Universidad
techniques are fast, cost effective and San Sebastián, Lago, Panguipulli 1390, Puerto
complementary to the existing experimental Montt 5501842, Chile.2. Facultad de Medicina
techniques of chemical biology. In this y Ciencia, Universidad San Sebastián,
presentation, we will discuss some examples Santiago,Lota 2465, Chile 3. Laboratorios de
of strategies that combine different structural Desarrollo de Fármacos, Facultad de Ciencias
bioinformatics approaches with chemical Químicas y Farmacéuticas. Universidad de
biology tools to successfully discovery of Chile. Santiago, Chile.
novel drugs, focusing on the analysis of the
inherent strengths and limitations on the use The rapid emergence of resistant bacteria is
of structural bioinformatics tools, as well as occurring worldwide, endangering the efficacy
of antibiotics, reducing the therapeutics arsenal
complementary biological assays.
for treatment of infectious diseases. According
this, it is a urgent need the development of
DESIGN STRATEGIES FOR NEW
new antibacterial drugs. In this study, we
CLASSES OF ANTIBACTERIAL
develop a new class of compounds obtained
DRUGS, A HOPE IN A POSTwith a simple synthesis in two single-step.
ANTIBIOTIC ERA.
These compounds were screened for in vitro
Vásquez-Velásquez D.
Drug Development Laboratory, Facultad antibacterial activity against ATCC strains and
de Ciencias Químicas y Farmacéuticas, clinical isolates, using the broth microdilution
Universidad de Chile, Sergio Livingstone method. In addition, a series of trials were
conducted to gather information about the
1007, 8380492, Santiago, Chile.
effectiveness and safety of derivatives, such as
Bacterial resistance is a growing problem how; toxicity in mammalian cells and galleria
worldwide and is estimated that deaths by mellonella, assays of potential for induction
infectious diseases associated with resistant of mutations, among others. The compounds
pathogens will generate 10 million deaths per exhibited MICs of 1-32 μg/ml against Gramyear in 2050. This problem becomes more positive ATCC strains. The MIC50 for
serious due to the low level of research and compound 7 against the MRSA isolates tested
development of new drugs, which has fallen were 2 mg/L, compound 16 exhibit 2 mg/L.
drastically in the last 40 years. For example, For the VREF isolates the compound 7 showed
in the last decade of a total of 293 new drugs MIC50 and MIC90 values of 2 and 4 mg/L,
approved by FDA, only 9 corresponded to and the compounds 16 obtain values of 4 and
antimicrobial drugs and none constituted 4 mg/L. The compounds were bactericidal
a new structural class. The majority of the in all isolates tested. Both compounds were
molecules in the clinical phase II or III, bactericidal in all clinical isolates tested.
coming from modifications of drugs in clinical Neither compound affected cell viability in
use, this strategy make easier the bacterial any of the mammalian cell lines and Galleria
susceptibility to generate resistance through mellonella larvaes, at any of the concentrations
the mechanisms expressed for their drug tested. These in vitro data indicate that
predecessors. Under this scenario, is urgent compounds 7 and 16 can advance in assay on
to generate the most novel strategies for the murine models of infection and determination
development of antibacterial compounds with of pharmacokinetics parameters.
new targets or mechanism of action, without
68
de Madrid. 2.Instituto de Investigaciones
Biomédicas “Alberto Sols” Departamento
de Bioquímica. Facultad de Medicina.
Universidad Autónoma de Madrid. España.3.
INTRODUCTION.
Instituto de Química Médica. CSIC. Madrid.
García, A.G.
Instituto Teófilo Hernando, Departamento España. 4. Instituto de Investigación La
de Farmacología y Terapéutica, Facultad de Princesa. Hospital Universitario La Princesa.
Medicina, Universidad Autónoma de Madrid, Madrid, España.
Spain.
Alzheimer´s disease (AD) is the most common
Alzheimer’s disease (AD) is becoming a form of dementia with still no effective
devastating health, social, and economical treatment. From a histopathological point
problem. Burden to society will increase of view, AD is characterized by extracellular
as population ages. The search of disease- aggregates of betaamyloid and, intracellular
tangles
composed
of
modifying drugs has focused on over neurofibrillary
30 distinct targets, most of them linked hyperphosphorylated tau protein. During
to amyloid beta (Aβ) aggregation or the last twenty years great effort has been
hyperphosphorylated tau. Anti-oxidant, anti- made to develop therapeutic strategies,
inflammatories, neurotransmitter receptors, mostly based on beta-amyloid pathology,
or growth factors have been explored as but without success. On the other hand, AD
targets to develop a medicine to delay disease shares with other neurodegenerative diseases
progression. Ligands for those targets have pathological mechanisms like oxidative stress,
been explored in cell and murine models of subchronic inflammation, mitochondrial
AD. Although many of them have shown dysfunction and proteinopathy. Given this
efficacy in this preclinical set-up, they have scenario, our group seeks to identify new
failed in dozens of clinical trials performed therapeutic targets focused on the regulation
during the last 20 years in AD patients. It is of oxidative stress and neuroinflammation,
interesting that the Alzheimer’s Foundation processes that precede the accumulation of
for Drug Discovery (AFDD) is not supporting aberrant proteins and cognitive impairment.
any more clinical trials with compounds We have therefore centered out attention on
targeted to Aβ or tau. So, new targets and ideas two targets: (1) the NADPH oxidases enzymes
are urgently needed. In this symposium on (NOXs), which are the enzymes responsible
new targets and drugs for AD, four scientists for the production of reactive oxygen species
from the Institute “Teófilo Hernando for Drug such as superoxide and hydrogen peroxide,
Discovery”, at the Universidad Autónoma and more specifically, in its NOX4 isoform
de Madrid, Spain, will present their work and, (2) in the transcription factor NRF2
on new approaches to the search of new (Nuclear factor (erythroid-derived 2) -like 2),
targets beyond conventional (Manuela García master regulator of the antioxidant response,
López), multitarget compounds (Rafael León which also regulates the expression of genes
Martínez), and Phosphatase PP2 (Raquel that participate in the anti-inflammatory
López Arribas). A last communication focus response and autophagic processes. To
on altered neurotransmission processes in validate NOX4 as a possible target, we have
AD (Luis Gandía Juan). It is expected that used transgenic mice that do not express this
only with these and other new strategies, we enzyme and a model of taupathy by injecting
can find out the way to a medicine capable i.c.v. adeno viruses containing the human
of slowing down the natural course of the tau protein mutated in P2301L, under the
disease; and what it is even more challenging, promoter synapsin. In this model we have
if administered at presymptomatic AD stages, been able to determine that animals that
in patients at risk diagnosed with biomarkers, do not express NOX4 have less oxidative
stress and less neuroinflammation which
this medicine be capable of delaying disease
results in an improvement in the cognitive
NON CONVENTIONAL TARGETS FOR tests. For our second target, we are looking
for compounds that inhibit the interaction
THE TREATMENT OF ALZHEIMER´S
Keap-1 (NRF2 repressor protein) and NRF2.
DISEASE.
López M.G. 1, Luengo E. 1, Trigo P. 1, To do this, we have performed an in-silico
Fernández-Mendivíl C. 1, Franco F. 1, del screening of large libraries using docking and
Sastre E. 1, Cuadrado A. 2, Rodriguez-Franco molecular dynamics, as well as the synthesis
of new compounds. In the latter case, we
M. I. 3 y León R. 1 4.
1.Instituto Teófilo Hernando. Departamento want to obtain multitarget compounds with
de Farmacología. Universidad Autónoma complementary activities to the induction of
5. ALZHEIMER’S DISEASE: NEW
TARGETS AND DRUGS.
69
NRF2, with the aim of interfering on different
nodes of Alzheimer’s pathophysiology. In this
project we are following a sequential screening
protocol based on studying the Nrf2 induction,
antioxidant, anti-neuroinflammatory and
neuroprotective properties of the compounds.
As a last step, those compounds with a more
favorable toxicological, pharmacokinetic and
pharmacodynamic profile will be evaluated in
in vivo models of AD.
ALTERATIONS IN
NEUROTRANSMISSION RELATED
TO THE PROGRESSION OF
ALZHEIMER’S DISEASE.
Nanclares, C., Colmena, I., Baraibar, A.M.,
Muñoz-Montero, A., García, A.G and Gandía,
Ll.
Instituto
Teófilo
Hernando,
Depto.
Farmacología, Facultad de Medicina,
Universidad Autónoma de Madrid, Madrid,
Spain.
Alzheimer´s disease (AD) is the most common
form of dementia, being aging the main
risk factor for the development this disease.
The alteration of several neurotransmitter
systems has been reported in AD, which could
be correlated with changes in the synthesis,
storage or release of these neurotransmitters.
In this study, we tested how aging affects
ionic currents, cell excitability and last
steps of exocytosis under physiological and
pathological conditions. For this purpose, we
used a triple transgenic model of AD (3xTg-AD)
that contains mutations in the gene encoding
the amyloid precursor protein (APPSwe),
presenilin-1 (PS1M146V) and tauO301L,
which mimics the development of the disease
on Alzheimer’s patients, and B6129SF
mice (wild type). By using amperometric
techniques, we have found significant changes
in the exocytosis of catecholamines occurring
in mice of 6 and more than 12 months of age,
where the pathology is already established,
when compared with prepathological mice
(2 months), in particular, an increase of the
number of amperometric spikes, although
the quantal catecholamine content on
individual spikes is lower. Kinetic analysis
of secretory spikes shows that as the disease
progresses amperometric spikes are faster
in triggering and shorter in duration. Patchclamp technique was also used to measure
the different ionic currents involved in the
physiological release of catecholamines. We
observed a decrease in sodium currents and
an increase in potassium currents in 3xTg-AD
compared with controls. Nicotinic currents
exhibited a similar pattern throughout the
70
age in both control and transgenic mice.
Finally, we found an increase in calcium
currents in 3xTg-AD mouse with age that was
not observed in wild type mice. These findings
suggest that throughout the development of
3xTg-AD mice and as Alzheimer’s disease is
established there is a change in chromaffin cell
excitability, which causes neurotransmission
to accelerate. These alterations could have
an impact on the response that the organism
offers in a stressful situation.
SYNTHESIS AND
PHARMACOLOGICAL EVALUATION
OF NEW COMPOUNDS DIRECTED TO
PP2A, A PROMISING THERAPEUTIC
TARGET FOR ALZHEIMER’S
DISEASE.
R. López- Arribas1, L. Viejo de Navas1, 2, C.
de los Ríos1, 2.
1. Instituto Fundación Teófilo Hernando.
Departamento
de
Farmacología
y
Terapéutica, Dpto de Farmacología, Facultad
de Medicina, Universidad Autónoma de
Madrid. C/ Arzobispo Morcillo, 4, Madrid. 2.
Instituto de Investigación Sanitaria Hospital
Universitario de la Princesa. C/ Diego de
León, 62, Madrid.
Introduction: Alzheimer’s disease (AD) is the
most common cause of dementia. Nowadays,
there is no cure for AD or a way to stop or
slow its progression. Main histopathological
hallmarks of AD are senile plaques, and
neurofibrillary
tangles,
generated
by
aggregation of the microtubule associated
protein tau. Over the past two decades,
most scientific efforts in the development
of new drugs to treat AD have focused on
inhibiting the degradation of acetylcholine
and avoiding amyloidogenesis. However, less
interest has aroused the therapeutic approach
consisting in preventing neurofibrillary
death
by
inhibiting
the
abnormal
hyperphosphorylation of tau. In this sense,
pharmacological strategies have been almost
completely oriented to inhibit the activity of
tau kinase enzymes, with discouraging results.
Our research group proposes to address the
aberrant phosphorylation of tau by restoring
the activity of its main phosphatase enzyme,
protein phosphatase 2A (PP2A), which is
decreased in the brains of patients with AD,
mainly due to the increase in the expression
of the endogenous inhibitors I1PP2A and
I2PP2A/SET . Hypothesis: The study of the
structure-activity relationship of okadaic
acid (OA), a toxin with selective inhibitory
activity of PP2A, has allowed us to design
and synthesize new analogue molecules of
OA that lack such inhibitory capacity. In this
sense, our starting hypothesis states that
these compounds, due to their binding to the
catalytic subunit of PP2A, would be able to
compete with the endogenous inhibitors of
PP2A, and thus, to restore the compromised
phosphatase activity in AD. Material and
results: Our molecules are able to reduce OAinduced neurotoxicity and some of them also
present a good profile in a model of oxidative
stress in SH-SY5Y cells and in a model of
excitotoxicity in cortical neurons. The new
compounds maintained serine/threonine
phosphatase activity, depressed by the action
of two PP2A inhibitors: OA and cytostatin.
Molecular docking studies indicated that the
compounds studied are capable of binding
PP2A in a similar manner to OA, but does
not interact with the catalytic site, confirming
our initial hypothesis. Conclusions: Our
compounds have a potential indication for
the treatment of neurodegenerative diseases
based on the maintenance of PP2A activity,
which avoids tau hyperphosphorylation.
6. MEDICINAL CHEMISTRY:
RATIONAL DESIGN AND STRUCTURE
ACTIVITY RELATIONSHIP, A
SYNTHETIC APPROACH OF
NEW BIOLOGICALLY ACTIVE
SUBSTANCES.
SYNTHESIS OF NEW INDOL
DERIVATIVES AND THEIR
ACTIVITY ON CHOLINERGIC AND
SEROTONERGIC SYSTEMS AND IN
BETA-AMYLOID DEPOSITION. A
MULTIFUNCTIONAL APPROACH TO
ALZHEIMER’S DISEASE.
Pessoa-Mahana P. Departamento de Química
Orgánica y Fisicoquímica, Facultad de Ciencias
Químicas y Farmacéuticas, Universidad de
Chile. 3, Centro de Investigación Biomédica
y Aplicada (CIBAP), Escuela de Medicina,
Facultad de Ciencias Médicas, Universidad
de Santiago de Chile.
Alzheimer’s disease is the most diffuse form
of senile dementia, and is among the most
devastating brain disorder an individual can
face. It involves progressive and irreversible
decline in cognitive functions including
memory,
judgment,
decision-making,
orientation to physical surroundings and
language. Despite substantial efforts in drug
development and an increased understanding
of the underlying pathology of Alzheimer’s
disease, no effective treatment has yet been
achieved. The main goal of this research
is to contribute to the knowledge of the
medicinal chemistry in the neurochemistry
field, through the design of novel ligands
functioning as multitarget agents in
Alzheimer’ disease (AD). In this proposal, we
describe the synthesis and in vitro biological
evaluation of novel indole derivative as single
chemical entities to simultaneously modulate
multiple targets which comprises i) bindingaffinity and potential agonist properties of
serotonin 5-HT4R ii) acetylcholinesterase
inhibition, iii) serotonin transport re-uptake
inhibition and iv) inhibition of β-amyloid
deposition.
DESIGN OF NEW BENZIMIDAZOLES
WITH BETA-3 ADRENERGIC
AFFINITY.
Mella J.
Laboratorio de Química Medicinal, Instituto
de Química y Bioquímica, Facultad de
Ciencias, Universidad de Valparaíso.
The human receptor β3-adrenergic has
been the target of recent studies due to its
potential to modulate various physiological
aspects of the organism involved in numerous
pathologies such as diabetes, hypertension,
overactive
bladder,
heart
problems,
depression, and cancer, among others. In this
context, our efforts are focused on the design,
synthesis and biological evaluation of new
heterocyclic compounds capable of binding to
the human receptor β3-adrenergic. Since the
beta-3 receptor is not crystallized, we have
performed extensive studies based on ligands
(3D-QSAR, CoMFA, and CoMSIA), which
have allowed us to generate a pharmacophoric
model that we use as a basis for the rational
design of our compounds. The routes of
synthesis of the heterocycles proposed by our
group follow a similar route to that used to
obtain Mirabegrón, the only drug currently
available that acts on the beta-3 receptor
indicated in the treatment of overactive
bladder.
NEONICOTINOIDS, SEARCHING FOR
NICOTINIC RECEPTOR LIGANDS OF
ALPHA4BETA2 NACHR SUBTYPE
AND ITS APPLICATION AS NEW
ANTI-ADDICTIVE SUBSTANCES.
Iturriaga-Vasquez, P.
Laboratorio de Farmacología Molecular y
Síntesis Orgánica, Depto. Cs. Químicas y
Recursos Naturales, Fac. de Ingeniería y
Ciencias, Universida de La Frontera, Temuco.
Addiction is a chronic and compulsive drug
seeking, producing detrimental consequences,
and long-lasting changes in the brain. It is
71
considered a brain disorder and a mental
illness. Addiction is the most severe form of
substance use disorders, caused by repeated
misuse of a substance. Neuronal Nicotinic
Acetylcholine Receptors (nAChR) are
involved in nicotine addiction and emerging
evidence suggests that nAChR could be acts
as pharmacological target to be considered
in alcohol abuse. Two of the most common
addictive substances used and accepted by the
society. Nicotinic ligands have been designed,
synthesized and tested on nicotinic receptor
for decades, but the focus of the design has
been full and partials agonists with good
therapeutics results on nicotine addiction
(i.e. cytisine and varenicline). However, there
are little evidences indicating that nicotinic
antagonist could expert anti-addictive effects
over nicotine addiction and alcoholism. In
our lab, we had designed and synthesized
simple nicotinic analogues with agonist or
antagonist properties on alpha4beta2 nAChR
subtype and using zebrafish as a behavioural
model we have identified a new nicotinic
antagonist, named UFR2709 that revert the
effect of nicotine using a homologous CPP for
zebrafish. Additionally, we tested UFR2709
on Wistar-derived University of Chile alcoholpreferring UChB rats a well-known model to
evaluate ethanol consumption. Our results
show that UFR2709 are able to decrease
the ethanol intake using a two-bottle choice
paradigm assay. UFR2709, an alpha4beta2
nAChR antagonist shows an anti-addictive
effect on nicotine addiction and ethanol
consumption and open a new way for drug
design and the treatment of nicotine and
ethanol addictions.
7. ANTIMICROBIAL ACTIVITY OF
HERBAL EXTRACTS AGAINST
CLINICALLY RELEVANT
PATHOGENS
NATURAL EXTRACTS AND THEIR
ROLE IN THE SEARCH FOR NEW
THERAPEUTIC ALTERNATIVES TO
TREAT INFECTIONS.
Molina-Berríos A.
Laboratorio de Farmacología, Instituto de
Investigación en Ciencias Odontológicas,
Facultad de Odontología, Universidad de
Chile, Santiago, Chile.
In May 2015 the World Health Assembly
adopted a global action plan against
antibiotic resistance, since deaths related to
multidrug resistant bacteria have increased
in alarming speed in the last decades. One of
the goals of this plan is to support research
72
and development of new antimicrobial drugs,
since classic or conventional antibiotic drugs
have been proposed to become obsolete in a
few decades from now. This scenario is not
exclusive for bacterial infections, since the
lack of new clinically effective drugs is also a
problem for fungal and parasite infections. In
this context, natural products have emerged
as a valid alternative for the discovery of new
antimicrobial agents with new mechanisms
of action and in some cases even in absence
of current resistance mechanisms. Plants
are affected by several microorganisms, so
they count with high content of secondary
metabolites with antibacterial, antifungal
and antiparasitic effects such as flavonoids,
tanins, terpenoids and alkaloids. However,
herbal extracts can vary among the same
species due to different extraction methods,
different geographical location and even
season collection. So, it is important to count
with adequate characterization methods
in order to achieve reproducible results
and standardized extracts respect to their
chemical composition and the proportion of
active principles that can be related to their
antimicrobial activity.
REVEALING ANTIMICROBIAL
ACTIVITY OF NATURAL PRODUCTS
USING CHEMICAL SUBTRACTION
AS NEW STRATEGY TO PREPARE
KNOCK-OUT AND KNOCK-IN
EXTRACTS.
Pastene-Navarrete E.1,2.
1 Laboratory of Pharmacognosy, Faculty
of Pharmacy, Universidad de Concepción,
Concepción, Chile; 2 Laboratory of Synthesis
and Biotransformation of Natural Products,
Department of Basic Sciences, Universidad
del Bio-Bio, Chillán, Chile.
Chilean plants have biased and incomplete
chemical-pharmacological studies. The main
reason for that has been the low availability
of enough quantities to make biological
tests and structural elucidation. Moreover,
the isolation of specific constituents often
omits the residual complexity existing in
plants, in which it is not uncommon to find
highly active compounds. In this work is
presented the application of a new strategy to
investigate antimicrobial activity of medicinal
and food plants. This tool unifies different
pharmacological/phytochemical approaches
using the liquid-liquid methodology named
Centrifugal Partition Chromatography (CPC)
to obtain DESIGNER extracts. These extracts
could be “knock-out” (selective removal of
one or a group of compounds) or “knock-
in” (selective addition of one or a group of
compounds). In the first example, we prepare
“knock-out” from propolis and Buddleja
globosa (Matico) and assess their antimicrobial
activity. Propolis without caffeic acid phenyl
ester (CAPE) shown similar antimicrobial
activity compared to raw extract, suggesting
that other compounds present in its residual
complexity are responsible for such activity.
On the other hand, Matico “knock-out”
(selective removal of verbascoside), displayed
minimal antimicrobial activity. In this last
example, the re-incorporation of verbascoside
recovered the biological activity. Finally, we
perform a double knock-out in Peumus boldus
extract, removing cytotoxic compounds (e.g.
ascaridole) and isoquinoline alkaloids (e.g.
boldine). This Boldo DESIGNER extract
reduce significantly cell injury in H. pyloriinfected AGS cells without the cell toxicity
observed in the raw extracts. To confirm the
protective properties of this extract in vivo,
we used a continuous liquid-liquid separation
by True Moving Bed system (TMB-500).
Hence, a dose of 100 mg/kg/day of Boldo
DESIGNER extract was able to prevent H.
pylori SS1 infection in Mongolian gerbils.
PHYTOPHARMACEUTICALS AND
ANTIMICROBIAL RESISTANCE IN
VETERINARY MEDICINE.
Müller-Sepúlveda, A.
Instituto de Ciencias Agronómicas
Veterinarias. Universidad de O´Higgins.
y
There is an increase in fungal infections
owing to the appearance of resistant fungi to
different drugs. Candida albicans is part of the
resident microbiota of the oral cavity but is
also the most frequent fungal pathogen, whose
biofilms formation represents one of the main
resistance mechanisms. In the oral cavity,
Candida albicans biofilms are extremely
resistant to antifungals and together with the
absence of new, effective and safe antifungals,
the search for pharmacological alternatives
is warranted. It has been described that
essential oils from Lavandula dentata, and
endemic plant in Chile, possess antimicrobial
and antifungal activity against several
microorganisms including Candida albicans.
We described the antifungal and antibiofilm
effect of Lavandula dentata essential oil on the
inhibition of Candida albicans Fluconazoleresistant strain (ATCC 10231), to adhere to
abiotic surfaces and to form biofilms. After the
chemical characterized of the essential oil by
Gas Chromatography and the determination
of minimal inhibitory concentration (MIC),
we evaluated the effect of this essential oil
on the adhesion ability through crystal violet
assay and the antibiofilm effect through the
viability of biofilm formation and scratch
assay. The MIC was able to inhibit adhesion
and biofilm formation in an abiotic surface for
the resistant strains assayed (ATCC 10231). In
conclusion, this study demonstrates that this
essential oil from Lavandula dentata could be
a promising strategy against biofilms from
resistant Candida albicans strains. Since
phytodrugs present many active compounds,
who makes then difficult to generate
resistance, they can be used in conjunction
with conventional antifungal, sensitizing the
pathogens and decreasing its adhesion and
later formation of biofilms.
PARASITES AND PLANTS:
ELUCIDATING THE ANTIPARASITIC
ACTIVITY OF CICHORIUM INTYBUS
(CHICORY).
Peña-Espinoza M.
Instituto de Farmacologia y Morfofisiologia,
Facultad
de
Ciencias
Veterinarias,
Universidad Austral de Chile.
Parasitic helminths and protozoa affect
billions of people worldwide and are amongst
the most prevalent infections in livestock.
Due to increasing parasite drug resistance
towards the limited therapeutic arsenal
available, novel antiparasitics are urgently
needed. Plants with reported antiparasitic
activity have been traditionally used and
may provide new lead compounds. One
antiparasitic plant increasingly investigated
is chicory (Cichorium intybus; Asteraceae),
a perennial herb distributed worldwide and
commonly cultivated as crop for human and
livestock consumption. Chicory has attracted
research interest for its effects against
parasitic nematodes in livestock, which have
been linked with its content of sesquiterpene
lactones (SLs). Previous in vivo studies have
confirmed that chicory-fed animals have
a reduced parasite burden, but detailed
identification of responsible compounds has
not been, until recently, thoroughly explored.
By integrating parasitological studies and
metabolomic analyses, we have investigated
the anthelmintic activity and phytochemical
profile of SL-extracts from chicory material
sampled in different geographical regions.
The in vitro activity of chicory SL-extracts was
first evaluated using the free-living nematode
Caenorhabditis elegans model and further
confirmed in the parasitic pig nematode
Ascaris suum, which is closely related
with the human parasite A. lumbricoides.
Marked differences in anthelmintic potency
73
were observed between SL-extracts from
different chicory material. Bioactivity-based
molecular networking analyses suggest that
some but not all SLs are linked with the
anthelmintic activity of chicory. In addition,
we have explored the antiprotozoal activity
of chicory against Trypanosoma cruzi, the
etiological agent of Chagas disease. Chicory
SL-extracts induced potent concentrationdependent trypanocidal activity against T.
cruzi trypomastigotes at concentrations that
are not toxic to mammalian cells. Isolation
and testing of individual chicory SLs are
undergoing to evaluate their antiparasitic
mechanisms.
8. BIOTECHONOLOGY ASPECTS
OF ASPARAGINASE CLINICAL AND
INDUSTRIAL DEVELOPMENT
PRODUCTION OF EXTRACELLULAR
L-ASPARAGINASE: FROM
BIOPROSPECTING TO
THE ENGINEERING
OF AN ANTILEUKEMIC
BIOPHARMACEUTICAL.
Farias J. A.1; Pessoa A.2; Monteiro G.2; Effer,
B.1. 1.
1. Departamento de Ingeniería Química
Universidad de La Frontera, Chile; 2.
Biochemical and Pharmaceutical Technology
Department, School of Pharmaceutical
Sciences, University of São Paulo, Brazil.
In 2013 the production of L-Asparaginase,
a biopharmaceutical widely used in
the treatment of acute lymphoblastic
leukemia (ALL), was suspended by the
foreign manufacturer who supplied the
drug (Elspar®) to Brazil since the 1980s.
The interruption of this supply led to life
threatening delays in treatment which
forced the Brazilian Ministry of Health
to find emergency alternatives, including
importation of a more expensive alternative
(Aginasa®). Later, in 2017 and following
an international public tender, MS started
importing the medicine Leuginase®, from
China. Such frequent changes in the supply
of L-Asparaginase has provoked intense
and controversial debate in Brazil regarding
the quality of the imported medicine. This
motivated our group to develop a technology
platform for the production of L-asparaginase
with more advantageous characteristics
than the imported formulations. Brazil is
considered a weak player on the World stage
in biopharmaceutical discovery, development
and production of biopharmaceuticals,
and the present project proposes a union
74
between several scientific and technological
competences for the development of
industrially viable L-Asparaginase production
process. This new proposal is a continuation
of the FAPESP Thematic Project (2013 /
08617-7) that has provided promising results,
since it enabled the development of new
recombinant strains of bacteria and yeast
with the capacity to produce L-asparaginases
with longer half-life, greater stability, lower
toxicity, and lower side effects in comparison
to the biopharmaceuticals currently in
clinical use not just in Brazil, but worldwide.
As a continuation of the previously initiated
studies, this thematic project proposes
the development of processes for the
production, under GLP and GMP of 4
antileukemic
biopharmaceuticals
with
different characteristics and with important
potential to be produced nationally and
even for export, including: 1) Escherichia
coli BL21 ( DE3) – a recombinant wildtype E. coli ASNase, overexpressed in
epichomal vector pet28a with a resistance
marker for kanamycin; 2) Escherichia coli
BL21 (DE3) – a recombinant wild-type
ASNase from Erwinia chrysanthemi ASNase,
overexpressed in epimasomal vector pet28a
with a resistance marker for kanamycin; 3)
Escherichia coli BL21 (DE3) – a recombinant
E. coli ASNase resistant to human serum
proteases - overexpressed in epichomalvector pet28a with a resistance marker for
kanamycin; 4) Recombinant Pichia pastoris
– a recombinant wild-type E. chrysanthemi
crisantaspase with humanized glycosylation
(expressed in pJAG-s1 in the Superman5Glycoswitch yeast from Biogrammatics™). To
improve aspects of stability, bioavailability,
toxicity and hyperallergenicity, which
are problems observed with current
formulations, nanotechnological approaches
such as pegylation and encapsulation in
polymer vesicles will be used. The project
aims to develop a production process
from optimization of microbial cultures to
purification, pegylation and final formulation
(lyophilized product), in sufficient quantity
to carry out subsequent preclinical studies.
The entire study will be accompanied by
technical and economic evaluations (Quality
by Design).
DEVELOPMENT OF
BIOTECHNOLOGICAL PROCESS
FOR THE PRODUCTION
OF THE ANTILEUKEMIC
BIOPHARMACEUTICAL
L-ASPARAGINASE (ASNASE)
USING GENETICALLY MODIFIED
MICROORGANISMS.
Pessoa, A.
Faculty
of
Pharmaceutical
Sciences,
University of São Paulo, Brazil.
There is a strong global tendency to find
alternative ways to produce pharmaceutical
active principles from biotechnology process.
In this scenario, Latin American countries
show a small expression in both research
and production. Worsening the situation,
international suppliers of biopharmaceuticals
to this region are losing interest in the
market and discontinuing production,
especially those related to onco-hematologic
treatment. In this context, the union of
different scientific and technological skills
have joined to achieve a viable industrial
process to biotechnologically produce
L-Asparaginase, a biopharmaceutical broadly
used in the treatment of leukemia. Two major
research fronts are being studied with the
objective of finding a promising antileukemic
biopharmaceutical: the optimization of
endogenous and heterologous production
processes of the enzyme, with bioprospecting
groups of fungi of the most varied biomes;
and the rational engineering of proteins
that will utilize as scaffold the S. cerevisiae
and E. coli L-Aparaginases for comparative
studies with the bacterial isoforms currently
employed in therapeutics. To improve
aspects of stability, bioavailability, toxicity
and allergenicity, problems observed
with
bacterial
formulations,
several
nanotechnological approaches are being
used, such as pegylation and encapsulation
in polymeric vesicles, and the project aims
to generate a biopharmaceutical to be
produced industrially. Fungi from different
biomes, such as cerrado, caatinga, marine
environment, and Antarctica, have been
isolated and several of them have been
evaluated for the production of the enzyme
in shaker and in 3- or 7-Liter bioreactors, and
by solid state cultivation. Biochemical and
kinetic characteristics are being determined
for all isolated L-asparaginases. The studies
aim at obtaining recombinant E. coli and
Pichia pastoris to produce of L-asparaginase
with potentially improved characteristics
(longer half-life, higher stability, lower
toxicity and lower side effects) in comparison
those that are in clinical use in the World.
A recombinant E. coli with the capacity to
produce L-asparaginase resistant to two
plasma proteases was obtained and the
toxicity studies shows important potential for
starting preclinical studies. A recombinant
P. pastoris strain with the ability to produce
L-asparaginase with humanized glycosylation
was also obtained, with great potential
to reduce to immunogenic reactions and,
therefore, safer for patients. Pegylation
and nanoencapsulation studies of the novel
L-asparaginases are being conducted and
the results have shown that site-directed
pegylation has the potential to generate a
biopharmaceutical with better characteristics
than the pegylated form on the market. In
addition, polymer encapsulation studies
have been conducted with promising results,
especially since it is a new alternative in the
nanobiotechnology process. The project is
underway with the development of processes
of production, in GLP (good laboratory
practice) and GMP (good manufacturing
practice), of new L-asparaginases with higher
characteristics and with important potential
to be produced nationally and even for
exportation.
ASPARAGINASE ENGINEERING IN
THE OBTAINMENT OF BIOBETTERS
OF THIS ANTITUMOR ENZYME.
Costa I.M. 1; Costa-Silva T.A. 1; Effer B. 1,2;
Meira-Lima G. 1; Biasoto H.P.1; Silva C.1;
Pessoa A.1; Rangel-Yagui C.1; Farias J. A.2;
Monteiro G.1.
1.
Biochemical
and
Pharmaceutical
Technology
Department,
School
of
Pharmaceutical Sciences, University of São
Paulo, Brazil; 2. Departamento de Ingeniería
Química Universidad de La Frontera, Chile.
Asparaginase
(ASNase),
an
enzyme
biotechnologically produced in bacteria,
is one of the most important compounds
in the polychemotherapy to treat acute
lymphoblastic leukemia (ALL) in children.
There are only three options available as
medicine: native enzyme from Erwinia
chrysanthemi (ErA) or extracted from
Escherichia coli (EcA) and formulated as
native or PEGylated (PEG-EcA). However,
these options yet present some problems in
patients, such as to elicit hypersensitivity
and allergenic reactions, neurotoxicity,
and hyperammonemia. Aiming to avoid
some of these problems, our research group
has developed several different mutant
proteoforms, expressed in bacteria and yeast,
in periplasmic or secreted to extracellular
75
space; with improvement in specific activity,
kinetic parameters, and stability; different
oligomerization states, glycosylated or not,
through engineering of genes from E. coli
and E. chrysanthemi. We obtained mutants
from E. coli ASNase more resistant to human
proteases and less immunogenic. In relation
to E. chrysanthemi enzyme, our mutants
present higher asparaginase activity than the
native form, with improved kcat. In addition,
we obtained strains of Pichia pastoris that
express glycosylated ASNases from bacteria.
Our results suggest several biobetters options
developed in this study.
PRODUCTION OF NOVEL
GLYCOSYLATED L-ASPARAGINASE
AS AN ALTERNATIVE AGAINST
ACUTE LYMPHOBLASTIC
LEUKEMIA.
Effer, B.
Departamento de Ingeniería Química,
Facultad de Ingeniería y Ciencias, Universidad
de La Frontera.
L-asparaginase (L-ASNase) is an important
bacterial enzyme used as biopharmaceutical to
treat acute lymphoblastic leukemia (ALL). Its
use as medicine has important side effects such
as pancreatitis, abnormalities in coagulation,
hepatosplenomegaly,
immunogenicity,
among others. It has been counteracted by
PEGylation; however, immunogenicity has
been observed in PEG. Here we explore the
production of recombinant L-ASNase from
D. chrysamthemi glycosylated like-mammals
in a Glycoswitch® Pichia pastoris strain as
an alternative to PEGylation. In our results,
the recombinant Erwinase occurred in three
extracellular, glycosylated and biologically
active variants; two of them tetramerics
(Erw240) and (Erw160) with specific activity
of 15.71 and 302.02 U mg-1 respectively; and
one new monomeric version (Erw40) with
48.45 U mg-1. The lightweight tetramer and
the monomer showed catalytic efficiency of
7.7 x 105 and 1.05 x 106 respectively. Mass
spectrometry analysis of the more active
tetrameric and monomeric versions showed
mainly an oligosaccharide GlcNAc2Man7,
bound to Asn170, which is part of a predicted
immunogenic T-cell epitope. ELISA assay
in vitro showed a significant reduction
of antibody recognition in the Erw160,
suggesting the oligosaccharide bound to
L-ASNasa had a cloaking effect against
antibodies. The new L-ASNase versions
reported here could provide an alternative for
the treatment of ALL.
76
MINISYMPOSIA
1. CARDIOVASCULAR AGING
PREVENTING PREMATURE
ENDOTHELIAL CELL SENESCENCE:
THE ROLE OF ANGIOTENSIN-(1-7).
Peiró, C.
Departamento de Farmacología, Facultad de
Medicina, Universidad Autónoma de Madrid.
Vascular aging is a complex multifaceted
process displaying functional and structural
alterations that ultimately favour vascular
disease and atherosclerosis. Endothelial
cell senescence, which may be induced by a
wide variety of extracellular stressors, is one
of the major mechanisms contributing to
vascular aging. The senescent endothelial cell
acquire a phenotype characterized by growth
arrest and the acquisition of a senescenceassociated secretory phenotype (SASP), that
promotes the release of pro-inflammatory
mediators and the onset of sterile agerelated inflammation. In this context, the
identification of pharmacological tools to
interfere with endothelial senescence may help
retarding vascular aging and its complications.
Angiotensin (Ang)-(1-7) is a heptapeptide
belonging to the so-called protective arm of the
renin-angiontesin system (RAS). Ang-(1-7) is a
ligand for the G-protein-coupled receptor Mas.
In the vascular system, Ang-(1-7) has been
acknowledged as a physiological antagonist
for angiotensin II (Ang II), since it displays
vasorelaxant, anti-proliferative and antiinflammatory actions, among other. Here, we
tested the capacity of Ang-(1-7) to act as an
anti-senescence molecule. In human cultured
endothelial cells, Ang-(1-7) was capable to
attenuate the pro-senescence actions driven by
Ang II in terms of DNA damage, senescenceassociated beta-galactosidase (SA-beta-gal)
activity and SASP-related cytokine release.
Importantly, Ang-(1-7) also attenuated the
endothelial cell senescence induced by nonRAS stressors, such as the pro-inflammatory
cytokine interleukin (IL)-1beta. These
protective actions of Ang-(1-7) were mediated
by Mas receptors since they were blunted by
the Mas antagonist drug A779. Furthermore,
we demonstrated that Ang-(1-7) exerted its
anti-senescence actions by activating two
cytoprotective systems, i.e., the Nrf2/hemeoxygenase axis and the anti-ageing protein
klotho. Overall, the Ang-(1-7)/Mas receptor
axis may a valuable pharmacological target to
attenuate endothelial senescence and to delay
vascular aging induced by a variety of stressors.
ROLE OF NLRP3 INFLAMMASOME
IN VASCULAR DAMAGE INDUCED BY
ADIPOKINES.
Sánchez Ferrer C.F.
Departamento de Farmacología, Facultad de
Medicina, Universidad Autónoma de Madrid.
We have demonstrated that these adipokines
promotes vascular inflammation and
endothelial dysfunction. Moreover, our
data suggest that vascular deleterious
effects evoked by visfatin/eNampt or sDPP4
may involve the activation of the NLRP3
(nucleotide-binding domain, leucine-richcontaining family, pyring domain-cotainin-3)
inflammasome. Indeed, evidence from our
laboratory demonstrates the activation of
NLRP3 inflammasome by visfatin/eNampt,
while the adipokine-evoked endothelial
dysfunction is prevented by inhibiting its
enzymatic activity with FK866, as well as
by the antagonism of toll-like receptors-4
(TLR4) with CLI-095, the interference
of NLRP3 inflammasome assembly with
MCC950, or the IL-1 receptor blockade with
anakinra. Therefore, we propose that visfatin/
eNampt induces vascular damage by a
TLR4-mediated pathway, leading to NLRP3inflammasome activation and the paracrine
production of IL-1beta. On the other hand,
we have shown that sDPP4 induces vascular
alterations by activating proteinase-activated
receptors-2 (PAR-2) and upregulating
thromboxane-A2 (TXA2) release. Moreover,
the endothelial dysfunction evoked by sDPP4
is also dependent on its enzymatic activity,
being attenuated by its inhibitors K579 and
linagliptin, as well as by the specific PAR2 antagonist GB83 and the TXA2 receptor
blocker SQ-29,548. Interestingly, this
pathway also leads to NLRP3 inflammasome
activation,
while
the
sDPP4-evoked
endothelial damage is reduced by interfering
NLRP3 inflammasome with MCC950. We
conclude that vascular NLRP3 inflammasome
activation can be a common pathway for
different
pro-inflammatory
adipokines.
Indeed, targeting NLRP3 inflammasome and
some receptors linked to this pathway (TLR4,
PAR-2, or IL-1) may represent therapeutic
strategies to treat and/or prevent obesityrelated vascular dysfunction.
CARDIAC FIBROBLAST ROLE
ON INFLAMMATORY PROCESS:
INTERACTION WITH IMMUNE
CELLS.
Díaz-Araya, G.
Molecular Pharmacology Laboratory and
FONDAP ACCDiS, Universidad de Chile.
The abundance and strategic location of cardiac
fibroblasts and also macrophages in cardiac
tissue damage, suggest the possibility of a
highly coordinated interaction between both
cell types, in order to orchestrate the different
stages of cardiac remodeling. In particular
macrophage is able to adapt their phenotype
and activity according to the cytokine milieu
present in the local cardiac environment.
This phenomenon, known as macrophage
polarization, contributes to the accumulation
of pro-inflammatory M1 macrophages
during the onset of cardiac remodelling,
while also explaining the high levels of antiinflammatory/profibrotic M2 macrophages
found in the later stages of cardiac repair. While
the effects of macrophages on cardiac fibroblast
activity have been extensively studied, the
ability of cardiac fibroblasts to modulate
macrophage behavior is less understood. LPS,
and Heparan sulfate as pro-inflammatory
stimulus, triggers on cardiac fibroblast ICAM1 and VCAM-1 expression levels, which allow
spleen mononuclear cells and neutrophils
adhesion. LPS triggers high TNF-α/IL-10
ratio, whereas, TGF-β1 a profibrotic stimulus
triggers an increase on ICAM-1 and VCAM-1
expression levels, but low TNF-α/IL-10 ratio.
Consequently, cardiac fibroblast under LPStreatment promote monocytes-macrophages
M1 polarization. By contrast, cardiac
fibroblast under TGF-β1 promote monocytesmacrophages M2 polarization. Our results
demonstrate that cardiac fibroblasts interact
with immune cells and contribute to monocyte
recruitment and induce their differentiation to
M1 or M2 macrophages.
2. NOVEL MOLECULAR PATHWAYS
FOR SCHIZOPHRENIA
DYSREGULATION OF THE AMYLOID
PRECURSOR PROTEIN AND IRON IN
SCHIZOPHRENIA.
Opazo, C.
The Florey Institute of Neuroscience and
Mental Health, The University of Melbourne,
Parkville,
VIC,
Australia,
Melbourne
Neuropsychiatry Centre, Department of
Psychiatry, The University of Melbourne
& Melbourne Health, Carlton South, VIC,
Australia.
77
Schizophrenia (Sz) is a debilitating mental
illness that disrupts the functioning of the
mind. Impairments in certain cognitive
functions are core features of Sz, which
are not addressed for existing drug targets
and remain a crucial unmet therapeutic
need. Our hypothesis is that schizophrenia
is a complex disease resulting from a lossof-function of key pathways that govern
neurodevelopment, neurotransmission and
synaptic connectivity. The Amyloid Precursor
Protein (APP), which we have extensively
investigated in relation to Alzheimer’s
disease, is a key regulator of brain structure
and function. Our data indicate that iron is
elevated in autopsy orbitofrontal cortex from
individuals with Sz relative to age- and sexmatched controls. We hypothesize that these
changes are mediated by the downregulation
of APP, which also occurs in prefrontal
cortex region of individuals with Sz. Our
group have characterized the age-dependent
accumulation of iron in the brain of global
APP knockout mice. Remarkably, global APP
KO display features of Sz, including agenesis
of corpus callosum and increased seizure
activity. Therefore, we propose that down
regulation of APP function may represent a
common lesion that leads to inappropriate
neurotransmission, synaptic pruning and
synaptic function that are involved in the
clinical manifestation of Sz.
THE UBIQUITIN PROTEASOME
SYSTEM IN SCHIZOPHRENIA.
Luza, S.
The Florey Institute of Neuroscience and
Mental Health, The University of Melbourne,
Parkville,
VIC,
Australia,
Melbourne
Neuropsychiatry Centre, Department of
Psychiatry, The University of Melbourne
& Melbourne Health, Carlton South, VIC,
Australia.
The aetiology of schizophrenia remains
unknow. It has been linked to abnormalities
in dopaminergic, glutamatergic, GABAergic
and
serotoninergic
neurotransmission
as well as signalling pathways critical for
brain growth and maturation. A common
consequence of these pathway abnormalities
in schizophrenia is a loss in proteostasis of the
key components of these extra/intracellular
pathways. Proteostasis requires the control
of protein synthesis, folding, conformational
maintenance, protein-protein interaction,
trafficking and degradation. The ubiquitinproteasome system (UPS) is central to
proteostasis, suggesting it likely plays a
pivotal role in the onset and progression of
78
schizophrenia. UPS is a master regulator of
neural development and the maintenance
of brain structure and function. It
influences neurogenesis, synaptogenesis
and neurotransmission by determining the
localization, interaction and turnover of
scaffolding, presynaptic and postsynaptic
proteins. Although links between UPS
dysfunction
and
neurodegenerative
disorders have been known for some time,
only recently have similar links emerged
for neurodevelopmental disorders, such as
schizophrenia. In this presentation, we will
review the components of the UPS that are
reported as dysregulated in schizophrenia
by our group and others, and we will
discuss specific molecular changes to these
components that may explain the complex
aetiology of this mental disorder as a
syndrome.
DR. JORGE MARDONES
RESTAT AWARD
1. USE OF NPSI-ΒCD COMPOSITE
MICROPARTICLES FOR THE
CONTROLLED RELEASE OF CAFFEIC
ACID AND PINOCEMBRIN, TWO
MAIN POLYPHENOLIC COMPOUNDS
FOUND IN A CHILEAN PROPOLIS.
Guzmán-Oyarzo D.1; Plaza T.2; RecioSánchez G.2,3; Abdalla D.S.P.4; HernandezMontelongo J.2; Salazar L.A.1.
1 , Center of Molecular Biology and
Pharmacogenetics,
Scientific
and
Technological
Bioresource
Nucleus
(BIOREN), Universidad de La Frontera;
2, Bioproducts and Advanced Materials
Research Center (BioMA), Faculty of
Engineering, Universidad Católica de Temuco;
3, Department of Physical and Mathematical
Sciences, Faculty of Engineering, Universidad
Católica de Temuco; 4, Department of
Clinical and Toxicological Analyses, Faculty
of Pharmaceutical Sciences, Universidade de
São Paulo, Brazil.
Propolis is widely recognized for its
various therapeutic properties, which
are attributed to its rich composition in
polyphenols. Polyphenols exhibit multiple
biological properties, such as antioxidant,
anti-inflammatory, anti-angiogenic, and
others. Despite of its multiple benefits,
oral administration of polyphenols results
in bioavailability at the site of action. An
alternative to face this problem is the use
of biomaterials at nano-micro scale due to
its high versatility as carriers and delivery
systems of various drugs and biomolecules.
In that sense, the aim of this work is to
determine if microparticles of nanoporous
silicon conjugated with a beta cyclodextrin
polymer to form the nPSi-βCD composite are
available material for the controlled release of
the two main polyphenols of Chilean propolis,
caffeic acid and pinocembrin. Moreover,
it was studied their cytocompatibility with
HUVECs. Using different physicochemical
techniques, it was demonstrated that nPSiβCD microparticles successfully retained
and controlled release caffeic acid and
pinocembrin.
Furthermore,
nPSi-βCD
microparticles presented cytocompatibility
with HUVECs culture at concentrations
of 0.25 mg/ml. These results suggest that
nPSi-βCD microparticles can be safely used
to improve the bioavailability of caffeic
acid or pinocembrin –and eventually
other polyphenols– in the target site, thus
enhancing its therapeutic effect for the
treatment of different diseases.
total hepatic lipids were extracted. HF treated
rats showed a significant body weight gain
only until the end of the treatment compared
to control diet rats. Regarding fat tissue,
perigonadal fat was 77% higher in rat fed an
HF diet and the percentage level of lipids in
the liver increasing up to 8%. These results
together suggest that a HF diet generates
important physiological changes in the
animal, producing in a short period a state of
adiposity consistent with pre-obesity in rats.
2. EARLY EFFECT OF A HIGH-FAT
DIET ON PERIGONADAL AND
HEPATIC FAT IN RATS.
López-Aguilera A.1; Eyzaguirre-Velásquez
J.1; Escobar-Luna J.1; Bravo J.A1; JulioPieper M.1
1. Grupo de NeuroGastroBioquímica,
Laboratorio de Bioquímica de Sistemas.
Instituto de Química, Facultad de Ciencias,
Pontificia Universidad Católica de Valparaíso,
Valparaíso, Chile.
Non-small cell lung cancer (NSCLC) is the
most lethal and prevalent type of lung cancer.
NSCLC patients carrying mutations in the
Kirsten rat sarcoma viral oncogene homolog
gene (KRAS) still lack targeted therapies.
Also, the levels of polyamines (putrescine,
spermidine, and spermine) are increased
in cancer, playing a pivotal role in tumor
proliferation. Indomethacin increases the
levels of the polyamine-catabolic enzyme
spermidine/spermine-N1-acetyltransferase
(SSAT). Consequently, the aim of this study
was to compare the effect of indomethacin
in the polyamine metabolism of two NSCLC
cell lines, with different KRAS mutation
status. A549 and H1299 NSCLC cells (KRASmutated and wild-type, respectively) were
exposed to indomethacin. Evaluations
included SSAT expression and protein levels,
and metabolic analysis of cells by CG-MS
metabolomics. Moreover, the difference in
polyamine synthesis enzymes among cell lines
and the synergistic effect of indomethacin
combined with inhibitors of these enzymes
were investigated. Indomethacin increased
the expression and levels of SSAT in both
cell lines. In A549 cells, indomethacin
significantly impairs polyamine metabolism.
However, in H1299 cells, the impact of
treatment on the polyamine pathway was
non-significant. Evaluation of the levels of the
polyamine synthesis enzymes showed that
ornithine decarboxylase (ODC) is increased in
A549 cells, whereas S-adenosylmethioninedecarboxylase (AMD1) and polyamine
oxidase (PAOX), are increased in H1299
Obesity is a multifactorial disease of great
impact in Chile and worldwide. Its causes
are heterogeneous and there are no totally
effective therapeutic interventions; therefore
the study and advances in this subject are
of great relevance. The dramatic increase
in the levels of obesity in the population is
related to a progressive change to sedentary
lifestyles and excessive consumption of highly
caloric foods, such as high-fat diets (HF).
These factors lead to excessive accumulation
of adipose tissue, and in the long term may
result in ectopic fat storage. One way to study
this disease and its comorbidities is the use of
rodents fed with HF diet, as a model of dietinduced obesity. The aim of this work was
to study the early effects of a HF diet on the
accumulation of adipose tissue. For this, male
Sprague-Dawley rats were fed a HF diet (62%
of calories from fat) from postnatal day 30 for
either 15, 30 or 60 days, and were compared
to age-matched rats fed with a control diet
(14% calories from fat). At the end of each
treatment, perigonadal fat was weighed and
3. INDOMETHACIN IMPAIRS
POLYAMINE METABOLISM IN LUNG
CANCER CELLS: A KRAS MUTATIONASSOCIATED FEATURE?.
López-Contreras F1, , Muñoz-Uribe M1,
Perez-Lainez J1, Ascencio-Leal L1, RiveraDictter A1, Martin-Martin A1, Burgos Aguilera
R1, Alarcon Uribe P1, López-Muñoz R1.
1 Instituto de Farmacología y Morfofisiología,
Facultad
de
Ciencias
Veterinarias,
Universidad Austral de Chile.
79
cells. Finally, indomethacin demonstrated
a synergistic effect with the PAOX inhibitor
MDL72527 in A549 cells, whereas in H1299
had a synergistic effect with the AMD1
inhibitors SAM486. Collectively, these results
indicate that indomethacin alters polyamine
metabolism in NSCLC cells and enhances
the effect of polyamine synthesis inhibitors
such as MDL72527 or SAM486. However,
this effect varies depending on the basal
metabolic fingerprint of each type of NSCLC
cell. FONDECYT-1160807.
INCORPORATIONS
1. AMPK ACTIVATION ON
CARDIAC FIBROBLASTS: ROLE
IN AUTOPHAGY AND CELL
PROLIFERATION INDUCED BY
CATECHOLAMINES.
Pachecho N.; Portales J.; Aránguiz P.
Escuela de Química y Farmacia, Facultad
de Medicina, Universidad Andres Bello,
2520000 Viña del Mar, Chile.
Activation of the adrenergic system is
commonly associated with cardiac fibrosis
and remodeling, and cardiac fibroblasts are
key players in these processes. Interestingly,
adrenergic stimulation activates both, cardiac
fibroblasts autophagy and cell proliferation,
however, the underlying mechanisms have
not been elucidated. In the present study, we
assessed the effects of adrenergic stimulation
on autophagy and cell proliferation in
cultured adult rat cardiac fibroblasts,
which were treated with beta-adrenergic
agonists
and
antagonists.
Autophagy
was determined by electron microscopy,
subcellular distribution and protein levels of
LC3-II, and the signaling pathways involved
in its activation after stimulation with
catecholamines were evaluated by western
blot. Our results suggest that AMPK plays
a key role in the induction of autophagy,
through the inhibition of mTOR activity.
Indeed, the AMPK pharmacological inhibitor,
compound c, prevents the autophagy induced
by adrenergic agonists, acting downstream of
AKT in the beta2-adrenergic receptor/AKT/
mTOR pathway. AMPK activation was also
necessary for ERK1/2 phosphorylation and
cell proliferation. In addition, the increase
in autophagy correlates with intracellular
collagen degradation. In summary, here
we show that beta2-adrenergic stimulation
activates AMPK and this protein governs
both processes, autophagy and proliferation
in
cardiac
fibroblasts,
therefore,
a
80
pharmacological modulation in this pathway
could contribute to reducing the harmful
effects of adrenergic stimulation in cardiac
fibrosis.
2. THE EFFECT OF THE ALLOSTERIC
INHIBITOR OF RIPK1 (NEC-1) ON
OVARY FUNCTION: IMPORTANCE
OF NECROPTOSIS IN FOLICULAR
DEVELOPMENT.
Cuevas, F., Lara, H.E.
Laboratorio
de
Neurobioquímica,
Departamento de Bioquímica y Biología
Molecular, Centre for Neurobiochemical
Studies in Endocrine Diseases. Facultad
de Ciencias Químicas y Farmacéuticas,
Universidad de Chile.
The ovarian follicle develops between
proliferation and cell death process. Three
types of cell death have been reported:
apoptosis, phagocytosis and necrosis. A
fourth type of cell death, necroptosis, has
been recently associated to ovarian function.
However, the physiological relevance of
necroptosis or its involvement in follicular
development it is not yet well understood. In
the present work we will use pharmacological
tools (allosteric inhibitor necrostatin-1),
to study the role that Necroptosis would
have in follicular development and ovarian
function in vivo. We used two groups of
animals: Sham and NEC-1. Adults rats were
hemiovariectomized and implanted with a
miniosmotic pump with NEC-1 (20 μM), for 28
days, or remains without drug administration
(sham). At the end of the procedure, rats
were euthanized and the ovaries and plasma
were collected. The ovaries were fixed for
morphometric analyses. Plasma levels of
steroid hormones were measured by EIA.
We found that necroptosis inhibition did not
affect the number of secondary follicles, but
increased total antral follicles by accumulating
atretic antral follicles. The number of type III
precystic follicles was increased while the cyst
number did not change. Corpus luteum didn’t
change in number but decreased the new
(bigger size) CL. An increase in testosterone
plasma levels was found. In conclusion, NEC1 treatment by blocking necroptosis in vivo,
favored cyst formation and the permanence
of old corpus luteum thus necroptosis could
be involved in luteolysis and in the transition
to follicular cyst in the ovary. In vivo models
help to describe new pharmacological targets
to regulate follicular development and hence
fertility.
recognition to activate APCs. TLR4 role was
confirmed through a decrease in IL-12p40
and IL-6 secretion induced by FLH when a
TLR4 blocking antibody was used; a reduction
was also observed in DCs from C3H/HeJ
mice. Additionally, IL-6 secretion induced by
FLH was abolished in macrophages deficient
for TLR4. We further showed that KLH and
FLH induced ERK1/2 phosphorylation. Our
data showed the involvement of TLR4 in
the hemocyaninmediated proinflammatory
response in APCs, which could cooperate with
1Fundación Ciencia y Tecnología Para el MR in innate immune recognition of these
Desarrollo (FUCITED), Santiago, Chile; glycoproteins.
2Medical Research Council Centre for
Medical Mycology, University of Aberdeen, 4. DISCOVERY OF NOVEL TASK-3
Aberdeen, United Kingdom; 3Trinity POTASSIUM CHANNEL BLOCKERS.
Biomedical Sciences Institute, Trinity Ramírez D.; Zuñiga L.; Kiper A.; Rinné S.;
College Dublin, Dublin, Ireland; 4School Decher N.; Caballero J.; González W. and
of Life Sciences, University of Nottingham, Caballero J.
Nottingham, United Kingdom; 5Facultad de Computational Biophysics, Bioinformatics
Ciencias Químicas y Farmacéuticas, Facultad & Drug Design Lab, Instituto de Ciencias
de Medicina, Universidad de Chile, Santiago, Biomédicas - Universidad Autónoma de
Chile; 6Biosonda Corporation, Santiago, Chile.
Chile
TASK-3 is a two-pore domain potassium
Mollusk hemocyanins have biomedical (K2P) channel highly expressed in
uses as carriers/adjuvants and nonspecific hippocampus, cerebellum, and cortex.
immunostimulants with beneficial clinical TASK-3 has been identified as an oncogenic
outcomes. Hemocyanins have a multivalent potassium channel and it is overexpressed in
nature as highly mannosylated antigens. different cancer types. For this reason, the
We have shown that hemocyanins are development of new TASK-3 blockers could
internalized by antigenpresenting cells (APCs) influence the pharmacological treatment of
through receptor-mediated endocytosis by cancer and several neurological conditions.
innate immune receptors, such as mannose In the present work, we search for novel
receptor (MR). However, the contribution TASK-3 blockers by using a virtual screening
of other pattern recognition receptors to (VS) protocol that includes pharmacophore
the proinflammatory signaling pathway modeling, molecular docking, and free
triggered by hemocyanins is unknown. Thus, energy calculations (MM/GBSA). With this
we studied the roles of Dectin-1, Dectin-2, protocol, 19 potential TASK-3 blockers were
and Toll-like receptor 4 (TLR4) in the identified. These molecules were tested in
hemocyanin activation of murine APCs, both TASK-3 using patch clamp, and one blocker
in dendritic cells (DCs) and macrophages, (DR16) was identified with an IC50 = 56.8 ±
using
hemocyanins
from
Megathura 3.9 μM. Using DR16 as scaffold we designed
crenulata (KLH), Concholepas concholepas DR16.1, a novel TASK-3 inhibitor with an
(CCH) and Fissurella latimarginata (FLH). IC50 = 14.2 ± 3.4 μM. Our finding takes on
The results showed that these hemocyanins greater relevance considering that not many
bound to chimeric Dectin-1 and Dectin-2 inhibitory TASK-3 modulators have been
receptors in vitro. However, hemocyanin- reported in the scientific literature until today.
induced proinflammatory effects in APCs These two novels TASK-3 channel inhibitors
from Dectin-1 knock-out (KO) and Dectin-2 (DR16 and DR16.1) are the first found using a
KO mice were independent of both receptors. pharmacophore-based virtual screening and
Moreover, the phosphorylation of Syk rational drug design protocol.
kinase was not detected after hemocyanin
stimulation. On the other hand, we confirmed
a glycan-dependent binding of hemocyanins
to chimeric TLR4 in vitro. Moreover, DCs from
mice deficient for MyD88-adapter-like (Mal),
were partially activated by FLH, suggesting
a role of the TLR pathway in hemocyanin
3. TLR4, BUT NEITHER DECTIN-1
NOR DECTIN-2, PARTICIPATES
IN THE MOLLUSK HEMOCYANININDUCED PROINFLAMMATORY
EFFECTS IN ANTIGEN-PRESENTING
CELLS FROM MAMMALS.
JM Jiménez 1, ML Salazar 1, S Arancibia 1, J
Villar 1, F Salazar 1,2, GD Brown 2, EC Lavelle
3, L Martínez-Pomares 4, J Ortiz-Quintero 5,
S Lavandero 5, A Manubens 6 and MI Becker
1,6.
81
5. IMMUNOLOGICAL BASIS OF
AUTISM: COGNITIVE EFFECTS OF
AUTOANTIBODIES FROM AUTISTIC
CHILDREN IN MEMORY AND
LEARNING PROCESSES.
Sandoval R.1, Rossi G.1,; Cobarrubias A.1;
Arancibia M.1; Araya G.1; Uribe F.1; Gámiz
F.2; Pancetti F.1; Gonzalez-Gronow M.1
1, Environmental Neurotoxicology laboratory,
Department of biomedical Sciences, Faculty
of Medicine, Universidad Católica del Norte:
2, Universidad de Granada.
Autism spectrum disorders (ASD) involve
a range of complex neurodevelopmental
disorders,
characterized
by
social
impairments, communication difficulties,
and restricted, repetitive and stereotyped
patterns of behavior. ASD exerts a significant
physiological, emotional and financial
burden on the families of the individual
and society as a whole. Recently, beside the
knowledge about genetic factors involved in
this pathology, there is new evidence related
to immunological causes of ASD. Therefore,
it is of outmost importance to elucidate the
molecular and physiological mechanisms
of ASD pathology. Taking this into account,
we hypothesized that ASD autoantibodies
generates autoimmune-related cognitive
impairment characteristic of ASD pathology.
To achieve this aim, we used ex vivo
experiments using hippocampal slices and a
rat model where mothers were injected with
ASD autoantibodies during pregnancy and/or
breast milk period and the breeding was tested
after that period using learning and memory
test together with electrophysiological
and immunohistochemical studies. Our
results have shown that normal young rat
hippocampal slices incubated with purified
IgA autoantibodies from ASD patients and
breeding rats from pregnant mothers injected
with the same antibodies, impairs LTP as
well as disrupts learning and memory. We
also found that both LTP and learning and
memory were significantly impaired in female
but not male breeding rats and this alteration
are correlated with the presence of ASD
autoantibodies in hippocampal slices. These
results demonstrate that ASD autoantibodies
cross the transplacental barrier and also are
ingested through breeding milk, crossing
both intestinal and blood-brain barrier
and impairs learning and memory in a sexpreference fashion. The pharmacological
implicances of this research involve new
mechanisms and possible therapeutical
targets for ASD pathology
82
6. ANTI-STEROIDOGENIC EFFECT OF
THE RFRP-3 NEUROPEPTIDE AND ITS
PARTICIPATION IN THE FOLLICULAR
DYNAMICS IN THE RAT.
Squicciarini V.1, Bentley GE.2 y Lara HE.1;
1, Centre for Neurobiochemical Studies for
Endocrine Diseases, Facultad de Ciencias
Químicas y Farmacéuticas, Universidad
de Chile. 2, Laboratorio de Reproductive
Neuroendocrinology, UC Berkeley, California.
Ovarian function is highly regulated, either by
hormones, autonomic nerves and paracrine
signals produced by the same ovarian cells.
Changes of these signals modified the normal
functioning of the ovary. Gonadotrophin
inhibitor hormone (GnIH) is a neuropeptide
that block the GnRH secretion at hypothalamic
level and therefore the gonadotropic axis
regulating the ovary. The recently described
receptor (GPR147 or NPFF1, homologous
in mammals) and RFRP-3 (mammalian
homologous peptide of GnIH) in the ovary
open the possibility to suggest that the local
presence of the peptide and its receptor
could participates as regulator of ovarian
function. We studied whether RFRP-3 and
NPFF1 receptor are present in the rat’s ovary
and the local effect of this neuropeptide on
hormone production and follicular dynamics.
We determine the presence of RFRP-3 in
the rat’s ovary. RFRP-3 was mainly in the
granulose cells of antral follicles and corpora
lutea. Then, we studied the effect of 10 ng/mL
RFRP-3 on the production of ovarian steroids
ex vivo. RFRP-3 inhibited the hCG-induced
ovarian progesterone and testosterone
secretion. In order to know if the chronic
presence peptide in the ovary modified the
follicular development and its function, we
designed a local chronic treatment in vivo
with RFRP-3. After 4 weeks of treatment
there was a decrease in serum testosterone
and an increase in size and number of corpora
lutea suggesting the appearance of new
corpora lutea and hence increased ovulation.
No changes appeared in secondary, antral,
cyst or atretic follicles. Data indicate a local
effect of RFRP-3 that positively affect ovarian
steroidogenesis and follicular dynamics. This
study opens new pharmacological targets,
such as neuropeptides, to treat disorders in
ovarian function.
ORAL COMMUNICATIONS
1. THE N-ACETYLCYSTEINEINDUCED REDUCTION OF CHRONIC
ALCOHOL COMSUMPTION IS
ASSOCIATED TO THE ACTIVATION
OF THE NRF2/ARE PATHWAY IN
HIG-ALCOHOL-DRINKING UCHB
RATS.
Alvarado-Rosas, R1, Morales P1,2, Farfán N1,
Herrera-Marschitz M1, Israel Y1, Quintanilla
ME1
1 Molecular & Clinical Pharmacology Program,
ICBM, 2Department of Neuroscience, Faculty
of Medicine University of Chile.
Current
evidence
suggests
that
neuroinflammation and oxidative stress are
associated to chronic alcohol consumption
and relapse, suggesting that the modulation
of oxidative stress induced by chronic alcohol
drinking can be a therapeutic target in
alcoholism. There is evidence that oxidative
stress activates the Nrf2 (Nuclear factor
erythroid 2-like), that translocates into
nucleus where it promotes the transcription of
antioxidant genes containing the AntioxidantResponse-Element
(ARE)
including
hemoxigenase 1 (HO-1) and NAD(P)H
dehydrogenase quinone 1 (NQO1). Previously
we have demonstrated that N-acetylcysteine
(NAC), a cysteine precursor, with antioxidant
action,
inhibits
alcohol
consumption,
neuroinflammation and alcohol-induced
oxidative stress in chronic drinking rats (UChB).
However, the mechanism of the antioxidant
action of N-acetylcysteine it is not clear. The
present study determinates whether the NACreduction of chronic alcohol consumption is
associated to the activation of the Nrf2 /ARE
pathway in high-alcohol-drinking rats. Chronic
alcohol drinking (61days) female UChB rats
were administered for nine consecutive days
either (i) NAC (100mg/kg/day, per os); (ii)
NAC + all-trans-retinoic acid (ATRA, a Nrf2
pharmacological inhibitor (10 mg/kg/day
ip); (iii) ATRA+ saline); (iv) Saline. After
determining the rates of alcohol consumption,
all groups were euthanized for hippocampal
histological and Western blot analyses. It
was found that (i) N-acetylcysteine inhibits
chronic alcohol intake (ii) N-acetylcysteine
induced Nrf2 nuclear translocation (ii)
N-acetylcysteine-induced inhibition of chronic
alcohol intake was prevented by ATRA a Nrf2
pharmacological inhibitor. In conclusion these
results support the idea that Nrf2 activation
is the mechanism by which NAC inhibited
chronic alcohol consumption and relapse.
2. D-LACTATE INDUCES
NEUTROPHIL EXTRACELLULAR
TRAPS (NET) RELEASE BY
DISTURBING CELLULAR
METABOLISM.
Quiroga J.1,2; Manosalva C.3; Alarcón P.1,2;
Teuber S. 1,2; Ramírez1 F.; Carretta1 M.D.;
Hidalgo A.1, Conejeros I4; Hermosilla C4;
Burgos R.A. 1,2
1. Laboratory of Inflammation Pharmacology,
Faculty of Veterinary Sciences, Institute
of Pharmacology and Morphophysiology,
Universidad Austral de Chile, Valdivia,
Chile. 2. Laboratory of Immunometabolism,
Faculty of Veterinary Sciences, Institute
of Pharmacology and Morphophysiology,
Universidad Austral de Chile, Valdivia, Chile.
3 Faculty of Sciences, Institute of Pharmacy,
Universidad Austral de Chile, Valdivia,
Chile 4. Institute of Parasitology, Faculty of
Veterinary Medicine, Justus Liebig University
Giessen, 35392, Giessen, Germany.
D-lactate is produced during acute ruminal
acidosis (ARA), a well-known fermentative
disorder in cattle. Recently, we demonstrated
that heifers with ARA show aseptic
neutrophilic synovitis, characterized by the
presence of D-lactate, abundant neutrophils,
NET, and metabolic disturbances in synovial
fluid. It has been described that D-lactate
entry is required to induce NET-release.
Since D-lactate is slowly metabolized by
mammalian cells, we hypothesized that
D-lactate induces metabolic disturbances
in neutrophils, and so could induce NETrelease. Blood neutrophils isolated from
5 healthy heifers were treated with 5 mM
D-lactate in vitro. First, we performed a
GC-MS untargeted metabolomic analysis.
D-lactate altered galactose metabolism,
starch and sucrose metabolism, nucleotide
sugars metabolism and glycolysis. Using
JC-1 probe we observed by flow cytometry
that D-lactate reduced Δψm. In addition,
D-lactate favored the glycogen degradation,
and increased glucose-1-P and glucose-6-P
intracellular levels. Also, D-lactate increased
the AKT and GSK-3β phosphorylation. The
inhibition of theses pathways with LY294002
and CHIR99021, respectively, interfered
the decrease of glycogen and NET release.
Our results suggest that D-lactate induces
NET release by disturbing cellular metabolic
pathways, involved in glycogen degradation.
83
3. CHANGES PRODUCED BY
PREBIOTIC FIBERS IN THE
SURVIVAL OF LACTOBACILLUS
CASEI, SUBSP CASEI DURING THE
SHELF LIFE OF A NUTRACEUTICAL
DAIRY DRINK.
Gómez-Pliego R. 1; Espinosa-Raya J. 2;
Morán-Díaz J.R. 3, Guevara-Salazar J.A.
3; Rios-Guerra H. 4; Morales-Ríos E.I. 2;
Quintana Zavala D 4.
1 Laboratorio de Microbiología Industrial,
Sección de Ciencias de la Salud,
Departamento de Ciencias Biológicas, 4
Sección de Química Orgánica, Departamento
de Ciencias Químicas, Facultad de Estudios
Superiores Cuautitlán, Universidad Nacional
Autónoma de México, Av. 1 de Mayo s/n,
Santa María las Torres, Cuautitlán Izcalli,
54740, Edo., México, México; 2 Farmacología
Conductual, Sección de Estudios de
Posgrado e Investigación, Departamento
de Farmacología. México, D.F., México; 3
Departamento de Farmacología, Escuela
Superior de Medicina, Instituto Politécnico
Nacional, Plan de San Luis y Díaz Mirón, S/N,
11340, México, D.F., México; 5 Laboratorio de
Química Orgánica, Centro de Investigación
en Ciencia y Tecnología Avanzada Unidad
Legaria, Instituto Politécnico Nacional,
Legaria No. 694, 11500, México, D.F., México.
production of lactic acid and syneresis over
time (1 to 4 weeks). Conclusions. The results
showed that although there was a decrease in
the survival of L. casei in the two fibers under
study (apple and potato), they preserved its
probiotic properties (1x108 CFU / mL), while
the physical and chemical changes observed
over time (1-4 weeks) did not modify the
sensory properties of fermented dairy drinks.
4. SYNTHESIS, CHARACTERIZATION,
THEORETICAL STUDY AND IN VITRO
EVALUATION OF BETA-LACTAMIC
COMPOUNDS AND IMINES WITH
POTENTIAL ANTIBACTERIAL
ACTIVITY.
Morán Díaz J.R. 1; Ávila Melo J.L. 2;
Quintana Zavala D. 3; Gómez Pliego R. 4;
Jiménez Vázquez H.A. 2; Guevara Salazar
J.A. 5; Trujillo Ferrara J.G. 1
1. Laboratorio de Investigación de Bioquímica,
Escuela Superior de Medicina, Instituto
Politécnico Nacional, 2. Departamento de
Química Orgánica, Escuela Nacional de
Ciencias Biológicas, Instituto Politécnico
Nacional, 3. Laboratorio de Química Orgánica,
Centro de Investigación en Ciencia Aplicada
y Tecnología Avanzada, Instituto Politécnico
Nacional, 4. Laboratorio de Microbiología
Industrial L-502-Anexo, Facultad de Estudios
Superiores Cuautitlán, Universidad Nacional
Introduction. The accelerated current Autónoma de México, 5. Laboratorio de
lifestyle has generated important changes Farmacología, Escuela Superior de Medicina,
in the population’s diet at global level. Instituto Politécnico Nacional.
There are reports that indicate that the use
of prebiotics and probiotics are important One of the most serious problems worldwide
factors in the modulation and restoration of is the resistance of the main pathogenic
gastrointestinal tract microbiota (MTGI), bacteria to the antibiotics used today. The
since by decreasing intestinal permeability evolution of the resistance seen in the light
and gastrointestinal inflammation modify of the Darwinian and Lamarckian theories of
homeostasis of immunity related to glucose adaptation gives rise to the understanding of
and lipid absorption, decreasing factors the causes of resistance and if the causes are
correlated with diseases such as obesity known, solutions can be proposed, otherwise,
and overweight (Fukuda et al., 2014). what will be achieved is to amplify the
The formulation and development of new problem to such an extent that hospitals will
nutraceutical foods fermented with probiotic become the repertoire of microbial infections
microorganisms and added prebiotic fibers resistant to any chemotherapeutic treatment.
such as apple and potato could be a viable With the current biochemical knowledge, a
alternative in the treatment of emerging and rational design of antibiotics with in silico
high impact diseases such as those mentioned experiments of chloromonobactams was
above (Mishra et al., 2019; Gibson et al., 2017). proposed from imines p-substituted with
Objective. In the present work, the changes stereochemistry (E), which demonstrated
produced by prebiotic apple and potato that both sets of molecules comply with
fibers on the survival of Lactobacillus casei the Lipinski rule of 5, which offers a viable
subsp. casei were evaluated, as well as the pharmacokinetics towards the organism. The
physicochemical changes produced during synthesis of chloromonobactams was carried
the shelf life of nutraceutical dairy drinks. out in two phases. The first is the synthesis of
Results. Dairy drinks fermented showed p-substituted imines with (E) configuration;
changes in the survival of L casei, and in the the second is a [2+2] Staudinger
physical-chemical properties (viscosity, pH, cycloaddition to obtain chloromonobactams.
84
The characterization of all the synthesized
compounds was performed by physical
tests (determination of Rf, melting point,
and solubility tests), and spectroscopy (UVvisible and IR spectrophotometry, 1H and
13C NMR spectroscopy, and high-resolution
mass spectrometry). The evaluation of the in
vitro antibacterial activity was carried out by
the disk diffusion method.The study strains
were S. aureus sensitive to dicloxacillin, E.coli
and P. aeruginosa sensitive to aztreonam. The
results obtained so far show that the imines
have antibacterial activity against the bacteria
under study, with the p-iodo imine and the
beta-lactam without substituents showing an
activity similar to aztreonam on P. aeruginosa.
5. ROLE OF MITOCHONDRIAL
METABOLISM IN OXIDATIVE
RESPONSE AND NETS RELEASE
INDUCED BY PAF IN BOVINE
NEUTROPHILS.
Quiroga J. 1 ; Alarcón P. 1; Manosalva C. 2;
Teuber S. 1; Carretta M. D. 1; Hidalgo M. A. 1;
Burgos R. A. 1
1, Laboratory of Inflammation Pharmacology,
Institute
of
Pharmacology
and
Morphophysiology, Faculty of Veterinary
Sciences, Universidad Austral de Chile; 2,
Faculty of Sciences, Institute of Pharmacy,
Universidad Austral de Chile.
Neutrophils (PMN) constitute the main line
of cellular defense in the innate immune
response. Since they obtain energy primarily
through glycolysis, it is assumed that they do
not produce ATP by oxidative phosphorylation.
However, mitochondrion of PMN maintains a
transmembrane potential, which is normally
associated with respiratory chain and
oxidative phosphorylation for ATP synthesis.
PMN were isolated from healthy heifers and
stimulated in vitro with platelet activating
factor (PAF), a key biochemical mediator in
various inflammatory conditions. Incubation
with PAF 100 nM increased mitochondrial
transmembrane potential and mitochondrial
reactive oxygen species (mtROS) production.
While mtROS levels were reduced using
rotenone 10 uM (mitochondrial complex I
inhibitor), these were increased by oligomycin
10 uM (mitochondrial complex V inhibitor).
PAF 100 nM also stimulated respiratory burst
in PMN, which was reduced not only with
2-deoxy-D-glucose 2 mM (2-DG, glycolysis
inhibitor), but also with rotenone 10 uM,
oligomycin 10 uM and carbonylcyanide3-chlorophenylhydrazone 5 nM (CCCP,
oxidative
phosphorylation
uncoupler).
Finally, PAF 1 uM induced neutrophils
extracellular traps (NETs) release, which was
reduced by 2-DG and CCCP, but increased
by oligomycin. These results suggest that
PAF triggers respiratory burst and NETs
release through glycolysis and mitochondrial
metabolism-dependent mechanisms.
Multitarget drug design for the treatment of
Alzheimer’s Disease.
1,2Rafael León, 1,2Patrycja Michalska,
1,2Pablo Duarte, 1,2Paloma Mayo, 1,2Izaskun
Buendia, 1,2Enrique Crisman y 1,2Manuela
G. López
Instituto de Investigación La Princesa.
Hospital Universitario La Princesa. Madrid,
España.
Instituto Teófilo Hernando del Medicamento.
Departamento de Farmacología. Facultad de
Medicina Universidad Autónoma de Madrid.
España.
Neurodegenerative diseases (NDDs) are
currently considered a worldwide pandemia
with a prevalence of about 47 million people.
It is estimated that, in 2050, two billion
people will be over 60 years old, thus the
number of people affected is expected to
triple. Therefore, the search for effective
drugs capable of controlling neuronal cell
death is one of the great challenges of this
century.
AD is associated with several neuronal
abnormalities in energy metabolism such
mitochondrial
dysfunction,
a
decline
in glucose uptake, dysfunction in Ca2+
homeostasis. It has been shown that
oxidative damage occurs before the onset
of significant Aβ plaque formation. For
instance, the free radical theory of ageing
implies progressive ROS cell damage with
age, leading to enhanced mitochondrial DNA
mutations, futile mitochondrial Ca2+ cycling
with excess ATP consumption and ensuing
mitochondrial dysfunction. On the other
hand, it is now increasingly recognized that
inflammation also strongly contributes to
extensive oxidative stress found in AD brains.
We therefore hypothesize that mitochondrial
dysfunction could be the potential link between
neuroinflammation and neurodegeneration.
Another common characteristic is the
interconnection between these pathways
that causes feedback pathological loops
that accelerates the advance of the disease.
Therefore, their therapeutic approach must
be directed to several pathological nodes, as
the design of multitarget drugs capable of
stopping different pathological pathways at
the same time.
85
In this sense, the intrinsic cellular defense
pathway, the Nrf2-ARE pathway, has been
proposed as a therapeutic alternative for the
development of effective drugs. Therefore, we
are developing new multitarget compounds
that combine the Nrf2 induction activity with
other specific targets capable of reducing
oxidative stress, neuroinflammation and
the formation of protein aggregates, besides
activating neuronal survival pathways that
could be of potential therapeutic relevance
to afford neuroprotection in Alzheimer’s
disease.
Acknowledgments: We thank IS Carlos
III (Ref: PI17/01700), Fundación la Caixa
(CaixaImpulse CI17-00048), Fundación
FIPSE (FIPSE-12-00001344), BAYER AG
(T4D-2015-03-1282) and Comunidad de
Madrid y Fondos estructurales de la UE ref:
S2017/BDM-3827
6. THE CONSUMPTION OF A
CALAFATE EXTRACT MODULATES
THE ENERGY EXPENDITURE,
FUNCTION AND MITOCHONDRIAL
DYNAMICS OF BROWN ADIPOSE
TISSUE OF OBESE MICE.
Ramírez L.A.1; Quezada J.2; Elorza A.2; Cruz
G.3, R. Bravo-Sagua R.4; Garcia-Diaz D.1
1, Laboratorio de Bioquímica, Departamento
de Nutrición, Facultad de Medicina,
Universidad de Chile; 2, Bioenergética
Experimental, Departamento de Biología,
Facultad de Ciencias Biológicas; 3, Laboratorio
de Alteraciones Reproductivas y Metabólicas,
Instituto de Fisiología, Facultad de Ciencias,
Universidad de Valparaíso; 4, Laboratorio de
Nutrición Básica y Epidemiología Genética,
Instituto de Nutrición y Tecnología de los
Alimentos, Universidad de Chile.
Obesity is a public health problem of global
concern. In its pathogenesis, the White
Adipose Tissue has a crucial role. There
is a mitochondrial dysfunction and lower
oxidative capacity in adipocytes of obese
individuals, with modifications in their
morphology. In contrast, Brown Adipose
Tissue (BAT) has a thermogenic function
through UCP-1. A new approach proposes
to increase energy expenditure through diet.
Our objective in this work was to evaluate the
effect of a calafate extract rich in polyphenols
on mitochondrial energy, function and
dynamics (fusion / fission) of obese mice. The
analyzes were performed on adult C57BL /
6J male mice, which were subdivided (n = 10
each) into 4 dietary regimens / treatments:
Control Diet (C), Control / Calafate (extract:
50 mg total polyphenols / kg weight; CC), High
86
Fat Diet (HF) and High Fat Diet / Calafate
(HFC). The mice were subjected to indirect
calorimetry. Post-euthanasia was evaluated:
gene and protein expression of UCP-1, PGC1alpha, OPA1 (fusion), DRP1 (fission) (qPCR,
western blot or immunofluorescence),
mitochondrial Oxygen Consumption Rate
(OCR) (XF24 Seahorse), HSP70 (amount of
mitochondria) and mitochondrial activity
(with MTO). The consumption of calafate
extract produced an increase in energy
expenditure and a decrease in respiratory
quotient. The treatment presented differences
at the level of mitochondrial function, with an
increase in thermogenesis (UCP-1) a recovery
of OCR, and a significant effect on the MTO
/ HSP70 ratio. It did not substantially
modify the mitochondrial morphology. The
consumption of a calafate extract rich in
polyphenols increases energy expenditure
and improves mitochondrial function in obese
mice. Additional studies on mitochondrial
dynamics are required to complement these
hypothesis.
7. FROM HOMO SAPIENS TO HOMO
TECHNOLOGICUS, BIOETHICAL
CHALLENGE OF TRANSHUMANISM.
Rifo F. L.
Instituto Superior de Bioética, Facultad de
Medicina, UCSC
Transhumanism, an empiricist thesis whose
anthropology dispenses with metaphysics.
It explains the human dynamism from
functionalist
neurobiologicism,
which
underlines the human to the functioning,
of its neural connections, and that seeks
its sustenance in scientific perfection. In
the bioethical field it is founded is liberal
utilitarianism. There are authors who
argue that we are in the last stage of the
development of homo sapiens, and in the era
of homo technologicus, it has the possibility of
continuing the evolution of the human species
towards a superior, better and happier, using
technology to its scope. Transhumanism
raises many questions, among others. Has
neurobiological physicalism been proven?
Who tells me that the more perfect I am
physically and psychically, that the more
capacities I have, I will be happier? What is
happiness? What does it mean to be better
or more perfect, who determines it? We try
a response in the moral and ontological field.
Then there are issues of a practical nature
when implementing the transhumanist plan:
embryonic selection and eugenic elimination
of embryos and fetuses with defects, problems
derived from nanotechnology applied to the
brain and neuroethics, cryopreservation
problems, use of drugs that change
personality, resource distribution problems,
etc.
This study aims to address the ethical and
anthropological challenge that underlies
transhumanism.
8. SYNTHESIS AND EVALUATION
OF INDOLYL-BENZAMIDOPIPERAZINES AS POTENTIAL
MULTI-TARGET-DIRECTED LIGANDS
IN ALZHEIMER´S DISEASE.
Rodriguez-Lavado, J. 1, Mallea, M. 1,
Gallardo-Garrido, C. 2, Osorio, R. 1, Chung, H.
2, Pessoa- Mahana, C. 2, Iturriaga-Vásquez,
P. 3, Saitz-Barria, C. 1, Pessoa-Mahana, H. 1
1, Facultad de Ciencias Químicas y
Farmacéuticas, Universidad de Chile.
Alzheimer’s disease (AD) is a chronic,
progressive and fatal neurodegenerative
disorder affecting cognition, behavior, and
function, being one of the most common
causes of mental deterioration in elderly
people: Around 50-60% of the overall
dementias correspond to AD. World Health
Organization estimates that about 46.8
million of people worldwide currently suffer
from AD, thus becoming a major public health
concern as the world’s population ages. (World
Alzheimer’s report, 2018). Development of
Multi-Target Directed Ligands (MTDLs) has
emerged as a promising approach for targeting
complex etiology of Alzheimer’s disease
(AD). Following these approach, and given
our interest in the search and development
of novel drugs displaying affinity acting as
promiscuous ligands. In the present work,
a novel series of indolylpropylpiperazinyl
piperazinebenzamides were synthesized and
biologically evaluated as multifunctional
ligands
in
the
following
targets:
acetylcholinesterase, SERT, and betaamyloid peptides The synthesis involved
connection between Piperazine benzamides
with N-Boc substituted piperazine derivative.
Boc cleavage and further coupling with
indolylpropyl tosylates was achieved in two
step-one pot reaction, obtaining the final
compounds with good to excellents overall
yields. Finally, the obtained compounds
were evaluated in its capabilities for AChE
inhibition,
SERTaffinity,
β-amyloid
inhibition, and cell toxicity (viability) with
very promising results.
9. DEVELOPMENT OF A
RECOMBINANT VACCINE
CANDIDATE AGAINST HANTAVIRUS.
Starck-Méndez M.F. 1,2; Neira P.J. 2, Varas
N.M.J. 1,2; Toledo J.R. 1,3; Acosta J. 1,3;
Sanchez O. 1,2
1,Center for Biotechnology and Biomedicine
Spa., Concepción, Chile. 2, Department of
Pharmacology, School of Biological Sciences,
Universidad de Concepción. 3, Department
of Physiopathology, School of Biological
Sciences, Universidad de Concepción.
Andes virus is the main causative agent of
Hantavirus
cardiopulmonary
syndrome
(HCPS) in South America. There are currently
no vaccines or treatments against Andes
virus. However, there are several evidences
suggesting that antibodies against Andes
virus envelope glycoproteins may be enough
to confer full protection against HCPS. The
main goal of the present work was to develop
a vaccine candidate against Hantavirus,
based on the surface glycoproteins Gn and
Gc. With this purpose, the sequence encoding
the extracellular domains of both antigens
was introduced into the methylotrophic
yeast Pichia pastoris. After induction
with methanol, the recombinant antigens
accumulated intracellularly as insoluble
aggregates. After cell disruption, the
recombinant antigens were solubilized and
purified by metal-ion affinity chromatography.
The immunogenicity of both antigens was
determined in immunization assays in both
mice and Syrian hamsters. In both species it
was possible to detect the presence of specific
antibodies against Gn and Gc. Part of these
antibodies showed neutralizing activity. The
results obtained to date suggest that the Gn
and Gc antigens from Andes virus, produced
in P. pastoris, have the potential to become
the first commercial vaccine against HCPS.
10. ADHERENCE TO
ANTIHYPERTENSIVE
PHARMACOLOGICAL TREATMENT IN
ELDERLY PEOPLE FROM HUALPEN
SUBMITTED TO A TRANSMEDIAL
PSYCHOEDUCATIONAL PROGRAM.
Sepúlveda, M.J.1*; Pinto, R.1; Iturra, R.1;
Müller, H.2; Chamblás, I.3; Victoriano, M.4;
Casanova, M.P.5; Guevara, P.6; Aguilera, R.7;
Cid, P.8
1 Departamento de Farmacología, Facultad
de Ciencias Biológicas. 2 Departamento de
Medicina Interna, Facultad de Medicina. 3
Departamento de Trabajo Social, Facultad
87
de Ciencias Sociales. 4 Departamento
de Nutrición, Facultad de Farmacia. 5
Departamento de Estadística, Facultad
de Ciencias Físicas y Matemáticas. 6
Departamento de Ingeniería Eléctrica,
Facultad de Ingeniería. 7 Departamento de
Economía, Facultad de Ciencias Económicas
y Administrativas. 8 Departamento de
Fundamentos de Enfermería y Salud Pública,
Facultad de Enfermería. Universidad de
Concepción, Concepción, Chile.
POSTERS
1. POSTER RETIRADO”
2. SEARCH FOR ANALOGS OF M554
AND M890 FOR INTERACTION
WITH THE GΒΓ DIMER IN REGIONS
INVOLVED IN THE REGULATION OF
THE GLYCINE RECEPTOR.
Argel A. Y.; Guzmán G. L.; Jiménez C. V.
Molecular
neurobiology
laboratory,
physiology department, biological Sciences
In Chile, the number of elderly people Faculty, Universidad de Concepción.
has steadily increased, with 20% of the
population projected by 2025. This change Ethanol is the drug with highest consumption
associated with a sedentary lifestyle is levels, with effects at different levels of the
linked with an increased prevalence in Central Nervous System. Accute consumption
chronic pathologies such as Hypertension. at high levels can induce coma and death.
The national prevalence is 27.6%. The lack This molecule modulats the activity of Glycine
of pharmacological adherence constitutes receptor (GlyR), a ligand gated ion channel
one of the main problems in the control that belong to the cys-loop subfamily of ion
of the disease, considering that only channels. Recently it has been identified that
approximately 50% of the patients adhere Gbg protein as a modulator of the channel
properly. Given this problem, a Transmedial interacting with the cytoplasmic domain
Psychoeducational Program (PST) was and potentiating the activity of the channel.
developed to support primary care treatment, With determined structure of Gbg and GlyR
focused on knowledge of the disease, as well cytoplasmic domain, it has been able to
as promoting the benefits of pharmacological identify chemical entities to inhibit the etanol
treatment and a healthy lifestyle.
effects. Initially, peptides were designed and
The PST was evaluated in two CESFAM in then peptidomimetic small molecules were
the commune of Hualpén with three levels developed, like M554 and M890. These
of intervention: Group A (n = 104) through a molecules interact with Gbg and inhibit
mobile application “AFAM-Health”, Group B etanol effects in vitro and in vivo. After that
(n = 97) using video capsules and Group C (n new molecules were designed applying
= 98) as control.
bioisosteric changes in the original molecules
The average age of the 299 elderly was 72 M554 and M890. Through bioinformatic
± 7.6 years, 83.7% have as their source of technics like docking , molecular dynamics
income the retirement salary, 88.6% lives, and free energy calculations (MM-GBSA),
accompanied, 74.3% attend their health it has been identified the derivatives
checks alone and 68.6% of them walk. (R,S)-M554_3, (S)-M554_13, (R)-M554_13,
After one year of intervention, group A was M890_4 y M890_5 which interact in the Gbg
significantly more adherents (Morisky-Green hotspot surface with conserved aminoacids.
test) over time with values of 52, 73, 64 and Finally, cytotoxicity assays were performed
64% of adherents at the beginning, third, in HEK cells determining that the molecules
sixth and twelfth month, respectively. Group were not toxic for cells.
A had 4.5 ± 1.8 medications / day, 43.4%
corresponded to antihypertensives, Losartan 3. BIOGUIDED ISOLATION OF
is the main one.
SECONDARY METABOLITES
From these results, it is determined that older PRESENT IN THE MEDICINAL
adults who use the “AFAM-Health” App as a SPECIES CALDCLUVIA PANICULATA
support increase pharmacological adherence (CUNONIACEAE) WITH INHIBITORY
unlike those who only receive the traditional ACTIVITY IN VITRO ON THE ENZYME
primary care treatment.
Α-GLUCOSIDASE.
Astudillo A. 1; Céspedes C. 3; Alvear M. 4;
Massri M. 4; Iturriaga P. 1,3; Schalchi H. 1;
Hormazábal E. 1,2.
1, Centro de Excelencia en Investigación
Biotecnológica Aplicada al Medio Ambiente,
Universidad de La Frontera. 2, Laboratorio
Química Ecológica, Departamento Ciencias
88
Químicas y Recursos Naturales, Universidad
de La Frontera. Temuco. 3, Laboratorio
de Farmacoquímica y Síntesis Orgánica,
Departamento Ciencias Químicas y Recursos
Naturales, Universidad de La Frontera.
Temuco. 4, Laboratorio Bioquímica de
Suelos, Departamento Ciencias Químicas
y Recursos Naturales, Universidad de La
Frontera. Temuco.
Diabetes mellitus (DM) is a metabolic
disease characterized by an increase in
blood sugar levels. According to the World
Health Organization, 422 million adults
worldwide had DM by 2014. There are
studies based on ethnobotanical knowledge
that support the use of medicinal plants with
hypoglycemic activity, ascribing their activity
to the presence of phenolic compounds.
Mapuche medicine suggests the consumption
of “Tiaca” (Caldcluvia paniculata) as a
hypoglycemic treatment. The objective of
the research was to evaluate in vitro the
inhibitory activity of secondary metabolites
present in C. paniculata on a-glucosidase. For
this purpose, the plant material was defatted
and macerated in a hydroalcoholic solution
for 7 days. The hydroalcoholic extracts were
dried, resuspended (MeOH:H2O - 70:30)
and partitioned by liquid extraction, with
solvents of increasing polarity. The activity of
the partitions was determined by inhibition
on a-glucosidase: Aqueous solutions of
the dry extract were prepared at different
concentrations
(1-100
ug/mL),
using
p-nitrophenyl-(1,4)-a-D-glucopyranoside as a
substrate and acarbose as positive inhibition
control. The positive control of inhibition
on the enzyme (acarbose) presented an
IC50 of 1288 ug/mL. Ethyl acetate partition
presented the lowest IC50, reaching values
of 13.6 and 14.5 ug/mL for leaf and stem
respectively. The most active partition was
fractionated by column chromatography
using silica gel and eluted with solvents of
increasing polarity. The 15 groups obtained
were evaluated for their inhibitory capacity
on a-glucosidase. Group G. 9 presented better
inhibition with an IC50 of 20.2 ug/mL. The
chromatographic profiles of leaves and stems
were analyzed by HPLC, observing similar
profiles and the presumptive presence of
phenolic compounds. The results obtained
are conclusive regarding the hypoglycemic
property of C. paniculata.
4. DETECTION AND IDENTIFICATION
OF ANTIBACTERIAL COMPOUNDS
IN LIQUID FERMENTATIONS OF
FUNGUS STEREUM SP. BY HPTLCBIOASSAY-MS.
J. Avendaño-Godoy 1, 2; P. Aqueveque; M.
Aranda 2; K. Henríquez-Aedo 1.
1 Laboratory of Biotechnology and Genetics
of Food. Department of Food Science and
Technology, Faculty of Pharmacy, University
of Concepcion, Barrio Universitario s/n,
Concepcion, Chile; 2 Laboratory of Advanced
Research on Foods and Drugs. Department
of Food Science and Technology, Faculty
of Pharmacy, University of Concepcion,
Barrio Universitario s/n, Concepcion, Chile;
3 Laboratory of Microbiology and Applied
Mycology. Department of Agroindustries,
Faculty
of
Agricultural
Engineering,
University of Concepción, Campus Chillan,
Chile.
Basidiomycetes belonging to higher fungi,
offer an exciting field to obtain new structures
with high potential for medical applications.
Higher fungi have an important advantage as
producers of bioactive secondary metabolites:
they release them to liquid media. Then,
the objective of this work was to detect and
identify compounds with antibacterial activity
in liquid fermentations of fungus Stereum sp.
Pure mycelial cultures were produced from
impressions of spores of fruiting bodies,
which were then cultured in YMG medium
(glucose, malt extract, yeast extract and agar)
previously sterilized. For liquid fermentation,
the following was performed: small sections
(15-10) of 5 mm diameter plug were cut
under sterile conditions and transferred to
an Erlenmeyer flask containing liquid YMG
medium. The flasks were incubated at 20-22
°C on a Shaker orbital shaker with constant
shaking. The cultures were stopped when
abundant mycelia were observed, the glucose
source was emptied, and the pH was about
7. The liquid culture was filtered to separate
the broth and the mycelium. Bioactive
compounds were extracted with ethyl acetate
from culture media. The total extract was
concentrated to dryness in a rotary evaporator
(45 °C), weighed and stored at 4 °C. The
extract dissolved in methanol was seeded on
HPTLC plates silica gel 60 F254. Separation
was performed using the mixture of tolueneethyl acetate (3.15 : 1.85 v/v) as a mobile
phase. The extract was seeded in triplicate
by dividing the HPLTC plate into three
sections: the first section was used for the
bioassay (direct bioautography), the second
section for the chemical derivatization and
89
the third section for the mass spectrometry
analysis (MS). After chromatography, the
first section was dried and a buffer solution
was atomized. The plate was immersed in
Bacillus subtilis bacteria suspension and
incubated at 37 °C for 2 hours. Subsequently,
the plate was atomized with a solution of
methylthiazolidiphenyl-tetrazolium bromide
(MTT), incubated at 37 °C for 30 min and
finally dried completely on a heating plate
at 50 °C for 5 min. A zone of inhibition was
detected on the HPTLC plate as a colorless
zone/band on a purple background. Using the
third section of the plate (dried previously),
this bioactive/inhibitory zone was directly
eluted by means of the TLC-MS interface
coupled to the electrospray ionization
source (ESI) of a triple quadrupole mass
spectrometer. Full scan mass spectra (m/z
100-1000) were recorded in positive (ESI+)
ionization mode. The bioactive compound
tentatively corresponds to Himanimide C.
5. EFFECTS OF ARSENIC (AS)
EXPOSURE ON BLOOD-BRAIN
BARRIER AND COLONIC
PERMEABILITY IN HEALTHY YOUNG
RATS.
Barrera-Bugueño, C.1,2; Heresmann, I.1;
Quiroz, W.2; Julio-Pieper, M.1; Bravo, J.A.1.
1Grupo
de
NeuroGastroBioquímica,
Laboratorio de Química Biológica. Instituto
de Química, Facultad de Ciencias, Pontificia
Universidad
Católica
de
Valparaíso,
Valparaíso. Chile 2Laboratorio de Química
Analítica Ambiental, Instituto de Química,
Facultad de Ciencias, Pontificia Universidad
Católica de Valparaíso, Valparaíso, Chile.
Arsenic (As) is a toxic metalloid, which has
become a health burden worldwide. Growing
evidence indicates that As has harmful effects
on the central nervous system (CNS), as this
metalloid crosses the blood-brain barrier
(BBB). On the other hand, there is little evidence
suggesting that loss of intestinal permeability
might affect BBB permeability. The aim of
this study is to evaluate if oral exposure to As
affects intestinal and BBB permeability, as
this pollutant might have an impact on what
now is known as the brain-gut axis. Methods:
Female Sprague–Dawley rats (PND35) where
given 10 ppm of NaAsO2 in the drinking water
for 24h (n=5), and compared to control rats
(n=6). At 24h the following samples were
collected: brain, colon, lung, stool and liver
tissues. Each sample was lyophilized and then
microwave digested in order to determinate
total As concentration by HPLC-HG-AFS.
Additionally, colonic permeability to FITC-
90
dextran 4.4kDa (FD4) was evaluated ex
vivo for 120 and 180 min by everted gut sac
technique. Results: Gut permeability to FD4
is increased in animals exposed for 24h to 10
ppm of NaAsO2 in comparison to controls.
In addition, the metalloid concentration was
higher in every studied tissue of exposed rats,
in comparison to controls. In the brain, As was
found in hypothalamus and cerebral cortex.
This data suggest that As is able to cross the
BBB and increases gut permeability in the rat,
an effect that might lead to alterations in BBB.
In conclusion, a toxic pollutant such as As
might cause alterations in the brain-gut axis,
effects which gives a novel approach in the
study of As toxicity.
6. TWO CONSERVED ALPHAHELICES IN CORTICOTROPHIN
RELEASING FACTOR BINDING
PROTEIN CONTAINING A
HYDROPHOBIC PATCH
DETERMINES ITS SORTING TO THE
REGULATED SECRETORY PATHWAY.
Bastías C.P.1; Blanco E.H.2; Lagos C.F.3;
Gysling K.1
1: Depto de Biología Celular y Molecular,
Facultad de Ciencias Biológicas, Pontificia
Universidad Catolica de Chile. 2: Universidad
de Antofagasta. 3: Chemical Biology &
Drug Discovery Lab, Escuela de Química y
Farmacia, Facultad de Medicina y Ciencia,
Universidad San Sebastián.
Corticotrophin releasing factor binding
protein, (CRF-BP) is a 37 kDa glycoprotein
that binds CRF with high affinity. CRFBP in the periphery controls CRF levels in
plasma during pregnancy. In the central
nervous system, CRF-BP facilitates the traffic
of CRFR2alfa acting as an escort protein.
Previously, it has been shown that CRF-BP
enters the regulated secretory pathway (VSR).
However, the sorting signal(s) are presently
unknown. We decided to determine the sorting
signal(s) of CRF-BP to VSR. We used NPS @,
an in silico secondary structure prediction
tool and PEPWHEEL to draw predicted alpha
helixes and the in silico modeling of CRF-BP
protein structure. Additionally, we did studies
of sorting of chimeras containing the putative
sorting signals in PC12 cells over-expressing
the selected chimeras. In silico analysis and
modeling of CRF-BP protein structure showed
the presence of three alpha-helix domains,
(50-74), (128-149), (229-251). The alphahelixes domain (50-74) and (229-251) in
CRF-BP is highly conserved among different
mammalian species and has a hydrophobic
patch characteristic of other sorting domains
to the VSR. The results show that the alphahelix domain (50-74)-CRFBP is capable
of restore the sorting of a chimeric variant
of proCART precursor, without its sorting
domain to the VSR. Furthermore, the presence
of the alpha-helix domain (50-74)-CRF-BP in
the chimeric variant of proCART allowed its
secretion triggered by a depolarizing stimulus.
Our results show that the preserved alphahelix domain (50-74)-CRF-BP, present in the
amino terminal of CRF-BP, is responsible for
its destination to the VSR. Further studies are
needed to evaluate if the other alpha-helix
domains also play a role in the sorting of CRFBP to the VSR.
7. TTAGP 1.0: A COMPUTATIONAL
TOOL FOR THE SPECIFIC
PREDICTION OF TUMOR T CELL
PEPTIDES.
Beltrán J.F.; Herrera L.; Farías J.G.
Department of Chemical Engineering, Faculty
of Engineering and Science, Universidad de
La Frontera.
Endocrinas, Facultad de Ciencias Químicas y
Farmacéuticas, Universidad de Chile.
The ovary is an endocrine organ which is
regulated by hormonal and neural signals.
It is well known that noradrenaline controls
ovarian steroidogenesis and folliculogenesis.
Its source comes from sympathetic neurons
that innervate the ovary. In addition, evidence
suggests that acetylcholine enhances follicular
development and its intraovarian production
would be in granulosa cells which express
choline acetyltransferase (ChAT), vesicular
acetylcholine transporter (VAChT) for storage
and acetylcholinesterase. Besides, cholinergic
muscarinic receptors (M1, M3, M5) are
expressed in ovarian follicles. However, it is
not known how this intraovarian cholinergic
system is regulated. In vitro studies had
shown that human granulose cells incubated
with neuronal growth factor (NGF), a
neurotrophin produced by ovary, increased
ChAT. The objective of the present work was
to determine if NGF enhances acetylcholine
production in the rat ovary. 26 days old rats
ovaries were incubated with 100 ng/mL NGF
during 3 and 24 hrs. 7 days old rats were
treated with 50 mg/Kg guanethidine during
3 weeks to induce a chronic endogenous NGF
increment and, after 3 months, ovaries were
obtained. We measured mRNA levels by qRTPCR, acetylcholine levels by fluorometric
assay, NGF and noradrenaline by ELISA
kit, and NGF by western blot. After 3 hrs,
NGF produced an increment in ChAT and
VAChT mRNA levels, but a decrease in
acetylcholine in medium. After 24 hrs, we
found a modest but constant increase in
acetylcholine
production.
Guanethidine
treatment didn’t induce an endogenous NGF
increment despite that noradrenaline levels
decreased. Altogether these data suggest that
NGF regulates intraovarian acetylcholine
production and storage ex vivo. Further
research is needed to elucidate if a longer
time of NGF stimulation is needed to better
visualize the neurotransmitter.
Nowadays, cancer is considered a global
pandemic and millions of people die every year
because this disease remains a challenge for
the world scientific community. Even with the
efforts made to combat it, there is a growing
need to discover and design new drugs and
vaccines. Among these alternatives, antitumor
peptides are a promising therapeutic solution
to reduce the incidence of deaths caused by
cancer. In the present study, we developed
TTAgP, an accurate bioinformatic tool
that uses the random forest algorithm for
antitumor peptide predictions, which are
presented in the context of MHC class I.
The predictive model of TTAgP was trained
and validated based on several features of
922 peptides. During the model validation
we achieved sensitivity = 0.89, specificity
= 0.92, accuracy = 0.90 and the Matthews
correlation coefficient = 0.79 performance
measures, which are indicative of a robust
model. TTAgP is a fast, accurate and intuitive
software focused on the prediction of tumor T
9. AMPHETAMINE AND TEMPOL
cell antigens.
MODULATE EXTRACELLULAR
CONCENTRATION OF DOPAMINE
8. NGF INCREASED CHOLINE
AND THE PHOSPHORYLATION
ACETYL TRANSFERASE AND THE
LEVEL OF THE DOPAMINE
VESICULAR ACETYLCHOLINE
TRANSPORTER EXPRESSION IN RAT TRANSPORTER.
Blanlot C.1; Zegers J.A.1; Yarur H.E.1; Gysling
OVARY EX VIVO.
K.1.
Benitez A. 1; Lara H.E. 1.
1,
Laboratorio
de
Neurobioquímica, Pontificia Universidad Católica de Chile.
Departamento
de
Bioquímica
y
Biología Molecular, Centro de Estudios Amphetamine (AMPH) is a highly
Neurobioquímicos
para
Enfermedades reinforcing, widely abused stimulant drug
91
that increases dopamine extracellular levels
in the mesocorticolimbic system, in neurons
projecting from the Ventral Tegmental
Area to Nucleus Accumbens and Prefrontal
Cortex. AMPH-stimulated efflux of dopamine
through the dopamine transporter (DAT) only
happens if DAT is previously phosphorylated.
Some kinases that act on DAT are PKC, ERK
and PKA and their activity is modulated by
different signaling pathways. The increase
in the production of reactive oxygen species
(ROS) after the intake of AMPH, besides
producing brain damage, can modulate the
action of these kinases. Scavenging ROS
may be a way to avoid the toxic effects of
oxidative stress induced by AMPH. Tempol
is an antioxidant that has neuroprotective
activity, diminishing the presence of oxidative
markers in the brain. It has been shown that
Tempol interferes with the development of
behavioral sensitization induced by cocaine.
We evaluated the effect of Tempol and AMPH
in the phosphorylation level of DAT using
rat Nucleus Accumbens synaptosomes that
were stimulated with AMPH, Tempol and
Tempol before AMPH. We also measured the
concentration of dopamine in the medium
where the synaptosomes were incubated by
microdialysis and electrochemical detection.
Western Blot analysis showed that AMPH
increased the phosphorylation of DAT and
that the presence of Tempol avoided this
increase. The extracellular concentration of
dopamine in the presence of AMPH alone
was significantly increased. No changes were
observed in the presence of Tempol alone.
Interestingly, AMPH in the presence of
Tempol induced a lower increase in dopamine
concentration. Taken together, these findings
suggest a role of ROS in the mechanism
by which AMPH increases dopamine
extracellular levels in Nucleus Accumbens.
10. COLCHICINE COMPETITIVELY
ANTAGONIZES THE ALPHA
3 SUBUNIT OF THE GLYCINE
RECEPTORS.
Burgos C.F. ; Lara C.O. ; Riquelme C. ; San
Martín V. ; Flaig D. ; Soto P. ; Aguayo L.G.
; Fuentealba J. ; Castro P.A. ; Guzmán L.
; Muñoz-Montecino C. ; Yévenes G.E. ;
Moraga-Cid G.
Department of Physiology, Faculty of
Biological Sciences, University of Concepción,
Chile.
Glycine receptors (GlyRs) are anion-selective
neurotransmitter-gated
ion
channels,
member of the pentameric Ligand Gated
Ion Channels (pLGICs) family. GlyRs are
92
essential players in the physiology of the
central nervous system and impairment of its
function underlie many neurological diseases,
including epilepsy, autism, chronic pain,
anxiety and schizophrenia among others.
The function of GlyRs containing the alpha1
or alpha2 subunits, can be competitively
inhibited by colchicine independently of
microtubule depolymerization. Interestingly,
a recent report showed that colchicine binds
directly to the GlyRs containing the alpha3
subunit, suggesting that the alpha3 GlyRs
mediated the suppression of the inflammatory
pain exerted by colchicine. However, the
functional effects on the alpha3 GlyRs function
elicited by colchicine are still undefined.
Using electrophysiological techniques and
molecular docking simulations, here we
show that colchicine is an inhibitor of the
alpha3 GlyR function. Colchicine, elicited
concentration-dependent inhibitory effects
on alpha3 GlyRs at micromolar range.
Single-channel recordings show that the
colchicine inhibition is associated with a
decrease in the open probability of the ion
channel. Molecular docking assays suggest
that colchicine preferentially bound to the
orthosteric site in an agonist-free, closed state
of the ion channel. Our results thus define the
pharmacological modulation of colchicine on
alpha3 GlyRs.
11. CHARACTERIZATION OF THE
NEUTROPHIL/LYMPHOCYTE RATIO
IN A SAMPLE OF PATIENTS WITH
RHEUMATOID ARTHRITIS.
Cáceres B. 1; Flores D. 1; Saez K. 2; Ormazabal
V. 3; Castro I. 4,5; Nova-Lamperti E. 1;
Lamperti L.1.
1Facultad de Farmacia,
2Facultad de Ciencias Físicas y Matemáticas,
3Facultad de Ciencias Biológicas, 4Facultad
de Medicina, Universidad de Concepción
y 5Hospital Guillermo Grant Benavente,
Concepción.
Introduction: Rheumatoid arthritis (RA) is
a systemic disease, still unknown etiology
and autoimmune character characterized
by chronic inflammation in the synovial
membrane. The activity of the disease in RA is
determined with DAS28 index, which allows
evaluating the efficacy of the drug therapy
administered. Monitoring the evolution of the
disease activity is essential to avoid disability
in long term. The increase in the neutrophil/
lymphocyte ratio (NLR) has been described as
a parameter of inflammation associated with
a predominance of neutrophils and a decrease
in lymphocytes. This NLR relationship has
proven to be a good predictor of inflammation
in chronic diseases such as diabetes and
cancer. Objective: To characterize the NLR
relationship in patients with RA. Methods:
11 AR patients and 11 controls of similar
ages and same sex were recruited and signed
an informed consent approved by Ethical
Scientific Committee. A blood sample was
performed on a Sysmex XS-1000i device. The
absolute values of neutrophils were divided
by lymphocytes. The values of DAS28 and
erythrocyte sedimentation rate (ESR) were
analyzed. Statistical tests were used for
variables. Results: The NLR was higher in
RA patients, 2.72±0.68 versus controls with
1.53±0.38 p<0,0001. The classification of
disease activity according to DAS28 shown
that NLR was 3.69 ± 0.25 for high activity,
2.49 ± 0.29 for moderate and 2.23 ± 0.32
for low and the coefficient of HSV-NLR
correlation was 0.34 and DAS28-NLR was
0.815. Conclusion: The NLR was higher in AR
patients than in the control group. It is shown
that NLR correlates positively with DAS28
and HSV. In addition, the NLR was higher for
patients with high disease activity, compared
to moderate and low.
12. GPER ANTAGONISM OF
THE POTENT ANTHOCYANININDUCED VASODILATION AND NO
PRODUCTION IN A VASCULAR BED
AND ISOLATED ENDOTHELIAL
CELLS.
Calfío C.; Huidobro-Toro JP.
Laboratorio de Farmacología, Facultad de
Química y Biología y Centro de Nanociencia y
Nanotecnología de la Universidad de Santiago
de Chile, Santiago, Chile.
Anthocyanins are colored water-soluble
flavonoids present in red-bluish berries,
fruits and vegetables. Flavonoids conserve
structural similarities with other molecules
such as steroids. Genistein is recognized as a
natural phytoestrogen with potent estrogenic
activity. Considering the potent vasodilation
elicited by the glycosylated or anthocyanin
aglycones is strictly endothelium-dependent,
we proposed that the anthocyanin-induced
vasodilation is mediated by the estrogen
receptor coupled to a trimeric G protein
(GPER) or estrogen receptor alpha (ERa),
both associated to a rapid, non-genomic
mechanism. We evaluated the vascular
response induced by the anthocyanin
delphinidin and its 3-O-glucoside derivative
(D3G) in pre-contracted mesenteric vascular
beds in the presence or absence of 1μM-G36, a
purported GPER receptor antagonist, or 1μM
fulvestrant a recognized ER α/β antagonist.
Also, we quantified the NO production elicited
by anthocyanidins by chemiluminescence.
Both and D3G elicit concentration-dependent
vasodilation as potent as acetylcholine used
as a standard control and 10 to 100 times
more potent than 17-beta-estradiol (E2) and
genistein respectively. The anthocyanins
activity is dependent on the endothelium
and eNOS enzymatic activity (97-98%, p
<0.001). G36 significantly reduced the
anthocyanins vasodilation (p <0.01) such as
G-1, GPER agonist (p <0.001). However, the
anthocyanins response was not blocked by
fulvestrant, which decreased E2 activity by
28%. The perfusion of the mesenteric vascular
bed with 100 nM or 1 μM of delphinidin
and D3G, or its application to endothelial
cells culture, increased NO production
compared to the controls. We conclude that
anthocyanins, glycosylated or not, induce
a potent vasodilator response mediated by
NO production that may be linked to GPER
activation at the vascular level. In addition,
the anthocyanin mechanism is rapid and nongenomic in nature.
13. MOLECULAR
CHARACTERIZATION OF HEAD AND
NECK SQUAMOUS CELL CARCINOMA
(HNSCC) 3D MODEL AND
METASTATIC CAPACITY EFFECTS OF
OLD DRUGS WITH NEW ANTITUMOR
ACTIVITY.
Carrasco J.1; Martínez D.1; Jara J.1.
1, Laboratory of Pharmacology, Faculty of
Dentistry, Institute for Research in Dental
Sciences (ICOD), Universidad de Chile.
Head and neck squamous cell carcinoma
(HNSCC) has an incidence in worldwide of
more than 350000 people and the mortality
associated with this cancer is 50%. Although
the treatment for these patients generally
consists of surgical, pharmacological and
radiation therapies, survival at 5 years is only
53%. One of the reasons for high mortality
and low survival is due to the presence
within tumor mass a subset of cells called
cancer stem cells (CSCs), which represent
the most tumorigenic subpopulation. This
population grows like spheroids and in
its center has hypoxic cells that are highly
resistant to chemotherapies. Here, the goal
was to obtain in vitro spheroid CSC from
HNSCC (HNCSCs) and determine expression
protein level involving on metabolism,
hypoxia, autophagy, and antitumor target, in
addition to assess the effects of Itraconazole
and hydroxychloroquine on invasion capacity
on spheroid cultures. Spheroid formed from
93
Cal27 and HEp-2 cell lines using culture
selecting conditions and protein expression
levels was determine by immunoblotting.
Spheroid was treated with Itraconazole and
hydroxychloroquine and then seeded over
matrigel to Boyden chamber 3D invasion
assays. Successful formation of spheroid
from the Cal27 and Hep-2 cell lines, where
needed 4500 and 3500 cells respectively to
obtain sizes greater than 300 μm. Spheroids
were also subjected to hypoxic conditions
with oxygen concentrations below 5%,
VDAC, PDK1, Hexokinase II, Hif-1 and LC3B
protein expression levels were different
between cell line and monolayer or spheroid
cultures. HNCSCs exposure to Itraconazole
and hydroxychloroquine modulate invasive
capacities, compared with control conditions.
Here we showed that protein expression
patterns are different between monolayer
and spheroid cultures and the effects of two
different drugs on metastatic capacity of
HNCSCs.
14. MESENCHYMAL STEM
CELL SECRETOMES (MSCSS)
ADMINISTRATION IMPROVES
BEHAVIOURAL DEVELOPMENT,
MOTOR AND COGNITIVE
IMPAIRMENTS INDUCED BY
PERINATAL ASPHYXIA.
Carril J.1; Obrecht S.2; Contreras N.2; Araya
M.1; Monzón E.1; Farfán N.1; Alvarado R.1;
Tapia-Bustos A.1;Vásquez R.1; Bustamante
D.1; Quintanilla M.E.1; Ezquer F.3; Israel
Y.1; Valdés J.L.2; Herrera-Marschitz M.1;
Morales P.1,2.
1Molecular & Clinical Pharmacology Program,
ICBM, 2Department of Neuroscience, Faculty
of Medicine University of Chile. 3Center for
Regenerative Medicine, Faculty of MedicineClínica Alemana, Universidad del Desarrollo,
Santiago, Chile.
Perinatal asphyxia (PA) induces deficits
in neurological reflexes, development,
motor coordination, emotional behaviour
and cognition. At present, no treatment
significantly attenuates or prevents these
sequelae. Mesenchymal stem cells (MSCs)
have been proposed as a therapeutic tool for
several CNS diseases, since MSCs display
remarkable antioxidant, anti-inflammatory
and repairing features (neurogenesis,
synaptogenesis, myelination). MSCs exert
paracrine effects by secreting a combination
of nano-vesicles and soluble factors
(referred to as secretomes). Preconditioning
MSCs with pro-inflammatory cytokines
(TNFalfa plus INFgamma) or hypoxia-like
94
environment (deferoxamine) improves their
effectiveness. The aim of this study was to
determine whether intranasal administration
of secretome derived from preconditioned
human MSCs prevents the (i) behavioural
development, (ii) motor and (iii) cognitive
disabilities resulting from PA. PA was induced
by immersing foetuses-containing uterine
horns into a water bath at 37 °C for 21 min.
Two hours after birth and at postnatal day (P)7
MSCSs (6 ug/16 ul, obtained from 1x106
preconditioned-MSCs) or 16 ul of vehicle
were administered intranasally to asphyxiaexposed or control rats. Neurobehavioral
development was evaluated by monitoring
the righting reflex (at P1, P4, P7 and P14);
negative geotaxis (P7, P14 and P21), and
cliff aversion (P7, P14 and P21). Locomotor
activity (P7) and motor coordination (P60)
were evaluated by open field and rotarod,
respectively. Anxiety (P30), by open field and
novel object recognition memory (P30). All
the PA induced effects were positively affected
by MSCSs treatment, including improvements
in: (i) the remarkable developmental delay in
the performance of righting and cliff aversion
reflexes; (ii) the decrease in locomotor
activity and deficits in motor coordination
and balance; (iii) an increase in anxiety and
novel object recognition memory deficits.
15. INHIBITORY EFFECT OF
COUMARINS DERIVATIVES ON
THE VIABILITY OF HELICOBACTER
PYLORI ATCC 43504 AND
RECOMBINANT CARBONIC
ANHYDRASE ACTIVITY.
Carvajal, R.C. 1,2,3.; García, A1.; Zúñiga,
F3.; Alarcón, J5; Ormazábal, V.4 & PasteneNavarrete, E. N.5.
1Laboratorio de Patogenicidad Bacteriana,
Departamento de Microbiología, Facultad
de Ciencias Biológicas, Universidad de
Concepción. 2Laboratorio de Farmacognosia,
Departamento de Farmacia, Facultad de
Farmacia, Universidad de Concepción.
3Departamento de Biología Clínica e
Inmunología,
Facultad
de
Farmacia,
Universidad de Concepción. 4 Departamento
de Farmacología, Facultad de Ciencias
Biológicas, Universidad de Concepción.
5Laboratorio de Síntesis y Biotransformación
de productos Naturales, Departamento
de Ciencias Básicas, Facultad de Ciencias,
Universidad del Bio-Bío.
Helicobacter pylori (Hp) is a Gram negative
pathogen that affects more than 50% of the
world population, and it is responsible for
different gastric pathologies. Since increasing
antibiotics resistance cause serious failure in
Hp eradication therapy, new pharmacological
targets need to be identified to affect the
viability of this bacteria. Carbonic anhydrase
(CA, EC 4.2.1.1) is an interesting new Hp
target since it is involved in neutralizing the
acid pH of the human stomach cooperatively
with urease. It has been described that
specific coumarin derivatives can inhibit CA.
Besides, several coumarins exhibit a strong
antibacterial activity. Objective: to clone
and characterize carbonic anhydrase from
the highly aggressive strain Hp ATCC 43504
and to determine the coumarins derivatives
effects on both, the recombinant protein and
Hp viability. Methodology: a-CA was cloned,
expressed in cell line HEK 293 and purified.
Recombinant α-CA was characterized by
esterase activity and protonography. We
determined the inhibitory effect of coumarins
derivatives against recombinant α-CA by
esterase activity and on Hp to determine its
MIC and MBC. Results: A 50 kDa functional
recombinant Hp a-CA was cloned that forms a
dimer structure in solution. The recombinant
Hp a-CA it is closely related to F78 strain,
with a 99.1% of identity. Esterase activity was
determined, complemented by protonography
analysis, and kinetic parameters. Overall, six
coumarins showed inhibitory activity against
Hp, with MIC ranging from 125 ug/mL to 15
ug/mL, and two coumarins were inhibitors
of the recombinant protein. Conclusion: The
characterization of this highly aggressive Hp
CA strain and the determination of inhibitory
effects of coumarins, makes them a potential
candidate for Hp therapy and eradication
of several genes. Potential HIF-1a activated
genes were identified in the rat genome
following hypoxic conditions, by extracting
promoter sequences of Rattus Norvegicus
from the UCSC database Genome Browser.
8762 genes with the HIF-1a binding sequence
(5`-RCGTG-3`) were identified using the “R”
software. These genes were introduced to the
Gen Ontology platform for performing an
enrichment analysis, selecting the following
processes linked to PA: (i) Hypoxia (865
genes); (ii) Glucose Metabolism (330 genes);
(iii) Neurogenesis (1243 genes); (iv) Apoptosis
(814 genes); (v) Angiogenesis (165 genes),
and (vi) Regulation of Gene Expression
(2076 genes). 865 hypoxia-associated genes
were further selected and compared with
experimental data by ChIP-Seq, with 772 and
98 genes from human and zebrafish genomes,
respectively, identifying 79 genes for the
three species. The 8762 genes were then
analysed by the Kyoto Encyclopedia of Genes
and Genomes (KEGG) platform, selecting
the HIF-1 pathway, identifying 47 genes.
The 79 genes filtered for human, zebrafish
and rat were compared with the 47 genes
obtained by the KEGG platform, yielding
12 genes. Finally, 12 genes were compared
with 47 genes referred by the literature to
be associated to PA, identifying 5 genes: (i)
Bcl2; (ii) Hif-1a; (iii) Ldha; (iv) Pdk1, and
(v) Vegfa. The pharmacological inhibition of
HIF-1a to establish the gene expression levels
of candidate genes in an in vivo model of PA
is studied, providing a proof-of-principle for
the participation of HIF 1a on the regulation
of gene expression following PA.
16. IDENTIFICATION OF POTENTIAL
CANDIDATE GENES RELATED TO
HYPOXIA INDUCIBLE FACTOR 1
ALPHA (HIF-1 ALPHA) INVOLVED IN
PERINATAL ASPHYXIA IN RATS.
Emmanuel Casanova 1; Pablo Baéz 2, Luis
Valenzuela 2, Rodrigo Assar 2, Andrea Tapia
1, Paola Morales 1, Katherine Marcelain
Cubillos 2, Mario Herrera-Marschitz 1.
1 Programme of Molecular & Clinical
Pharmacology, ICBM, Medical Faculty,
University of Chile and 2 Cancer Genomics
Laboratory,
Basic-Clinical
Oncology
Department, Medical Faculty, University of
Chile, Santiago, Chile.
17. DEVELOPMENT AND
CHARACTERIZATION OF A
NANOPARTICULATE SYSTEM
FOR NASAL ADMINISTRATION OF
CURCUMIN DERIVATIVE.
Salas M.J.1; Castillo E.2; Gómez C.1; von
Plessing C.1. 1 Departamento de Farmacia,
Facultad de Farmacia, Universidad de
Concepción, Concepción, Chile. 2 Facultad
de Medicina y Ciencia, Universidad San
Sebastián, Concepción, Chile.
Perinatal asphyxia (PA) is characterized
by interruption of oxygen bioavailability at
birth. Hypoxia implies HIF-1 alpha (HIF-1a)
activation, a key sentinel protein, which, upon
translocation to the nucleus, binds to response
elements (HREs), promoting transcription
In the present work, the nanoparticles (NPs)
of chitosan were elaborated and characterized
by means of the ionic gelation method,
using for this the magnetic stirring and as a
crosslinking agent sodium tripolyphosphate
(TPP) and / or a derivative of thiamine. The
optimization of the NPs of the chitosan the
response has been carried out by means of
a central composite design, in which the
different parameters have been evaluated as
95
the relationship between the mass between
the chitosan/derivative of thiamine/TPP,
different speeds and times of agitation, pH
of the solutions and order of addition of the
components, with respect to the average
diameter, polydispersity and zeta potential
of the NPs. The physical characteristic of the
best formulation gave spherical shapes, with
an average diameter of 136,12 ± 3,60 nm, a
polidispersion index of 0,42 ± 0,02 and a zeta
potential of +31,06 ± 0,97 mV for white NPs.
The NPs loaded with curcumin have a stability
of 90 days at 5 °C ± 3 °C and a stability of 6
days at 25 °C ± 2 °C with 60 % ± 5 % relative
humidity. A cell viability study was carried
out, using the THP-1 cell line, the results
indicated that the NPs with a concentration
of 11 μg/mL curcumin equivalents, the
viability is higher than 80 %. Finally, the
encapsulation efficiency of curcumin in the
final NPs formulation was 24,4 ± 4,48 %, with
a load of 455 ± 0,06 ug%, with a size, PDI and
zeta potential of 218,3 nm, 0,134 and +31,9
mV, respectively. Keywords: nanoparticles,
chitosan, curcumin, cell viability.
18. RESOLVIN D1 INCREASES
COLLAGEN-1 SYNTHESIS ON
RAT CARDIAC MYOFIBROBLAST
THROUGH ALX/FPR2 RECEPTOR
ACTIVATION.
Esteban Castro-Carrasco1; José Miguel Lillo1;
Guillermo Díaz-Araya1,2.
1Laboratory of Molecular Pharmacology,
Department
of
Pharmacological
and
Toxicological Chemistry, Facultry of Chemical
and Pharmaceutical Sciences, University of
Chile, Santiago, Chile. 2Advanced Center
for Chronic Diseases (ACCDiS), FAculty of
Chemical and Pharmaceutical Sciences &
Faculty of Medicine, University of Chile,
Santiago, Chile.
that the CF-to-CMF differentiation increases
ALX/FPR2 protein levels, and consequently,
ALX/FPR2 activation by RvD1 enhances
collagen-1 synthesis. Methods: Secondary
culture of adult rat CF was starved for 24
hours, stimulated with TGF-b1 (10 ng/mL)
for 72 hours on fetal bovine serum (FBS)
1% to induce CMF differentiation and then
treated with RvD1 at different intervals
in presence and absence of PD98059,
LY294002 (ERK ½ and AKT inhibitors) and
WRW4 (ALX/FPR2 antagonist). The proteins
levels of p-EKR ½, p-AKT and collagen-1
were measured by western blot. Results: After
CMF differentiation, it was found an increase
of ALX expression. On CMF, RvD1 activated
the ERK ½-AKT pathways and enhanced the
expression of collagen-1. These effects were
blocked by PD98059, LY294002 and WRW4.
On the other hand, RvD1 did not decrease
α-SMA protein levels, which suggests that
it does not reverse CMF differentiation.
Conclusions: Our results suggest that the CFto-CMF differentiation increases the protein
levels of ALX/FPR2, and that RvD1 enhances
collagen-1 synthesis through ERK ½ and AKT
pathways.
19. PROCYANIDINS-RICH EXTRACT
NANOEMULSIONS O/W AS A
POTENTIAL ANTITUMORAL TOOL:
EVALUATION IN MELANOMA CELL
LINE.
Cerda-Opazo P. 1,2,3; Gotteland M. 4;
Oyarzun-Ampuero F. 2, 3; Garcia L. 1, 3.
1, Depto. Bioquímica y Biología Molecular,
Facultad
de
Ciencias
Químicas
y
Farmacéuticas, Universidad de Chile,
Santiago, Chile; 2, Depto. Ciencia y Tecnología
Farmacéutica, Facultad de Ciencias Químicas
y Farmacéuticas, Universidad de Chile,
Santiago, Chile; 3, Advanced Center for
Chronic Diseases (ACCDiS), Santiago, Chile;
Background: Cardiac fibroblast (CF) to 4, Depto. de Nutrición, Facultad de Medicina,
cardiac myofibroblast (CMF) differentiation Universidad de Chile, Santiago, Chile.
is mainly mediated by TGF-b1 released from
immune cells by angiotensin II (Ang II) effect, During the last 10 years, the incidence of
or in vitro conditions due to mechanical melanoma in Chile has incremented 27.2%.
stress. CMF are able to produce extracellular Approximately 80% of all skin cancer-related
matrix proteins, mostly collagen, in the deaths are attributed to melanoma and longscar formation proccess. Also, in the CF-to- term survival of patients is only 5%. This
CMF process it has been demonstrated the type of cancer is considered a multifactorial
upregulation of Kinin-B1 and AT1R receptor, disease related to environmental interaction
among others. However, the presence of and genetic susceptibility that trigger a
Resolvin D1 (RvD1) receptor, ALX/FPR2, constitutive activation of several signaling
has not been elucidated. RvD1 is an anti- pathways, promoting proliferation and
inflammatory lipidic mediator that regulates survival of tumoral cells. Current therapies
matrix proteins like a-SMA and collagen in cause a considerable number of side effects,
various cell types, yet there is not evidence of still representing a global human health issue.
this effect on CMF. Purpose: To demonstrate Recently, the use of polyphenols has been
96
reported for both prevention and treatment
of melanoma. Some evidence demonstrates
that different extracts of polyphenols (grape,
pomegranate, among others) are high
potential candidates to treat various types
of cancer. Of note, the main limitations of
these molecules are the low bioavailability
and the necessity of a high concentration
dose to cause a biological effect. The aim of
this study was to utilize the nanotechnology
to encapsulate procyanidins and to evaluate
its antitumoral activity in B16F10 melanoma
cell line. To achieve the proposed objective,
an avocado peel procyanidin-rich extract was
used and cellular viability, proliferation and
migration in B16F10 cells were evaluated.
Nanoemulsions were elaborated by solvent
evaporation and were in a nanometric range
of 170 ± 2 nm and showed low polydispersity
(between 0.1 and 0.2). The nanoformulations
showed negative zeta potential (−44 ± 4 mV),
realizing a stable system. The administration
of procyanidin-rich extract nanovehicles was
more efficient in reducing cellular viability,
proliferation and migration compared to free
extract. Altogether, our results suggest that
encapsulation of procyanidins significantly
improves its effect on melanoma therapy.
20. MATERNAL HIGH-FAT
DIET CONSUMPTION DURING
PREGNANCY AND LACTATION
IMPAIRS THE INHIBITORY
SYNAPTIC TRANSMISSION OF CA1
REGION OF HIPPOCAMPUS OF THE
YOUNG OFFSPRING.
Cerna C.1, Valero V. 1,2, Santander O.1,3
García F.1,3, Guiffa F.1,4, Cruz G. 1 and
Fuenzalida M. 1*
1Centro de Neurobiología y Fisiopatología
Integrativa (CENFI), Instituto de Fisiología,
Facultad de Ciencias, Universidad de
Valparaíso, Valparaíso, Chile. 2Programa de
Doctorado en Ciencias e Ingeniería para la
Salud, Universidad de Valparaíso, Valparaíso,
Chile. 3Programa de Doctorado en Ciencias
Mención neurociencia, Universidad de
Valparaíso, Valparaíso, Chile.4Programa de
Magister en Ciencias, mención neurociencia,
Universidad de Valparaíso, Valparaíso, Chile.
Before birth and early in life the brain
development is acutely sensitive to its
environment. Experimental and clinical
data indicate that maternal obesity can
predispose the offspring to suffer metabolic
and neuronal alterations. Most studies
have focused on the functional relationship
between maternal obesity and hypothalamus
alterations. However, the impact of maternal
nutrition on other brain areas remains
elusive. Recently, it has suggested that
fetal exposure to maternal obesity causes
decreased neurogenesis and impaired
hippocampal learning. The hippocampus is
important for learning and memory, and its
development is sensitive to the metabolic
environment in utero. In a model of maternal
obesity induced by a high-fat diet (HFD,
60Kcal in fat) consumption we study whether
this adverse prenatal environment impairs
the hippocampal synaptic transmission. In
offspring mice, during adolescence, using
electrophysiological recordings in the CA1
area of mice hippocampus, we observe
that maternal HFD consumption increases
the frequency and amplitude of Inhibitory
postsynaptic currents. This increase in
inhibition level onto pyramidal neurons
could have important consequences in the
excitation/inhibition balance, being able to
modify the hippocampal cognitive function in
juvenile offspring mice.
21. PHARMACOKINETIC
VARIABILITY IN PATIENTS WITH
KIDNEY TRANSPLANTATION,
TREATED WITH CYCLOSPORINE.
Cerpa L.1,4; Rodríguez M.S.2; Corvalán F.1;
Contreras S.1; Cayún JP.1,4; Llull G. 1,3;
Sandoval C.1; Farías N.2; Alvarez C.2; Plubins
L.2; Ñuñez G.2; Castro L.2; Espinoza R.2;
Chavez R.2; Goic I.2; Mur P.2; Cordero C.2;
Acuña P.2; Moya C.2; Varela N.1,4; Quiñones
L.1,4.
1. Laboratory of Chemical Carcinogenesis
and Pharmacogenetics (CQF), Department
of Basic and Clinical Oncology, Faculty
of Medicine, University of Chile. 2. Adult
Nephrology Unit, San Juan de Dios Hospital.
3. Clinical Laboratory, San Juan de Dios
Hospital. 4. Red Latinoamericana para
la Implementación y Validación de Guías
Clínicas Farmacogenómicas (RELIVAFCYTED).
Introduction: Cyclosporine (CsA) is an
immunosuppressive drug, used to prevent
organ rejection in transplant patients.
However, this drug is characterized by
having a narrow therapeutic range and high
inter and intra-individual pharmacokinetic
variation. There are some genetics variants
involved in pharmacokinetic variability,
both at the level of cyclosporine absorption
and metabolization. However, genetic
variants of relevant impact are not yet
identified. Objective: to evaluate the genetic
variants involved in the pharmacokinetics of
cyclosporine and to establish an association
97
between the pharmacogenetic profile, and the
safety and efficacy of the treatment during
the first three months after transplantation.
Methodology: One hundred and seven
patients (107) with kidney transplants from
San Juan de Dios Hospital (Project No.
028-13) were retrospectively included and
were genotyped for the genetic variants
CYP3A4
*1B
rs2740574,
CYP3A4*22
rs25599367, CYP3A5*3 rs776746, POR*28
rs1057868, MDR1 3435 rs1045642, MDR1
2677 rs2032582 and MDR1 1236 rs1128503.
Genotypic frequency were associated with
blood concentrations of cyclosporine (CsA),
creatinine value and blood pressure within the
first three months post-transplant. Results:
Heterozygous patients for CYP3A4*1B had a
lower creatinine value from the second posttransplant week. In relation to cyclosporine
levels, an increase was observed, in patients
with at least one altered allele for CYP3A5*3
from the second post-transplant week.
Conclusions: The results show that genetic
variants can account for variations in the
pharmacokinetic parameters of cyclosporine,
which affect the efficacy and safety of
cyclosporine treatment. It is expected
that, based on the results found, a genetic
predictive panel of response to CsA will be
setted-up to be used before transplantation.
22. ANTIFUNGAL AND ANTIBIOFILM
EFFECT OF OREGANUM VULGARE
ESSENTIAL OIL AGAINST CANDIDA
ALBICANS AND NON-ALBICANS.
Cid, C.1; Mûller, A.; Díaz, M. 2; Jara, J.;
Molina-Berríos, A.1.
1, Laboratorio de Farmacología, Instituto
de Investigación en Ciencias Odontológicas,
Facultad de Odontología, Universidad de
Chile. 2, Laboratorio de Nanobiomateriales,
Instituto de Investigación en Ciencias
Odontológicas, Facultad de Odontología,
Universidad de Chile.
Oral candidiasis is the most common fungal
infection in humans. The most frequent
causative agent isolated is Candida albicans (C.
albicans), but the number of resistant strains
such as Candida krusei (C. krusei), Candida
tropicalis (C.tropicalis) and Candida Glabrata
(C.glabrata) has increased. It is treated locally
with miconazole and nystatin; however,
patients present high recurrence rates due
to the formation of biofilms. Biofilms are
biological communities adhered to a surface
(oral mucosa, dental prostheses) with high
resistance to antifungals, which has driven
the search for natural alternative therapies,
such as essential oils. Oregano essential oil
98
(OE-O) is effective and a therapeutic option
on bacterial biofilms, but its activity on fungal
biofilms is unknown. Methods. The minimum
inhibitory
concentration
(MIC)
was
determined on reference strains for C.Albicans
(fluconazole sensitive and resistant) and
non-albicans strains (C.tropicalis, C.krusei
and C.glabrata). The antibiofilm effect was
evaluated by: a) morphogenesis inhibition
assay: an inoculum was incubated for 5 hours
at 37°C in the presence of the OE-O MIC,
then the percentage of filamentous cells was
counted using a Neubauer chamber; and b)
inhibition of biofilm adhesion: an inoculum
was incubated in a 96-well plate in the
presence of the CIM of OE-O for 4 hours at
37°C, the adhered biofilm was stained with
crystal-violet and absorbance was measured
in microplate reader. Fluconazole and
nystatin were used as controls. Results. AE-O
significantly inhibited both biofilm adhesion
and morphogenesis of the strains studied
compared to controls. Conclusion. These
results indicate that OE-O has significant
antibiofilm activity in both C. albicans and
non-albicans strains.
23. EFFECTS OF MODAFINIL
ADMINISTRATION ON SOCIAL
PLAY BEHAVIOUR AND DOPAMINE
TRANSMISSION IN JUVENILE RATS.
Cid-Jofré V.1,2; Gárate M.1; SotomayorZárate R.3, Cruz G.2; Renard GM.1.
1 Laboratorio de Conductas Sociales y
Adictivas, Escuela de Medicina, Centro de
investigaciones Biomédicas y aplicadas,
Facultad de Medicina, Universidad de
Santiago de Chile. 2 Laboratorio de
Alteraciones Reproductivas y Metabólicas,
Centro de Neurobiología y Fisiopatología
Integrativa (CENFI), Instituto de Fisiología,
Facultad de Ciencias, Universidad de
Valparaíso. 3Laboratorio de Neuroquímica y
Neurofarmacología, Centro de Neurobiología
y Fisiopatología Integrativa (CENFI),
Instituto de Fisiología, Facultad de Ciencias,
Universidad de Valparaíso.
Modafinil (MOD) is a stimulant used to
enhance wakefulness and vigilance. The
mechanism of action of MOD has not
been completely elucidated but a blockage
of dopamine (DA) and norepinephrine
transporters has been observed. Some clinical
trials are testing MOD for the treatment of
attentional deficit disorder (ADD). In view of
a reported over diagnostic of ADD, evaluating
the effects of MOD in healthy individuals is
important. Herein, we evaluate the effects
of MOD on social play behaviour (SPB) and
DA extracellular levels in juvenile rats. 35
juvenile male Sprague-Dawley rats were
treated from PND25 with MOD (75 mg/
kg i.p.) or vehicle for 14 days. Locomotor
and social exploration were tested 24 hours
after de last injection. Nucleus Accumbens
(NAc) and ventral tegmental area (VTA)
were dissected to measure DA content by
HPLC coupled to electrochemical detection.
We observed a decrease in the “pinning
events” (responses to play behaviour) and
tendency to decrease in the pouncing latency
(the events of solicitation to play) in the
MOD group. Also, there was a decrease in
DOPAC/DA and DOPAC content in VTA. No
differences in social exploration time and DA
content were observed. SPB is fundamental
to establish social and cognitive development
in highly social animals like rats and humans.
These preliminary results show that MOD
could affect SPB by altering the rewarding
effects of socialization. Importantly, DA levels
are almost the same in both NAc and VTA,
although there is a tendency for lower levels
in MOD group. More studies are needed to
unravel the effects of stimulants, specially on
young population, over important social skills
like playing, social interactions and memory
24. HIGH-FAT DIET EXPOSURE
INCREASES THE EXPRESSION OF
KEY PROTEINS IN DOPAMINERGIC
NEUROTRANSMISSION OF RAT
LATERAL SEPTUM.
Collio, V.1; Martínez-Pinto, J.2; Cruz, G.2;
Bonasco, C.2; Renard, G.M.3; SotomayorZárate, R.2. 1Programa de Magíster en
Ciencias Biológicas mención Neurociencias,
Facultad de Ciencias, Universidad de
Valparaíso, Valparaíso, Chile. 2Centro de
Neurobiología y Fisiopatología Integrativa
(CENFI), Instituto de Fisiología, Facultad
de Ciencias, Universidad de Valparaíso,
Valparaíso, Chile. 3Centro de Investigación
Biomédica y Aplicada (CIBAP), Escuela de
Medicina, Facultad de Ciencias Médicas,
Universidad de Santiago de Chile, Santiago,
Chile.
Obesity is a global pandemic that must be
studied from many points of view, such as
social, preclinical, clinical, economic, etc.
At the level of the central nervous system,
there are several structures involved in
the control of food intake, being one of the
most important to promote the feeding
the lateral hypothalamus (LH). The main
brain area that controls the neural activity
of LH is the lateral septum (LS), which it
sends GABAergic projections towards LH,
controlling feeding behavior. In addition, LH
projects glutamatergic/orexinergic neurons
to the ventral tegmental area (VTA), which
it sends dopaminergic projections to the
nucleus accumbens and LS. This circuit is
very important for the intake of rewarding
foods, but it is not known the effects of
chronic exposure to high-fat diet (HFD) on
LS neurotransmission. For this work we
used 2 groups of male Sprague-dawley rats
exposed from weaning to postnatal day 60
(PND 60) to chow diet (control) and HFD.
At PND 60 the animals were euthanized
and the LS was microdissected to measure
by western blot key proteins involved in
LS dopaminergic neurotransmission. Our
results demonstrate that exposure to HFD
results in a significant weight gain at the end
of the experimental period together with
an increase in retroperitoneal fat levels. In
LS the chronic exposure to HFD resulted in
an increase in the expression of the type 2
dopamine receptor (D2) and the dopamine
transporter (DAT) compared to control rats.
These results suggest that these proteins
functionally reduce the dopaminergic tone in
LS, which would affect their inhibitory control
over LH activity. However, the implication of
these results will be evaluated in subsequent
experiments.
25. BOLDINE PREVENTS TGF-Β1INDUCED DIFFERENTIATION
OF CARDIAC FIBROBLASTS BY
INHIBITING FOXO1.
Contreras A. 1, Anfossi R. 2., Suarez C. 2,
Cárdenas, S. 1., Vivar, R 2.
1.- Department of Biology, Faculty of
Basics Sciences, Metropolitan University of
Educational Sciences; 2.- Faculty of Medicine,
University of Chile.
Diabetes and myocardial infarction promote
the development of cardiac fibrosis. Normally,
cardiac fibroblasts (CF) are responsible for the
synthesis and maintenance of extracellular
matrix components (ECM), whereas in
pathological conditions CFs differentiate
into cardiac myofibroblasts, generating
an imbalance in the secretion of the ECM
proteins. TGF-beta1 plays a crucial role in the
development of cardiac fibrosis by regulating
the expression of FoxO1, a transcription factor
that is involved in functions such as apoptosis,
oxidative stress and cell differentiation. On
the other hand, boldine, a natural alkaloid,
has been shown to exert antifibrotic effects in
experimental models of diabetes. Therefore,
this work attempted to demonstrate the
antifibrotic effect of boldine in a model
99
effects, and generation of polluting waste
or expensive raw material. Therefore, due
to their nature and structural diversity they
could have great potential for cosmetic use,
for example, as hair dyes. To characterize
the fungal pigments and evaluate their
antioxidant effect, Penicillium murcianum
species was selected. This fungus is an ecotype found in Chilean native forests, which
was cultivated under conditions previously
optimized for the production of brownyellow pigments. To facilitate the chemicalbiological analysis, the extract obtained
from the culture broth was fractionated
by Centrifugal Partition Chromatography
(CPC) with a phase system of ethyl acetate:
butanol: water. The above allowed to purify
several fractions, which were evaluated for
their antioxidant activity in the DPPH assay.
These fractions recorded an inhibition close
to 90% and an IC50 value of 4.64 mg / mL for
the crude extract. In the case of the purified
fractions, two were selected with the highest
antioxidant activity corresponding to an IC50
of 1.17 mg / mL and 0.708 mg / mL, which were
subsequently analyzed by HPLC / PAD / MS.
Our preliminary results revealed the presence
of azafilones such as monashexenone,
26. CHEMICAL-BIOLOGICAL
monankarin and monaphilol and the
CHARACTERIZATION OF
ANTIOXIDANT PIGMENTS PURIFIED anthraquinoids sterigmatocystin, endocrocin
and flavokermesic acid, which would be
FROM EXTRACTS AND FRACTIONS
responsible for the yellow pigmentation of the
OF PENICILLIUM MURCIANUM
extract. These compounds have bibliographic
AND ITS POTENTIAL COSMETIC
antecedents related to antioxidant and
APPLICATIONS.
Contreras-Machuca P.1, Avello M.1, Pastene antimicrobial activities, among others,
E.1,6, Machuca A.2, ArandaM.3, Hernández which highlights a great opportunity for
future research and applications for these
V.4 & Fernández M.5.
1
Laboratorio
de
Farmacognosia, metabolites of P. murcianum.
Departamento de Farmacia, Facultad de
Farmacia, Universidad de Concepción, 2 27. IN VITRO PROPAGATION
Laboratorio de Biotecnología de Hongos, OF RODOPHIALA PRATENSIS
Departamento de Ciencias y Tecnología AND ITS TOXICITY IN VITRO ON
Vegetal, Universidad de Concepción, Campus EPITHELIAL CELLS OF GASTRIC
Los Ángeles, 3 Laboratorio de Espectrometría ADENOCARCINOMA (AGS).
de Masas, Departamento de Ciencia y Correa D.I. 1; Pastene-Navarrete E.R. 1,2;
Tecnología de los Alimentos, Facultad de Bustamante L. 2; Baeza M. 3; Alarcón-Enos
Farmacia, Universidad de Concepción, 4 J.I 4.
Centro de Biotecnología, Universidad de 1Laboratorio de Farmacognosia, Dpto. de
Concepción, 5 Laboratorio de Tecnología Farmacia, Facultad de Farmacia, P.O. Box
Farmacéutica, Departamento de Farmacia, 237, Universidad de Concepción, Concepción,
Universidad de Concepción, 6 Laboratorio Chile; 2Dpto. de análisis instrumental,
de Síntesis y Biotransformación de Productos Facultad de Farmacia, Universidad de
Concepción, Concepción, Chile; 3Dpto.
Naturales, Universidad del Bío-Bío.
Botánica, Facultad de Ciencias Naturales y
In order to find new sources of active Oceanográficas, Universidad de Concepción,
metabolites as functional ingredients for Concepción, Chile; 4Laboratorio de Síntesis
the cosmetic industry, the idea of studying y Biotransformación de Productos Naturales,
filamentous fungi that produce natural Dpto. Ciencias Básicas, Universidad del Biopigments arises. These compounds meet the Bio, Chillan, Chile.
requirements of having low toxicity, adverse
of cardiac fibrosis in vitro. To respond to
this hypothesis, the differentiation of adult
Sprague-Dawley rats CF by TGF-beta1 was
used as in vitro model of cardiac fibrosis. The
differentiation of CF was determined by the
expression of alpha-SMA and CTGF, through
western blot (WB) and immunocytochemistry
(ICQ). On the other hand, the activation of
FoxO1 was evaluated by analyzing phosphoFoxO1 (WB) and nuclear location of FoxO1
(ICQ). AS1842856 a FoxO1 activity inhibitor
was used to evaluate the role of FoxO1. TGFbeta1 10ng/ml increased the expression of
alpha-SMA and CTGF at 48h, which was
corroborated by an ICQ against alpha-SMA.
In addition, TGF-beta1 increased the activity
of FoxO1, which was determined by decreased
phosphorylation of FoxO1 and increased
nuclear localization, whereas inhibition of
FoxO1 prevented the differentiation of CF
induced by TGF-beta1. Finally, boldine 50uM
and 100uM abolished the differentiation of
CF and the activation of FoxO1 induced by
TGF-beta1. Collectively ours results suggest
that boldine prevents TGF-beta1-induced CF
differentiation by inhibiting FoxO1.
100
Amaryllidaceae is a family of bulbous
plants, producers of alkaloids which are
biogenetically related and exhibit high
pharmacological activity. Lycorine and
homolycorine alkaloids have been studied as
potent antitumor agents. However, the study
of these molecules is difficult due to the low
availability and production in the plant in the
wild, so the objective of this study is by in vitro
propagation to obtain biomass of R. pratensis
in an efficient and sustainable way, to identify
the type of Alkaloids that are produced and
assess their cytotoxic potential on tumor
cells. Methodology: Rhodophiala prantesis
bulbs, were sterilized and cut into twin-scales,
to sow them in Murashige-Skoog growth
medium, supplemented with sucrose and
different combinations of naphthalenacetic
acid and 6-benzylaminopurine, the alkaloid
analysis was performed by CG-MS. AGS
cells were cultured in DMEM medium
supplemented with SBF (10%), antibiotic
(1%). The assay was performed in 96-well
plate using resazurin at 6 and 24 hours after
exposure of the alkaloid extract. R. pranthesis
callus were obtained in in vitro culture in
semi-solid medium. In addition, 25 alkaloids
were identified in the bulb’s alkaloid extract
which decreased the viability of AGS cells
at 6 and 24 hours of exposure. Conclusion:
Hormonal combinations were evaluated for
the production of callus of R. pratensis, in
addition the alkaloids have cytotoxic activity
on AGS as a function of exposure time.
toxicity and many side effects. Therefore,
new therapies directed to the metastatic cells
of the CRC are urgently needed, with high
pharmacological efficacy, reducing the side
effects and treatment costs. In recent years,
neoplastic mitochondria are an attractive
pharmacological target for cancer therapy,
since they have higher mitochondria potential.
In our laboratory, gallic acid derivatives linked
to an aliphatic chain of ten carbons associated
with triphenylphosphonium (TPP+C10), a
lipophilic cationic molecule that induces the
uncoupling of the electron transport chain
was synthesized and evaluated in CRC cells
as well as gentysic derivative (GA-TPP+C10).
The objective of this study is to evaluate the
synergistic effect of the compounds, GATPP+C10 and TPP+C10 in combination
with conventional drugs for the treatment
of CRC, 5-FU and oxaliplatin, using the
COLO 205 metastatic human CRC cell line.
Through the MTT assay, the cytotoxicity of
the combinations was evaluated after 48 h of
treatment and by flow cytometry the synergy
of the combinations inducing apoptosis was
evaluated. The observed results showed
that bothTPP+C10 and GA-TPP+C10 are
synergistic with low concentrations of 5-FU
and Oxaliplatin, inducing greater apoptosis of
the colorectal cells. In conclusion, the results
suggest that these new compounds could
synergize the effects of conventional therapy
against colorectal cancer.
29. CHANGES IN DOPAMINE
RECEPTOR TYPE 2 EXPRESSION IN
PREFRONTAL CORTEX INDUCED
BY EARLY-LIFE INTESTINAL
DYSBIOSIS.
Covarrubias MJ.1; González-Arancibia C.2,3;
Martinez-Pinto J.2; Sotomayor-Zárate R.2;
Julio-Pieper M.1; Bravo JA.1.
1Grupo
de
NeuroGastroBioquímica,
Laboratorio de Química Biológica. Instituto
de Química, Facultad de Ciencias, Pontificia
Universidad
Católica
de
Valparaíso,
Valparaíso. Chile. 2, Laboratorio de
Neuroquímica y Neurofarmacología, Centro
de Neurobiología y Fisiopatología Integrativa
(CENFI), Instituto de Fisiología, Facultad
de Ciencias, Universidad de Valparaíso. 3,
Programa de Doctorado en Ciencias mención
Neurociencias,
Facultad
de
Ciencias,
Colorectal cancer (CRC) is the third leading Universidad de Valparaíso.
cause of cancer death in the world. The
standard drugs currently used for the Intestinal microbiota has been shown to
treatment of CRC are 5-fluouracil (5- modulate central nervous system function.
FU), oxaliplatin and irinotecan. These For instance, a reduction in the gut’s
chemotherapeutic agents are effective in the microbiota richness and diversity through
early stages of the disease, presenting high the use of antibiotics, sensitizes mice to drug
28. SYNERGISTIC EFFECT
OF GENTISIC AND GALLATE
DERIVATIVES WITH STANDARD
THERAPY FOR COLORECTAL
CANCER CELLS.
Cortés, G. 1,2, Ramírez, D.1,2, Rojas, D. 1,
Escobar, B.1,3, Catalán, M.1.
1 Laboratory of Biochemistry, Metabolism
and Drug Resistance, ICBM, Facultad de
Medicina, Universidad de Chile, Santiago,
Chile. 2 Department of Biology, Faculty of
Basic Sciences, Metropolitan University
of Education Sciences, Santiago, Chile.
3 Pharmacology Laboratory, Research
Institute of Dental Sciences (ICOD), School
of Dentistry, University of Chile, Santiago,
Chile.
101
seeking behaviors. Furthermore, this behavior
is driven by changes in dopamine receptor
expression within the mesocorticolimbic
system, which strongly supports the
relevance of the microbiota-gut-brain axis in
the development of addictive behaviors. In
most animals, early-life gut colonization by
microbial symbionts occur begins at birth,
with bacteria coming from the mother, and
is a process that happens in parallel with
early stages of brain development. Therefore,
changes in maternal gut microbiota
richness and diversity would impact on the
development of neuropsychiatric diseases
later in life, including addiction. To test this,
we administered pregnant Spraque-Dawley
dams a cocktail of oral wide-spectrum
antibiotics (neomicyn, bacitracin, vancomycin
and pimaricin) from embryonic day 18 till
post-natal day (PND) 7, and then evaluated
dopamine receptor 2 (D2) expression in the
prefrontal cortex (Pfx) of female and male
offspring at PND60, and compared with the
offspring of pregnant dams given saline.
The results show that Pfx D2 expression
in female offspring of antibiotic exposed
dams is reduced (although not significant)
when compared to age matched controls,
but no differences were observed in the
male offspring. These results suggest that a
reduction in the diversity and richness of gut
microbes during early-life provokes changes
in PFx D2 expression in females, but not
males, and furthermore, this changes might
impact the female’s drugs seeking behaviors.
30. EARLY-LIFE EXPOSURE TO
ORAL WIDE-SPECTRUM NONABSORVABLE ANTIBIOTICS AND
THEIR EFFECTS ON DOPAMINE
RECEPTOR 2 EXPRESSION IN THE
SUBSTANTIA NIGRA OF SPRAGUEDAWLEY RATS.
Da-oliveira, Y. M.1; Urrutia-Piñones, J.1;
Martinez -Pinto, J2; Sotomayor-Zarate, R2;
Julio-Pieper, M.1, Bravo, JA.1.
1Grupo
de
NeuroGastroBioquímica,
Laboratorio de Química Biológica. Instituto
de Química, Facultad de Ciencias, Pontificia
Universidad
Católica
de
Valparaíso,
Valparaíso. Chile. 2, Laboratorio de
Neuroquímica y Neurofarmacología, Centro
de Neurobiología y Fisiopatología Integrativa
(CENFI), Instituto de Fisiología, Facultad
de Ciencias, Universidad de Valparaíso,
Valparaíso. Chile.
development of gut, immune and brain
physiology. We have previously shown that
exposing pregnant Sprague-Dawley dams
to an oral cocktail of wide-spectrum non
absorbable antibiotics (neomycin, bacitracin,
vancomycin all three at a dose of 100 mg/
kg and pimaricin at 5microg/kg) from
embryonic day 18 until post-natal day (PND)
7, lowers intestinal microbial richness and
diversity in the male offspring at PND35.
Additionally, early-life exposure to antibiotics
lowers dopamine receptor 1 (D1) expression
in key areas of the mesocorticolimbic circuit
of male offspring when compared to age
matched controls that were not exposed to
antibiotics perinatally. This suggest that
early-life exposure to antibiotics, which
affects gut microbial ecology, impacts on
dopaminergic circuits related to reward.
However, another question was raised:
what happens in substantia nigra (SN),
another major source of dopamine that is
also involved in reward and motor function.
Thus, we evaluated D2 expression in the SN
of males (PND35) from Sprague-Dawley
dams exposed to the aforementioned cocktail
of antibiotics, and compared it with control
rats. Immunohistochemical analysis revealed
that early-life exposure to antibiotics does not
affect D2 expression in comparison to control
rats. This result suggest that reduction of
microbial diversity and richness in early life,
affects specifically the mesocorticolimbic
circuit, with no effects on the D2 expression
in the SN. Such specific effect further
suggests that within the microbiota-gutbrain communication, there are very specific
pathways that may in part underlie the
basis of neuropsychiatric disorders, such as
addiction.
31. MECHANICAL STIMULATION
INCREASES EXTRACELULLAR
ATP AND NO SECRETION TO
THE MEDIA OF CELL CULTURES;
PHYSIOLOGICAL IMPLICATIONS.
Donoso F.; Donoso M.V.; Huidobro-Toro J.P.
Laboratorio de Nucleótidos, Departamento
de Biología, Facultad de Química y Biología,
y Centro de Nanociencia y Nanotecnología,
CEDENNA, Universidad de Santiago de Chile.
As a requirement of many protocols it is
common to add drugs to cell cultures to
investigate cell processes in the presence
of a determined pharmacological agent.
However, if this procedure is not well
Microbial gut colonization begins at birth, controlled by appropriate standards, it may
where the transfer of intestinal microbes cause additional experimental variability.
from mother to infant is key in early-life Our working hypothesis proposed that
102
pipetting/agitation of the culture medium
causes extracellular ATP secretion, which in
the case of endothelial cells is also related
to NO secretion. Endothelial cells of the
rat mesentery, fibroblasts, and oocytes of
Xenopus Laevis were used. In endothelial cell
cultures, 100 uL of Tyrode buffer is gently
applied; extracellular fluid sample is collected
to measure ATP and NO production. ATP
and metabolites are quantified as fluorescent
ethene purines; separated and quantified by
HPLC procedures. NO was determined by a
fluorescent probe as the [DAF-NO] complex.
Buffer application increases medium ATP
from 117 ± 75.7 to 317 ± 25 pmoles ATP/
mg protein (p <0.001); the signal peaked
by one minute, thereafter, ATP decays.
This stimulus also rapidly and significantly
increases NO production (p <0.0001). This
increase is blunted by 150 μM L-NAME, an
eNOS inhibitor, and by apyrase (4 U/mL),
suggesting the participation of extracellular
ATP. The agitation of fibroblast culture
medium in culture increases 4-fold ATP
secretion, a transient effect that decreased
in minutes. Likewise, Xenopus oocytes
agitation by a variable inclination agitator
increased 3.8 times extracellular ATP (p
<0.01). In conclusion, different mechanical
stimuli secrete extracellular ATP in different
cell types, suggesting that cells respond to
chemical and sensory stimuli by increasing
nucleotide secretion. The nucleotide surge
may cause unexpected variations in the final
cellular response due to indirect or direct
purinoceptor activity.
32. MODIFIED SYMPATHETIC
NERVOUS TERMINALS AND
ENDOTELIAL CELLS FROM THE
MESENTERIAL ARTERIAL BED
BY STREPTOZOTOCIN-INDUCED
DIABETES.
Donoso, M.V., Huidobro-Toro, J.P.
Laboratorio de Farmacología, Departamento
de Biología, Facultad de Química y Biología,
CEDENNA, Universidad de Santiago de Chile.
Respiratory, intestinal and / or vascular
epithelial cells modify the activity of its
adjacent smooth muscle layer. We studied
whether the prostatic vas deferens epithelium
modulate the motor response induced by
electrical stimulation by releasing an epithelial
messenger. To this aim, the isometric
muscular contraction of the prostatic segment
of the rat vas deferens intact or mechanically
denuded of its epithelium was recorded using
a force displacement transducer. The tissues
were placed in a super fusion bath with Tyrode
buffer at 37°C, 95% O2-5% CO2. Muscle
tension was recorded by a Grass polygraph
coupled to a transducer; contractions were
induced by electrical field depolarization
or with chemical stimuli. The muscular
contraction induced by the exogenous
application of 100 uM norepinephrine
decreased following epithelium removal
(1.5±0.2g versus 0.8±0.1g, n =10, p<0.0038),
but not those elicited by ATP or KCl.
Exogenous ATP, at concentrations that do
not induce contractions (1-100 uM), reduced
the motor effect induced by electric trains
of 0.15 Hz. Epithelial removal reduced this
ATP effect (p <0.05). ATP reduces muscle
contraction induced by 4 Hz trains, in the
phasic component, epithelial removal causes
a greater inhibitory effect of ATP (p <0.05).
The inhibition of NO synthesis with 100 uM
N-omega-nitro-l-arginine does not modify
the inhibitory effect of ATP on the electrical
stimulus. In contrast, indomethacin increases
the inhibitory effect of ATP on 0.15 Hz in the
absence of epithelium, and on 4 Hz, both
in the presence and absence of epithelium.
Altogether, present results suggest that a
non-identified arachidonic acid derivative,
but not NO, sensitizes the motor response of
the duct, demonstrating that the epithelium
participates and modulates its motor activity.
33. CHANGES IN THE
EXTRACELLULAR LEVELS
OF DOPAMINE IN NUCLEUS
ACCUMBENS OF ADULT RATS
NEONATALLY EXPOSED TO
SEX HORMONES: STUDIES OF
BRAIN MICRODIALYSIS USING
METHYLPHENIDATE.
Elgueta-Reyes, M.1,2; Renard, G.M.3;
Sotomayor-Zárate, R.1.
1
Laboratorio
de
Neuroquímica
y
Neurofarmacología, Centro de Neurobiología
y Fisiopatología Integrativa (CENFI),
Instituto de Fisiología, Facultad de Ciencias,
Universidad de Valparaíso. 2 Programa de
Magíster en Ciencias Biológicas mención
Neurociencia,
Facultad
de
Ciencias,
Universidad de Valparaíso. 3 Laboratorio
de Neurobiología de Conductas Sociales y
Adictivas, Escuela de Medicina, Centro de
Investigaciones Biomédicas y Aplicadas,
Facultad de Medicina, Universidad de
Santiago, Chile.
Sex hormones produce several effects
in reproductive and non-reproductive
tissues. In this sense, brain expresses sex
hormones receptors in cortical and limbic
areas. Nigrostriatal and mesocorticolimbic
103
pathways are modulated by sex hormones,
affecting the expression of key dopaminergic
proteins in adult animals. However, few
studies have been focused in long-term
effects produced by early exposure to sex
hormones. Last time, our lab has been shown
some neurochemical and behavioral changes
produced by neonatal administration of sex
hormones in brain dopamine areas such as
higher levels of dopamine (DA), expression
of tyrosine hydroxylase and addictive-like
behaviors induced by morphine. Last time,
we published that adult rats exposed during
first hours of postnatal life to sex hormones
had a lower locomotor activity induced by
methylphenidate (MPD) than control rats
and this effect was associated with a lower
expression of the dopamine transporter
(DAT) in nucleus accumbens (NAcc).
Therefore, the aim of this work was studied
the basal and stimulate (MPD: 5 mg/kg)
extracellular levels of DA, Glutamate and
GABA in NAcc of adult rats exposed during
the first hours of postnatal life to estradiol
valerate (EV: 0.1 mg/50μL). Our results
showed a lower NAcc DA release induced by
MPD in EV rats compared to control rats. In
control rats we observed in NAcc a reduction
in extracellular levels of GABA after MPD
administration, however this effect was not
observed in EV rats. In addition, basal and
stimulate glutamate levels in NAcc were
not different between experimental groups.
These results suggest that neonatal exposure
to EV affect the neurochemical response to
psychostimulants in adulthood, which could
be a vulnerability factor to increase the doses
of abuse drugs.
(Ab) peptide. Experimental evidence has
shown that the oligomeric form is able to induce
reactive oxidative species (ROS). Ascorbic
acid (AA) is an essential micronutrient with
a preponderant role in oxidative stress and its
main transport system in the brain is SVCT2,
that incorporates the reduced form of vitamin
C (ascorbic acid, AA), which is the prevalent
form in vivo. Recent works show the relevance
of AA in AD’s progression by the inhibition
of SVCT2, nevertheless, these studies didn’t
analyze how vitamin C is acquired by neurons
and compartmentalized within organelles.
In this work, we studied the subcellular
localization of the SVCT2 transporter in
primary cultures of mouse hippocampal
neurons, and the changes in localization and
expression levels associated to oligomeric
Ab treatment. Material y methods: 18 days
embryos hippocampus of C57BL/6 mice were
dissociated and plated. Were cultured in vitro
by 9 days until its treatment with oligomeric
Ab. At 24 and 48 hours of treatment the
subcellular localization of SVCT2 was
evaluated, though immunofluorescence.
Results: It was observed that hippocampal
neurons show expression of SVCT2 which
is located mainly at the mitochondria.
Treatment with Ab oligomers increases
the colocalization of SVCT2 with this
organelle. Discussion: Our results suggest
that mitochondrial AA might be relevant for
neuronal survival in response to ab damage
and suggest that this transporter would be a
new therapeutic target to treat this disease.
35. NOVEL CAFFEIC ACID
DERIVATIVES AGAINST ORAL
CANCER CELLS.
Escobar B.1, Rojas D.2, González D.2, Cortés
34. MITOCHONDRIAL EXPRESSION
G.2, Jara JA.1, Catalán M.2.
OF SVCT2 IN HIPPOCAMPAL
1.-Laboratory of Pharmacology, ICOD,
NEURONS TREATED WITH
OLIGOMERIC AB.
Facultad de Odontología, Universidad
Escobar-Acuña K.1; Panes J.1; Saavedra de Chile 2.- Laboratory of Biochemistry,
P.1; Moraga G. 1; Fuentealba J.1; Rivas C.2; Metabolism and Drug Resistance, ICBM,
Facultad de Medicina, Universidad de Chile.
Muñoz-Montesino C.1.
1, Laboratorio de Neuropatología Molecular,
Departamento de Fisiología, Facultad The cancer is a cellular process characteristic
de Ciencias Biológicas, Universidad de by uncontrolled growth and dissemination,
Concepción. 2, Laboratorio de Antioxidantes, being the second cause of death worldwide.
Departamento de Fisiopatología, Facultad Likewise, the oral cancer is one of the ten
de Ciencias Biológicas, Universidad de most common neoplasm in the world,
mainly concern the tongue. The gold
Concepción.
standard therapies are surgery, which can
Introduction: Alzheimer’s disease (AD) is a be complemented with radiotherapy and/or
neurodegenerative disorder characterized chemotherapy. The latter, represent the firstby progressive memory impairment and line therapies, involving decreasing tumor size
cognitive dysfunction. The most distinctive and abolishing microscopic disease. However,
phenomenon correlating with AD is the this cancer has small average of survival,
presence of aggregates form of the β-amyloid several side effects and drug resistances.
104
Therefore, new drugs are currently being
studied, such as novel caffeic acid derivatives
reporter before with antitumoral effect
in breast cancer. We evaluated these new
compounds by measurements of cytotoxic
effect by MTT assay, mitochondrial effects by
evaluation of mitochondrial-transmembrane
potential by flow cytometry and ATP levels
through luminescence assay. We showed
that the derivates have cytotoxic effect in
human cancer cell lines Cal27 and Hep-2.
We found that the compounds generated the
decrease in cellular ATP levels and decrease
the measuring ATP levels on human cancer
cell line Cal27 and Hep of the mitochondrialtransmembrane potential. In addition, we
evaluated the selectivity of these compounds
by exposing normal epithelium cells to their
action by evaluation of cytotoxicity. Our
results demonstrated the novel caffeic acid
derivates exert a selective cytotoxic effect by
mitochondrial mechanism.
36. CARDIOMYOCYTES
HYPERTROPHY INDUCED BY
CHRONIC STIMULATION WITH
FRUCTOSE: BENEFICIAL EFFECT OF
METFORMIN.
Escorcia L.A.1; Muñoz-Rodríguez C.1;
Labraña P. 1; Catalán M. 2; Olmedo I.1.
1, Programa de Farmacología, Instituto de
Ciencias Biomédicas, Facultad de Medicina,
Universidad de Chile. 2, Programa de
Farmacología Molecular y Clínica, Instituto de
Ciencias Biomédicas, Facultad de Medicina,
Universidad de Chile.
Fructose intake has been increased in
recent decades. Studies have indicated
that high fructose consumption would be
associated with an increase in the incidence
of cardiovascular diseases (CVD) such as
cardiac hypertrophy. Cardiac hypertrophy
is an adaptive response to chronic work
overload imposed on the heart, where
cardiomyocytes
undergo
numerous
morphological and functional changes.
According to literature, fructose is able to
decrease cell redox defenses and reduce
adenosine monophosphate-activated protein
kinase (AMPK) activation. AMPK activation is
related to cardioprotective effects, and during
hypertrophy allows the heart to change its
metabolism. The objective of this work was to
study the effect of fructose on cardiomyocyte
hypertrophy and AMPK activation in the
presence of metformin, a cardioprotective
drug widely used in the treatment of type
2 diabetes mellitus. Cultured neonatal rat
cardiomyocytes (1-3 days) were treated with
25 mM fructose at different times. The mRNA
levels of hypertrophic markers (Beta-MHC,
ANP, BNP and RCAN1.4) were determined
by qRT-PCR. Phosphorylated -AMPK, totalAMPK and Beta-MHC protein levels were
determined by inmunowestern blot (WB).
The results showed that fructose increased
mRNA levels of the hypertrophic markers
and Beta-MHC protein levels. Further, it was
observed that metformin was able to increase
AMPK activation even in the presence of
fructose which could prevent the progression
of cardiomyocyte hypertrophy induced by
this carbohydrate.
37. INDUCTION OF CELLULAR
SENESCENCE IN PRIMARY ADULT
MOUSE CARDIAC FIBROBLASTS.
Espitia-Corredor, J.A.1,2; Peiró-Vallejo, C.2;
Díaz-Araya, G.A.1.
1, Laboratory of Molecular Pharmacology,
Department
of
Pharmacological
and
Toxicological Chemistry, Faculty of Chemical
and Pharmaceutical Sciences, University
of Chile. 2, Laboratory L-5, Department
of Pharmacology, Faculty of Medicine,
Autonomous University of Madrid.
Introduction: Cellular senescence – a
hallmark of aging – is related with the
biomarkers appearance and up-regulation,
such
as
senescence-associated
betagalactosidase activity (SABG), activation of
tumor suppressor proteins (p53, p21CIP1)
and increase of DNA damage associated
proteins (gammaH2A.X). Recent studies
suggested that interleukin-1beta (IL-1b) and
doxorubicin (Dox) promote cardiac aging
through an inflammatory process. Cardiac
fibroblast (CF) keeps the extracellular matrix
homeostasis and actively participates on
damage-associated inflammatory and scaring
processes that are altered in cardiac aging.
Few studies have evaluated the increase of
these biomarkers on CF by pro-inflammatory
molecules such as IL-1b and Dox as
potential inducers of cardiovascular damage.
Objective: The objective is to evaluate IL-1b
and Dox effects upon SABG, levels of tumor
suppressor- and DNA damage-associated
proteins as biomarkers of cellular senescence.
Methods: CF were isolated from 8-10-weekold C57BL/6 male mice. CF were serumstarved by 24 hours prior to stimulation with
IL-1b (2,5 ng/mL) and Dox (10 nM) for 24
hours. SABG was measured by microscopy
with a commercial kit (Cell Signaling
TECHNOLOGY®) and protein levels of p53,
p21CIP1, and gammaH2A.X by immuneblot. Results: IL-1b and Dox increase the
105
percentage of SABG positive cells as long
as the protein levels of p53, p21CIP1, and
gammaH2A.X. Conclusions: IL-1b and Dox
show an inductor effect of cellular senescence
– featured by increased SABG and p53,
p21CIP1 and gammaH2A.X levels – on CF.
threshold. Taken together, the activation of
this receptor within MSNs of the Nacc has
effects on excitability parameters of these
cells and also a possible modulation of
serotonin and dopamine transmission within
this nucleus.
39. MESENCHYMAL STEM
CELL SECRETOME (MSCSS)
ADMINISTRATION REDUCES
OXIDATIVE STRESS AND
NEUROINFLAMMATION INDUCED
BY PERINATAL ASPHYXIA IN RAT
HIPPOCAMPUS.
Farfán N1, Araya M1, Monzón E1, Carril J1,
Alvarado R1, Tapia-Bustos A1, Vásquez R1,
Santapau D3, Bustamante D1, Quintanilla
ME1, Ezquer F3, Valdés JL2, Israel Y1,
Herrera-Marschitz M1, Morales P1,2.
Programa de Farmacología Molecular y
Clínica, Instituto de Ciencias Biomédicas,
Serotonin is a neurotransmitter implicated Facultad de Medicina, Universidad de Chile.
in most processes related to mood, sleep
regulation, sexual behavior and cognitive Perinatal asphyxia (PA) is an important
modulation, being important in synaptic obstetric risk occurring at the time of delivery.
transmission as well. While, serotonin Surviving children develop long-lasting
transmission is not completely, new motor and cognitive deficits. PA implies a
evidences showed an important role for primary energy crisis resulting from oxygen
serotonin receptor activation in prefrontal interruption, followed by a secondary insult
cortex (PFC), dorsal raphe nucleus (DRN) linked to the required re-oxygenation, leading
and ventral tegmental area (VTA), specifically to oxidative stress, neuroinflammation
the activation of serotonin receptor 5HT2A. and cell death, affecting basal ganglia
hippocampus.
Neuroinflammation
Electrophysiological studies in PFC showed and
that serotonin has effects on excitability activates NF-kappa b signalling, which
profiles of pyramidal cells of layer V implies p65 nuclear translocation and prothrough the activation of 5HT2AR where inflammatory gene expression, resulting
its expression pattern is very high. These in glial activation and apoptosis. Oxidative
cells project to nucleus accumbens (Nacc; stress activates Nrf2, the antioxidant defense
another nucleus with high expression of master regulator, promoting antioxidant
5HT2AR), where the specific role of 5HT2AR gene transcription, including hemoxigenase
activation is not known regarding synaptic 1 (HO-1) and NAD(P)H dehydrogenase
transmission. To address this issue, we used quinone 1 (NQO1). MSCs have been proposed
electrophysiological whole cell patch clamp as potent agents to treat several conditions
and current clamp techniques to record associated with neuroinflammation and
in acute coronal slices from Nacc of adult oxidative stress. Preconditioning of MSCs
Sprague Dawley rats, MSNs in presence of with pro-inflammatory cytokines or hypoxic
serotonin and TCB-2 (agonist of 5-HT2AR). conditions improve their effectiveness.
The experiments were made in presence of The aim of this study was to determine
picrotoxin and tethrodotoxin to determine whether a single intranasal administration
and characterize the specific activation of of secretome derived from preconditioned
5-HT2AR in MSN only of the excitatory human MSCs to asphyxia-exposed rats
inputs. Our results show a decrease in the activate (i) the Nrf2 pathway, (ii) reducing
amplitude of mEPSCs (miniature excitatory oxidative stress, (iii) neuroinflammation
postsynaptic currents), which is related with and (iv) cell death in rat hippocampus.
a postsynaptic event, and no significant Two hours after birth MSCSs (6 ug/16 ul,
changes in the frequencies of mEPSCs. obtained from 1x10-6 preconditionedAlso, we observed changes in firing rate and MSCs) or 16 ul of vehicle were administered
action potential threshold parameters in the intranasally to asphyxia-exposed or control
presence of TCB-2, augmenting the firing rats. Animals were euthanized at day P7.
rate and diminishing the action potential Oxidative stress was monitored by the GSSG/
38. NEUROPHYSIOLOGIC
MODULATION OF NUCLEUS
ACCUMBENS BY THE ACTIVATION
OF SEROTONIN RECEPTOR 5-HT2A
Estay, C.1,2; Bonansco, C.2; Fuenzalida M.2;
Sotomayor-Zárate R.2.
1Programa de Doctorado en Ciencias
mención Neurociencias, Facultad de Ciencias,
Universidad de Valparaíso, Valparaíso, Chile.
2 Centro de Neurobiología y Fisiopatología
Integrativa (CENFI), Instituto de Fisiología,
Facultad de Ciencias, Universidad de
Valparaíso, Valparaíso, Chile.
106
GSH ratio, Nrf-2 nuclear translocation and
HO-1 and NQO1 protein levels by Western
blots (WB) and immunofluorescence (IFI).
Neuroinflammation by microglial reactivity
(anti-Iba-1, IFI) and p65 nuclear translocation
(WB). Cell death by cleaved caspase-3 protein
level (WB). The administration of MSCSs:
(i) lowered the PA increased GSSG/GSH
ratio, and (ii) increased both Nrf2 nuclear
translocation and HO-1, NQO1 levels, (iii)
reduced the microglial reactivity and nuclear
P65 and (iv) reduced cleaved caspase levels.
40. A COMPUTATIONALLY GUIDED
ANTIBODY AFFINITY OPTIMIZATION
METHOD BASED ON MACHINE
LEARNING SINGLE-POINT MUTANTS
SELECTION.
Fica-León V.1; Garrido, J.L.3; Barría, M.I.2;
Salas-Burgos, A.1.
1, Nanocell Laboratory, Pharmacology
Department, Biological Science Faculty,
Concepción
University.
2,
Virology
Laboratory,
Microbiology
Department,
Biological Science Faculty, Concepción
University. 3, Ichor Biologics LLC, New York,
NY 10065, USA.
Antibodies are the biomolecules with
therapeutic applications (TAbs) with highest
growth and great effectivity. Currently, we
know 78 monoclonal antibodies approved
by the FDA, and many others are in last
clinical stages. The applicability of antibodies
depends on two properties: specificity, and
affinity. Antibody affinity maturation is a
natural process and can be experimentally
emulated from diversity libraries and epitope
selection, process called optimization. This
selection is affected by external factors
and unexplored conformational states, the
discovered sequences have variable affinities
with values we can improve. On the other
hand, the next generation sequencing, the
molecular three-dimensional modelling, and
molecular docking have enabled perform
rational antibody design without protein
crystal complex. This process is assisted by
computers and complex algorithm based on
biophysical properties of the intermolecular
forces that drive the binding energy, the
interface between the epitope on the antigen,
and the complementary determinants
region on the antibody are dependent of
the residues type and position in this no
continuous loop. To improve and optimize
the affinity we need explored the residues
substitution and evaluate the energy free
changes. The combinatory is about 10^9
unique sequences, while the free energy
methods are computational requirement
extensive. We develop a Abpred, an algorithm
based in protein interfaces features and
machine learning selection to propose a
point-mutations combinatory to optimize the
affinity. We train Abpred using an artificiallybalanced dataset derived from SKEMPI-2.0;
1392 single-point mutations on 50 Ab-antigen
complexes. Evaluation on blind-test (20% of
dataset) achieved an RMSE of 1.66 kcal/mol,
and 0.593 correlation. Abpred enable the
affinity optimization in silico to accelerate the
design of news TAbs.
41. SELECTION OF MOLECULES FOR
THE FUNCTIONALIZATION OF GENECHARGED-GOLD-NANOPARTICLES
THAT ACHIEVE TARGETING OF
PROSTATE CANCER CELLS.
Fleitas-Salazar N.; Godoy E.; PedrosoSantana S.; Cerro R.P., Oliva R.; Fernández
K, Gonzalez I.; Toledo J.R.
Universidad de Concepción.
Metal nanoparticles (NPms) have attracted
the interest of biomedical researchers due to
their potential application in the diagnostic
and treatment of diseases like cancer. These
NPs can enter the cell depending on their
size, charge, and surface functionalization.
NPs between 10 to 100 nm have a large
surface area that can be functionalized with
different molecules for targeting (antibodies,
ligands of cell surface proteins), detection
(fluorescent probes) and treatment (drugs,
proteins, nucleic acids) of affected cells. In
this way, NPs could allow detection of target
cells and be a therapy, at the same time. Such
NPs has been described as ¨theranostic NPs¨.
Among molecules used to successfully target
cancer cells are folic acid, RGD peptides, and
EGF-receptor antibodies. In this work, we
show the formulation and characterization
of gold nanoparticles designed for gene
delivery treatment of prostate cancer cells.
Selectivity for prostate cancer cells of NPms
functionalized with folic acid or anti-LOX-1
antibody were compared. These NPms were
tested in four prostate cancer cell lines:
LnCap, Du-145, C4-2B, and PC3 while RWPE1 was used as a control cell line. Molecules that
mediate cell targeting of NPs are determinant
in the success of theranostic nanoparticles.
These NPs could favor cancer early detection,
improving treatment response and/or
achieving a complete recovery.
107
42. INFLUENCE OF DIVERSE
FACTORS ON ADHERENCE TO ORAL
ANTIDIABETIC MEDICATION IN
MEXICAN PATIENTS WITH TYPE 2
DIABETES.
Flores-Durán D. M.; Guzmán-Trujillo M.;
Briones- Aranda A.
Facultad de Medicina Humana de la
Universidad Autónoma de Chiapas, México.
Several factors can affect adherence to
pharmacological treatment in patients with
type 2 diabetes (T2D), including gender,
the time of evolution of the disease, and
the beliefs of the patient about health. The
aim of the present study was to examine the
main factors that influence adherence in T2D
patients affiliated with a public hospital in
Chiapas, Mexico. A cross-sectional study was
carried out on a sample of 200 patients being
treated with oral hypoglycemic agents, of
which 50% were women and the average age
of the women was 61.2 ± 7.7 years and of the
men 65.2 ± 1.4 years. The patients were asked
to provide the most recent result of fasting
glycemia and to fill out a sociodemographic
questionnaire, the Morisky-Green test and
the Beliefs about Medicines Questionnaire
(BMQ). The possible associations among the
variables were analyzed by the Chi-squared
test, while the comparison of the results of the
BMQ between compliant and non-compliant
patients was examined with the MannWhitney U test. The results show that 77% of
the women and 85% of the men presented a
lack of adherence to medication. The habit
of smoking had a significant association
with the lack of adherence. The men and
women who were compliant mentioned a
greater need for the medication (U1=369.50,
U2=553; p<0.005). However, only the
compliant women had levels of glycemia
significantly lower than the group of noncompliant patients (t=757; p<0.05). Hence, it
is possible that the responses of the men were
more prone to falsehoods than those of the
women. This could justify a future revision of
the instruments used to test the adherence of
men.
108
43. CYTOTOXICITY OF
NITROFURANS AND C-5
SUBSTITUTED FURANS.
EVIDENCE OF NITROREDUCTIONINDEPENDENT CYTOTOXIC
EFFECTS.
Gallardo C.A. 1,2, Pessoa-Mahana H. 2,
Faúndez M. 1.
1,
Laboratorio
de
Farmacología
y
Toxicología Molecular, Facultad de Química,
Pontificia Universidad Católica de Chile. 2,
Departamento de Química Orgánica y Físico
Química, Facultad de Ciencias Químicas y
Farmacéuticas, Universidad de Chile.
Nitrofurans constitute a family of drugs that
have antibacterial and antiparasitic effects.
The consumption of these causes various
adverse effects which are mainly attributed to
the monoelectronic reduction of the 5-nitro
group mediated by host enzymes. This triggers
the generation of nitroanion radical which,
upon entering redox cycling with molecular
oxygen, results in the formation of superoxide
anion radical and the regeneration of the
nitro derivative. The generated oxidative
stress allows to explain, at least in part, the
adverse effects attributed to nitrofurans.
However, not all side effects are attributable
to that mechanism. In order to demonstrate
that the cytotoxic effects of nitrofurans
are not solely due to the reduction of the
5-nitro group, derivatives of 3 commercial
nitrofurans (nitrofurazone, nitrofurantoin
and nifuroxazide) were synthesized in
which the 5-nitro group was substituted by
Methyl, Bromo or Hydrogen, maintaining the
parental skeleton. The synthesis was carried
out in aqueous solution through ultrasound,
mixing the aldehydes with the corresponding
hydrazines. Subsequently, the effect of these
derivatives on cell viability and their ability
to generate intracellular ROS was evaluated.
The results show that the cytotoxic effect
of furan derivatives is dependent on the
presence of electron withdrawing groups
in position 5 of the furan ring; while the
generation of intracellular ROS does not
depend exclusively on the presence of the
5-nitro group. This suggests new toxicity
mechanisms independent of oxidative stress
induced by redox cycling of nitrofurans.
44. ELEPHANT BLACK
GARLIC EXTRACT PREVENTS
MITOCHONDRIAL DYSFUNCTION
INDUCED BY BETA-AMYLOID
PEPTIDE IN MOUSE HIPPOCAMPAL
SLICES.
Gavilán J.1; Panes J.1; Salgado G.1; Godoy
P.A.1; Muñoz N.2; Ramírez-Molina O.1; Varas
P.4; Pérez C.3; Yévenes G.2; Fuentealba J.1.
1, Laboratorio de Screening de Compuestos
Neuroactivos, Departamento de Fisiología,
Facultad
de
Ciencias
Biológicas,
Universidad de Concepción. 2, Laboratorio
de Neurofarmacología, Departamento de
Fisiología, Facultad de Ciencias Biológicas,
Universidad de Concepción. 3, Laboratorio
de Química de Productos Naturales,
Departamento de Botánica, Facultad de
Ciencias
Naturales
y
Oceanográficas,
Universidad de Concepción. 4, Agrícola
Melimei, Manao, Ancud, Chiloé.
Soluble oligomers of the beta-amyloid
peptide (SO-Aβ) are central elements in the
pathogenesis of Alzheimer’s Disease (AD).
It has been demonstrated that SO-Aβ forms
pores in the plasma membrane which leads
to Ca2+ influx and leakage of large molecules,
such as ATP. Cytosolic Ca2+ overload induces
mitochondrial dysfunction and synaptic
failure resulting in cell death. Some studies
suggest that common black garlic (Allium
sativum) prevents the toxicity of SO-Aβ due to
its enriched composition of sulfur metabolites.
However, there are no studies on the biological
activity of elephant black garlic (Allium
ampeloprasum), and recently in our laboratory
we identified new sulfur compounds that are
not present in common garlic. The objective
of this work was to evaluate neuroprotective
properties of elephant black garlic extract (BG)
against SO-Aβ toxicity. In mouse hippocampal
slices, BG (20 μg/mL) prevented the decrease
in cellular viability induced by SO-Aβ (2.5
μM) by 60±6%. In parallel, BG kept the
mitochondrial membrane potential stable
compared with SO-Aβ (SO-Aβ: 67±3%; SOAβ+BG: 96±5%), suggesting a direct effects
on mitochondrial function; additionally, the
intracellular (SO-Aβ: 70±6%; SO-Aβ+BG:
110±9%) and extracellular ATP levels (SOAβ: 180±23%; SO-Aβ+BG: 125±16%), were
recovery when BG was present. We also
observed that BG normalized the levels of the
mitochondrial fusion protein MFN1 (SO-Aβ:
49± 10%; SO-Aβ+BG: 91±13%). On the other
hand, our results showed that BG preserved
the synaptic structure and prevented the
decrease in SV2 (SO-Aβ: 64±4%; BG: 97±9%)
and PSD95 proteins (SO-Aβ: 45±8%; SO-
Aβ+BG: 76±7%) and also the decrease on
transient intracellular calcium induced by SOAβ (54±5%). Finally, our results suggest that
the bioactive compounds present in BG could
be new pharmacological tools to treat the SOAβ toxicity.
45. ROLE OF ROCK-1 AND ROCK-2
IN ADHESION AND MIGRATION OF
TRYPANOSOMA CRUZI- ACTIVATED
MACROPHAGES TREATED WITH
ATORVASTATIN.
González-Herrera, Fabiola 1; Cerutti,
Camilla2; Clayton, Natasha2; GuzmánRivera, Daniela1; Vivar, Raúl1; Ridley, Anne2;
Maya, Juan Diego1.
1, Programa de Farmacología Molecular
y Clínica - ICBM, Facultad de Medicina,
Universidad de Chile. 2, School of Cellular and
Molecular Medicine, University of Bristol.
Chagas Disease (CD) is caused by protozoan
Trypanosoma cruzi (T. cruzi). The most
severe CD clinical manifestation is Chronic
Chagas cardiomyopathy (CCC). Currently
treatment is benznidazole, a trypanocidal
therapy, ineffective against CCC. Therefore,
is interesting to develop new therapeutic
pharmacologic strategy. It has been observed
in autopsies of patients with CCC that present
an important leukocytes infiltration in cardiac
tissue, and more of 50% are macrophages.
The macrophage infiltration needs the
macrophages adhesion and migration from
the vascular endothelium to damage site.
Rho-associated protein kinase (ROCK) 1 and
2 are serine-threonine kinases activated by
small GTPase RhoA. ROCK phosphorylate
myosin light chain (MLC), promoting cellular
contraction and generating focal adhesion.
On the other hand, ROCK phosphorylate
cofilin, increasing actin polymerization.
Therefore, ROCK activation affects cellular
migration and adhesion. In our laboratory
we have observed, T. cruzi activates
ROCK in human macrophages, leading to
proinflammatory phenotype and atorvastatin
inhibited ROCK, changing the phenotype of
this cells. However, it has not been studied
ROCK-1 and ROCK-2 role in T. cruzi effect
and atorvastin over macrophages adhesion
and migration. Human macrophages U937
were used, with a knockdown for ROCK-1 and
ROCK-2 and with both proteins constitutively
active by nucleofection. T. cruzi-activated
macrophages were treated with atorvastin
and adhesion process to endothelial cells
(HUVEC) were determined by microscopy
and western blot. Also, migration was studied
by microscopy time lapse.
109
It was observed that both isoforms are involved
in adhesion and migration increase promoted
by T. cruzi and is inhibit by atorvastin. These
results allow us to propose atorvastin as a
therapy to decrease macrophages infiltration
in CCC.
of NMDAR that impair the GABAergic
interneurons maturation and function,
decrease the GABA release, which can reverse
the temporal dependence of STDP-LTD
modifying the synaptic plasticity and function
of mPFC network in adulthood mice.
46. IMPAIRMENT OF SPIKE
TIMING-DEPENDENT PLASTICITY
IN PREFRONTAL CORTEX IN A
KETAMINE TREATED MICE.
Guiffa F1,2, Morales K1, Sotomayor-Zárate
R1, Bonansco C1 and Fuenzalida M1.
1- Centro de Neurobiología y Fisiopatología
Integrativa (CENFI), Instituto de Fisiología,
Facultad de Ciencias, Universidad de
Valparaíso, Valparaíso, Chile. 2- Magíster en
Ciencias Biológicas mención Neurociencia,
Facultad de Ciencias, Universidad de
Valparaíso.
47. CHARACTERIZATION OF THE
ADIPOGENIC PROTEIN E4ORF1
FROM ADENOVIRUS 36 THROUGH
AN IN-SILICO APPROACH.
Gutierrez A. 1, Monteiro G.F. 2.3, Machuca J.
4, Venthur H. 4, Feres F. 3, Hirata M.H. 2.3,
Hirata R. 2.3, Cerda A. 1.5.
Centro
de
Excelencia
en
Medicina
Traslacional, CEMT-BIOREN, Temuco, Chile.
The medial prefrontal cortex (mPFC) is a
key structure involved in cognitive functions
like working memory and decision-making.
Long-term changes in synaptic plasticity, i.e:
long-term potentiation (LTP) and long-term
depression (LTD) have been proposed as the
cellular substrate of these cognitive processes.
During the postnatal development, the
maturation of mPFC circuits depends largely
on the network of inhibitory interneurons,
which modulate the pyramidal neurons
(PYN) function. The mPFC interneurons
are especially vulnerable to injury during
adolescence and the impairment of GABAergic
interneurons function is involved in several
neuropsychiatric
diseases,
including
schizophrenia (SZ). However, it is still
unknown how the disruption of GABAergic
interneurons development during adolescence
can affect the long-term synaptic plasticity in
the adult brain. Using electrophysiological and
pharmacological approaches, we evaluate the
efficacy of synaptic transmission and spiketiming-dependent plasticity in mPFC in an SZ
mice model based in the adolescence treated
with non-competitive NMDAR antagonist
ketamine (Ket). Through recording in PYN of
the layer II / III of mPFC slices in adulthood, we
found that the frequency of spontaneous and
miniature inhibitory post-synaptic currents
(sIPSC and mIPSC) was lower in Ket treated
animals than control. Also, we observe that
the paired-pulse ratio of eIPSC was higher in
Ket mPFC slice than control. Using the STDP
protocol we found that while the protocol in
t-LTP induced a similar potentiation that in
control, the t-LTD protocol was unable to
induce depression, conversely it can induce
LTP. These data suggest that hypofunction
110
Adenovirus 36 (Ad-36) is related to human
obesity due to its adipogenic activity mediated
by the Early 4 Open Reading Frame 1 (E4orf1)
protein. Mechanisms underlying adipogenic
effect of E4orf1 are not completely understood;
however, it has been characterized the
increased proliferation and differentiation
of fat cells through the activation of the
Phosphatidyl Inositol 3 Kinase pathway by
binding proteins containing PDZ-domains.
We aimed to characterize E4orf1 structure
and analyze its interactions with PDZ-domain
containing proteins in order to recognize
important residues with pharmacological
purpose. In-silico approaches such as
homology modeling, molecular dynamics and
molecular docking between E4orf1 and five
PDZ-domains from different proteins (PDZ1 and 2 from Disk Large Homolog 1; PDZ-3
from Membrane Associated Guanylate Kinase
1; and PDZ-7 and 10 from Multy PDZ-Domain
Protein 1) were performed. Mutagenesis
of selected residues was performed to
evaluate its importance in the stabilization
of E4orf1:PDZ complexes. We predicted the
first 3D model of E4orf1, which suggests a
key role of residues at c-termini region (114
to 125), demonstrating its importance in
initial stabilization. The complex formed by
predicted E4orf1 and PDZ10 was more stable
than others. Moreover, residues at “core”
region (residues 80 to 85) in E4orf1:PDZ10
complex were important in stabilization as
demonstrated by its electrostatic interactions.
Mutagenesis highlighted residues 80-85,
demonstrating its importance in complex
stabilization. In conclusion, E4orf1 forms a
stable complex with PDZ10 domain, being
residues 80-85 of particular importance.
Characterization of E4orf1 interactions
provides a first approach in discovering
druggable targets for Ad-36 induced obesity.
48. INTERFERON AS THERAPEUTIC
CANDIDATE FOR CANINE VIRAL
DISEASES.
Gutierrez N. 1; Espinoza F. 1; Hidalgo A. 1;
Lamazares E. 1;Toledo J.R. 1.
1, Laboratorio de biotecnología y biofármacos,
Facultad de Cs Biologicas, Universidad de
Concepción.
Chilean dog population is estimated in 3,2
million, with a continue growing in pet market
associated with care products. In health
expenditure, vaccines reach 25% of total cost
in pet’s care. Among pathologies that affects
pets, viruses are the main source of infectious
diseases, including distemper, parvovirus
and tracheobronchitis. Vaccines against
parvovirus are currently available, however
still is considered one of the most important
disease worldwide, with a high prevalence
level in our country. Nowadays, there is
not a large spectrum and specifics antiviral
drugs, having to use human antivirals or
from another species. Specifically, cytokines
used as therapeutics activates immune
system and helps to defense against virus,
preventing replication and infection. In this
work we developed a prototype drug for
canine specifically antiviral treatment, based
in a recombinant dog-derived interferon. The
molecule was produced in a E. coli expression
system. Besides, specific interferon activity
was measured by OAS-2 and PKR mRNA
quantification, in Madin Darby Canine
Kidney (MDCK) cell line and dog lymphocytes
primary cultures stimulated with Canine
recombinant interferon was obtained with
purity around 88% and the antiviral markers
were enhanced in canine cells, hence the
cytokine could be an option for antiviral
therapy in canine population.
where well-characterized morphological and
functional alterations occur. At present there
is no specific and early urinary marker that
allows determining the risk of developing
diabetic nephropathy once diabetes is
established. Therefore, the objective of this
work was to evaluate SGLT-2 as a specific
urinary marker of renal damage in a murine
model with early diabetic nephropathy. Type
1 diabetes was induced in Wistar rats by
administration of streptozotocin (60 mg / kg,
ip). Diabetic rats were sacrificed 21 days after
induction. Proteinuria and creatinine were
determined as renal function tests. Western
blotting analyzed the expression of SGLT-2 and
β2-microglobulin. An increase in the urinary
excretion of SGLT-2 was found in the third
week of damage with diabetic nephropathy
and the presence of β2-microglobulin in the
urine as a marker confirmed the presence
of damage. These results suggest SGLT2 as a new urinary marker of diabetic
nephropathy that allows predicting the risk of
developing diabetic nephropathy. Keywords:
Diabetic nephropathy, streptozotocin, β2microglobulin, SGLT-2
50. TMA-6(2,4,6TRIMETHOXYAMPHETAMINE)
MODULATES NEUROPLASTICITY
IN THE PREFRONTAL CORTEX
AND ENHANCES HEAD-SHAKES
BEHAVIOR IN SPRAGUE-DAWLEY
RATS.
Hernández, A.1; Klink, A.2,3; Castro-Castillo,
V.4; Barra, R.5,6; Sáez-Briones, P.2,5.
1 Laboratory of Neurobiology, Faculty
of Chemistry and Biology, Universidad
de Santiago de Chile. 2 Laboratory of
Neuropharmacology and Behavior, Faculty
of Medical Sciences, Universidad de Santiago
de Chile. 3 Studiengang Biophysik, Johann
Wolfgang Goethe Universität (Frankfurt am
Main, Germany). 4 Department of Organic
Chemistry and Physical Chemistry, Faculty
of Chemical and Pharmaceutical Sciences,
Universidad de Chile. 5 School of Medicine,
Faculty of Medical Sciences, Universidad
de Santiago de Chile. 6 CIBAP, Faculty of
Medical Sciences, Universidad de Santiago de
Chile.
49. THE GLUCOSE TRANSPORTER
(SGLT-2) AS A POSSIBLE URINARY
MARKER OF EARLY DIABETIC
NEPHROPATHY.
Hernández- Carmona Luis A.¹, RodríguezMuñoz Rafael², Namorado-Tónix María del
Carmen² Graciela-Cervantez Luz Graciela²
and Reyes-Sánchez José Luis²
1) Pharmacology Dept., 2) Physiology,
Biophysics and Neurosciences Dept. Center
for Research and Advanced Studies, National The search for new analogs of the
Polytechnic Institute (CINVESTAV-IPN)
entactogen MDMA (3,4-methylenedioxymethamphetamine, “ecstasy”) is of great
Hyperglycemia is characterized by high blood relevance for the development of new
glucose concentrations (≥126 mg / dL) and drugs useful in the treatment of prevalent
over time these glucose levels damage renal neuropsychiatric diseases. While the
microvasculature. Diabetic nephropathy central effects described for MDMA
is the final complication of this pathology, clearly differ from other psychotropic
111
compounds, it has been proposed that the
presumed low potency hallucinogen TMA6 (2,4,6-trimethoxyamphetamine) might
induce MDMA-like effects at low doses
in humans. Since both hallucinogens and
MDMA might exert different effects on
cognitive processes, one may anticipate
differential effects with respect to
neuroplasticity at key sites of the central
nervous system. Here we compared the
acute effects of TMA-6 with MDMA on (i)
in vivo induction of long-term potentiation
(LTP) in the prefrontal cortex of the rat, and
(ii) induction of paroxysmal head rotations
termed “head-shakes”, which is considered
a behavioral proxy in rodents for human
hallucinogenic-like effects. The results
obtained showed that 20 mg/kg TMA-6 not
only prevented the induction of LTP in the
prefrontal cortex but turns it into a longterm depression-like event. In addition,
TMA-6 significantly increased the number
of head-shakes, verifying the hallucinogenic
nature of this compound. In contrast, 10
mg/kg MDMA significantly increased the
prefrontal cortical LTP but fully abolished
the number of head shakes. Taken together,
and unlike the presumption based on the
subjective interpretation of its effects in
humans, the inverse electrophysiological/
behavioral profile of TMA-6 referred to
MDMA suggests that the former seems
to lack entactogenic-like effects, rather
supporting the hallucinogenic essence of this
drug.
51. SITE-SPECIFIC PEGYLATION OF
L-ASPARAGINASE: AN ALTERNATIVE
FOR THE TREATMENT OF ACUTE
LYMPHOBLASTIC LEUKEMIA.
Herrera L. 1,2; de Oliveira C.2; Beltrán J.F. 1;
Pessoa A.2 and Farías J.G. 1.
1 Department of Chemical Engineering.
Universidad de La Frontera, Temuco. Chile; 2
Department of Biochemical-Pharmaceutical
Technology. University of São Paulo, São
Paulo. Brazil.
L-asparaginase (ASNase) is a therapeutic
enzyme considered a cornerstone in the
treatment of Acute Lymphoblastic Leukemia
(ALL), the most common cancer in children
worldwide. Four formulations of bacterial
origin are available in the market for the
treatment of ALL. However, despite their
effectiveness, they generate immunological
reactions, decreasing the action of the drug
and damaging patient safety. PEGylation
is one of the strategies adopted to reduce
the immunogenicity of asparaginase,
112
which consists of the covalent binding of
polyethylene glycol (PEG) chains to the
enzyme, at specific or random sites. This allows
reducing the recognition of the enzyme by
the immune system and its early elimination
from the bloodstream. In this work, we
performed the N-terminal PEGylation of
E.coli ASNase, with methoxy polyethylene
glycol-carboxymethyl N hydroxysuccinimidyl
ester (mPEG-NHS 10kDa) in 100 mM PBS
at pH 7.5 and PEG: ASNase ratio of 25:1. As
a result, we obtained the monoPEGylated
ASNase with an activity of 134 ± 11.5 IU/
mg and acceptable kinetic parameters.
MonoPEGylated ASNase also showed more
stability at different pH, temperatures and
against human serum proteases than the
native enzyme, demonstrating its potential as
a less immunogenic biopharmaceutical in the
treatment of ALL.
52. CHARACTERIZATION OF CA125
ANTIGEN EXPRESSED IN HUMAN
CERVICAL CARCINOMA CELLS.
Hidalgo A.1; Meza, C.1; Benavente, B.1; Leiva,
MJ.1; Montesino, R.1; Toledo J.R1.
1, Laboratorio de Biotecnología y Biofármacos,
Dpto. de Fisiopatología, Facultad de Ciencias
Biológicas, Universidad de Concepción.
Epithelial ovarian cancer is the seventh
death cause related to cancer in women
worldwide. Due to the absence of early
stage clinical symptoms, patients are usually
diagnosed when decease had spread further
than ovary with an unfavorable prognostic.
Cancer antigen 125 (CA125) is a serum
marker extensively used in gynecology for
monitoring epithelial ovary cancer patients.
It’s a repetitive peptide antigen of the
membrane glycoprotein MUC16, whose
over-expression in ovarian cancer has been
linked with both pro-metastatic and protumorigenic properties. In the present study,
we characterized the N-oligosaccharides
bonded to the C-terminal region of MUC16,
expressed in cervical carcinoma cell culture
by adenoviral transduction. Enzymatic
deglycosylation, HPLC, and MADI-TOF
analysis showed mainly complex type of
oligosaccharide N-linked, with bi-antennary,
mono-sialylated
and
mono-focusylated
core structures, predominantly. It has been
previously reported that N-glycan profiles
from cancer patients shows an increase of
these structures, compared to healthy groups.
Furthermore, these N-glycan structures have
been described as part of innate and adaptive
immune response recognition by CA125
antigen. Concluding, the glycosylation status
of the CA125 may provide specific biomarkers in order to study steric effect into the
and therapeutic target for gynecologic cancer. binding site. Our results indicate that, some
compounds are able to displace radioligands
from nicotinic receptor and MAT, showing a
53. DESIGN, SYNTHESIS AND
promiscuous behavior. However, the ranges
BINDING AFFINITIES OF
of affinities were in the micromolar order.
CYCLOALKYLAMINES AND
PIPERIDINES ESTERS DERIVATIVES In addition, docking experiments were
performing in order to rationalize the binding
ON ALPHA4BETA2 NICOTINIC
mode and the similar interaction between
RECEPTOR AND MONOAMINE
nAChR, SERT and DAT with our compounds.
TRANSPORTERS (HSERT AND
HDAT).
Hodar, M.1; Guerra-Diaz, N.2 ; Pessoa- 54. RATIONAL DESIGN AND
Mahana, H.2; Reyes-Parada, M.3; Iturriaga- BIOLOGICAL EVALUATION OF
TRIAZOLOPYRIDINES AGAINST T.
Vásquez, P.1.
1, Laboratorio de Farmacología Molecular CRUZI.
y Síntesis Orgánica, Depto. Cs. Químicas Lapier M.1-2; Ramos-Aguayo A.1; Quintero
y Recursos Naturales, Fac. de Ingeniería H. 3; Maya J.2.
y Ciencias, Universida de La frontera. 2 1Laboratorio de Antioxidantes y radicales
Departamento de Química Orgánica y libres, Departamento de Química Inorgánica
Fisicoquímica, Facultad de Ciencias Químicas y Analítica, Facultad de Ciencias Químicas
y Farmacéuticas, Universidad de Chile. 3, y Farmacéuticas, Universidad de Chile.
Centro de Investigación Biomédica y Aplicada 2Laboratorio de Bioquímica, Metabolismo
(CIBAP), Escuela de Medicina, Facultad de y Resistencia a Fármacos, Instituto de
Ciencias Médicas, Universidad de Santiago Ciencias Biomédicas ICBM, Facultad
de Medicina, Universidad de Chile.
de Chile.
3Laboratorio de Productos Naturales,
The
monoaminergic
and
cholinergic Departamento de Química Farmacológica y
neurotransmitter systems exhibit, in the Toxicológica Facultad de Ciencias Químicas y
central nervous system (CNS), a wide range Farmacéuticas, Universidad de Chile.
of functional interactions and mutual
regulations. Furthermore, acetylcholine Since a while ago, Azole-containing
(ACh) actions mediated by nicotinic receptors compounds have been recognized for their
(nAChRs), as well as monoamines such as antifungal features and with well-known
serotonin (5-HT) and dopamine (DA), are applications in current clinical field. Some of
involved in the modulation of several brain features highlighting azole heterocycle come
functions, including (but not limited to) from its specific metabolism, those related
cognition, voluntary movement, motivation to its high binding affinity to heme-containg
and reward, mood, attention and learning, proteins such as cytochrome p450. The p450
as well as in the physiopathology of a complex has the function of metabolizing
variety of diseases. In addition, most of the drugs, exogenous and endogenous molecules
drugs currently used for the treatment of that could render in cell toxicity. Lanosterol
neurological and neuropsychiatric disorders 14α demethilase (CYP51) is the key enzyme
such as Parkinson`s and Alzheimer`s in sterol biosynthesis in Trypanosoma cruzi
diseases,
depression,
drug
addiction, (Tc), agent that causes Chagas disease.
schizophrenia, etc., have mechanisms of The inhibition of this enzyme will induce
action associated to the regulation of one or accumulation of toxic metabolites that cause
more of these systems. Indeed, there are some the death of the parasite. Recently, we have
examples of therapeutically useful drugs, synthesized and characterized derivatives
which act through simultaneous interactions of triazolo pyridines, based on a rational
with SERT/DAT and nAChRs. Therefore, study (structure-activity), where we found
it seems attractive to search/formulate that compound 1 has antiproliferative effect
ligands that show such a promiscuous profile against the replicative form. From this
fusing structural aspect of nicotinic ligands structure, we obtained 24 molecules with
and antidepressants. Here, we design and variations in positions 3 and 7 in the ring
synthesize cycloalkylamines and piperidines [1,2,3] triazolo [1,5-a] pyridine with different
esters derivatives. Binding experiments were electrophiles such as pyridines, thiophenes,
assessed for alpha4beta2nAChR on brain benzenes and pyrazines. Studies (structure/
synaptosomes and hSERT and hDAT from activity) of this new series have shown that
specific cell lines. The ester moieties were the compound 2 increased the trypanocidal
acetyl, propionyl and benzoyl derivatives potency. On the other hand, preliminary
113
docking studies showed that the association
energy of the compound to CYP51 are similar
to the binding energy values of fluconazole
to CYP51, our approximations have shown
that both the azole fraction and pyridine
are potentially related to the coordination
with hemo. This has led to design of a new
proposal of antichagasic drugs with a possible
action on CYP51.
Through a systematic analysis of 26 analogs
of 2,6-DTBP, the present work allowed the
identification of novel a3GlyRs PAMs with
improved efficacy. In addition, the generation
of a pharmacophore based on these findings
expand the possibilities for further design
and development of new GlyRs PAMs.
56. ATORVASTATIN AND
ROSUVASTATIN TREATMENT
55. EXPLORING NOVEL ALLOSTERIC CONTRIBUTES TO THE DECREASE
MODULATORS OF ALPHA3 GLYCINE OF PROLIFERATION IN HUMAN
RECEPTORS: STRUCTURE-ACTIVITY UMBILICAL ARTERY SMOOTH
MUSCLE.
RELATIONSHIP OF 2,6-DI-TERTLeal, K. 1 ; Saavedra, K. 1 ; Salazar, L.A. 1.
BUTYLPHENOL DERIVATES.
Lara, C.O. 1; Burgos, C.F.1, San Martin V.P.1, 1 Centro de Biología Molecular y
Universidad
de
La
Marileo A.M.1, Sazo, A.E.1, Moraga-Cid, G.1; Farmacogenética,
Frontera, Temuco.
Yévenes, G.E. 1.
1 Department of Physiology, Biological
The
treatment
of
choice
for
Sciences Faculty, University of Concepción.
hypercholesterolemia is the use of statins.
Glycine Receptors (GlyRs) are pentameric Enzymes that inhibit the enzyme 3-hydroxyanion-permeable ligand-gated ion channels methylglutaryl coenzyme A reductase,
highly expressed in spinal cord and brain which is a limiting factor in the biosynthesis
stem, where mediate processes such as motor of liver cholesterol. Despite this, clinical
coordination and sensorial processing. The studies have revealed that statins can exert
modulation of glycine receptors composed atheroprotective effects, beyond the decrease
by alpha3 subunit (a3GlyRs) by positive of serum cholesterol. These effects have been
allosteric modulators (PAMs) has been called pleiotropic effects and include antiassociated to generation of analgesia in proliferative and anti-migratory properties
chronic pain models. Previously, we shown in vascular smooth muscle cells, which are
that the non-sedative propofol analog a key cell type in atherosclerotic plaque
2,6-di-tert-butylphenol (2,6-DTBP) is a development. For this reason, Human
PAM of a3GlyRs. Here, we analyzed series Umbilical Artery Smooth Muscle cultures
of 2,6-DTBP analogs using bioinformatics were stimulated with 10 ng/ml of plateletand electrophysiological methods. Whole- derived growth factor and treated with 20
cell recordings shown that 2,6-DTBP (0,1 μM atorvastatin and 20 μM rosuvastatin
mM) potentiate a3GlyRs-evoked currents for 24 hours. The proliferative effect was
in a 171±21%. The first set of experiments subsequently evaluated for 48 hours by
evaluated the impact of the tert-butyl group spectrophotometry using the CellTiter 96®
positions around the phenolic core. These AQueous One Solution Reagent colorimetric
studies concluded that the presence of two assay. The migratory effect was also evaluated
tert-butyl groups at the position 2 and 4 for 4 hours by optical microscopy using
around the phenolic core (i.e. 2,4-DTBP) transwell with 8 μm pores. Migrated cells
were sufficient to generate an a3GlyR PAM were counted by means of ImageJ software
with a significantly higher efficacy. A second with automated macros function commands.
set of molecules evaluated the impact of It was observed that 20 μM statins treatment
additional chemical groups on the 2,4-DTBP reduces cell proliferation in a 48-hour period
scaffold (i.e. methyl, ethyl, oxime, amine, (p=0,05). However, there is no decrease in
carboxylic, sulfonamide groups). Whole- migratory cell capacity after being treated
cell recordings shown that ≈30% of these with atorvastatin (p=0,05) and rosuvastatin
compounds displayed improved efficacy in (p=0,05). For this reason, atorvastatin and
comparison to 2,6-DTBP (≈3-4 fold of a3GlyR rosuvastatin treatments might contribute to
potentiation). Bioinformatics analysis shown the proliferation reduction in smooth muscle
that all the compounds evaluated have an cells, which are involved in atherosclerotic
ADME profile compatible with drugs-like plaque formation. Financial support:
molecules. Pharmacophore modeling shown FONDECYT 1171765 & DIUFRO DI19-2018
that the localization of polar, hydrophobic
and charged groups within the 2,4-DTBP
is pivotal for the a3GlyR modulation.
114
57. EXPRESSION AND
CHARACTERIZATION OF A NOVEL
SINGLE-CHAIN ANTIBODY AGAINST
VASCULAR ENDOTHELIAL GROWTH
FACTOR (VEGF) IN GOAT MILK.
Leiva-Carrasco M. J1., Parra N2., Montesino
R1., Sánchez O2., Macaya-Zapata L2., Toledo
J.R1.
1, Laboratorio de Biotecnología y Biofármacos,
Departamento de Fisiopatología, Facultad
de Ciencias Biológicas, Concepción. 2,
Laboratorio de Biofármacos Recombinates,
Departamento de Farmacología, Facultad
de Ciencias Biológicas, Universidad de
Concepción.
Immunology Department, Pharmacy Faculty,
Concepción University.
Type II Diabetes Mellitus (T2DM) have an
estimated 8.5% of the world adult population
affected in 2014 and a projection of reaching
17.9% in 2060. Many different approaches
have been developed for tackling the noinsullinic management of hyperglycemia in
the diabetic patient, exploring the diverse
variety of targets that the multifactorial
nature of T2DM offers, which includes
classicals insulin sensitizers like metformin,
and also new pharmacological developments.
One new approach to control T2DM is
the use of sodium-glucose transporter 2
(SGLT2) inhibitors. While SGLT1 is involved
in intestinal glucose intake, SGLT2 is
located in the proximal tubules of kidneys,
and is responsible for most of the glucose
reabsorption (>90%). Inhibition of SGLT2
in combination with traditional treatments
have shown improvements in the control
of glycemia and a concomitant reduction
in cardiovascular risk. Challenges in the
development and extension of the chemical
space associated with the inhibitory activity
of SGLT2 are the high costs in time and
money of the in vitro research and the lack of
the human transporter atomic coordinates,
due to the difficulties to crystallizing
membrane proteins. These two problems
can be addressed through a methodology
that includes an exploration of molecular
dynamics from the protein transporter and
the structure-based virtual screening. We
have developed a robust pipeline perform a
virtual screening through massive molecular
docking against a diversity library of
molecules for the exploration of the chemical
space for the discovery of new inhibitors.
We use SGLT2 and known inhibitors to
perform a sequential refinement to optimize
the identification from a not supervised
methods and extend the methodology to the
other potential candidates transporter with
therapeutics applications.
Tumor angiogenesis is a hallmark of cancer
and plays a significant role in establishing
a vascular supply within the tumor
which is essential for tumor growth and
metastasis. The vascular endothelial growth
factor (VEGF) plays an important role in
angiogenesis process promoting endothelial
cell proliferation, migration, and invasion.
VEGF is overexpressed in many solid cancers
including lung cancer, one of the most
common cancers in the world. According
to this, different approaches have been
developed to suppress tumor angiogenesis,
including anti-VEGF monoclonal antibodies
as part of the cancer immunotherapy strategy.
However, high production costs limit the
widespread access to this treatment. In this
study, we designed a novel single-chain
monoclonal antibody (anti-VEGF) that can
bind to VEGF. This antibody can be efficiently
expressed in the mammary gland of goats by
adenoviral transduction and purified from
their milk. Results showed that anti-VEGF
was able to avoid cells migration in a wound
healing test and suppressed VEGF-induced
microvessel sprouting in rat aortic ring assay.
Furthermore, in vivo efficacy was evaluated
on a xenograft lung tumor model where antiVEGF treatment had an inhibitory effect
on tumor growth. Our findings suggest the
therapeutic potential of anti-VEGF as an antitumor agent correlated with suppression of
59. CHARACTERIZATION OF THE
angiogenesis.
INTESTINAL MICROBIOTA OF
PEOPLE TREATED WITH INFUSIONS
58. DIVERSITY LIBRARY
OF ACAENA SPLENDENS AND
SEQUENTIAL SCREENING TO
ITS ASSOCIATION WITH THE
IDENTIFY COMMON DESCRIPTORS
INFLAMMATORY STATUS.
OF THE SGLTS INHIBITORS.
Lema J.M.1; Ormazabal, V.A.1,2; Zuñiga, Tabilo B.; Pino J.A.; Vieytes M.P.
Biomedical Sciences Laboratory, Faculty of
F.A.2; Salas-Burgos, A.1.
1, Nanocell Laboratory, Pharmacology Medicine, University of Atacama.
Department, Biological Science Faculty,
Concepción
University.
2,
Exosome Given the importance and usefulness of
Laboratory, Clinical Biochemistry and medicinal plants as therapeutic agents,
115
the Chilean Ministry of Health, under the
guidance of the world health organization
(WHO), has been working in recent years
on the regulatory framework of the policy of
Complementary Medicine and Traditional
Herbal Medicines with clinical evidence
support. Acaena splendens (Amores secos)
has been used in Chilean traditional medicine
for the treatment of fever and inflammation.
In addition, an infusion of Acaena splendens,
similar to that recommended by folk healers,
exhibited highly significant antiinflammatory
activity (46.7%) in a murine model of
inflammation. Additionally, there is an
emerging body of work on the human gut
microbiome and how it mediates feedback
between the foods we eat and our bodies. The
gut microbiome is also an important mediator
of inflammation in the gut and systemically.
Considering that the mechanism(s) by
which Acaena splendens modulate systemic
inflammation are unknown, we propose that
infusions of Acaena splendens could decrease
systemic inflammation through changes in
human gut microbiome. To test this idea
thirty healthy volunteers will consume an
infusion prepared from Acaena splendens
for six and twelve days and blood/fecal
samples will be obtained before and after the
treatment for microbiome analysis (Illumina
16S rRNA sequencing and Oxford Nanopore
MinIon) and inflammatory markers analysis
(cytokine array kits and ELISA cytokine kits).
The results from this proposed study related
with the effect of medicinal plants on the
composition of the intestinal microbiota and
its association with systemic inflammation
could provide new therapeutic strategies for
treatment of inflammation-related diseases
(e.g. colorectal cancer) a condition of high
prevalence in the Atacama region.
60. STUDY OF A MOLECULE THAT
INTERFERES IN GΒΓ BINDING WITH
THE CYTOPLASMIC DOMAIN OF
GLYCINE RECEPTOR α1.
López A.D.E; Guzmán L.
Laboratorio de Neurobiología Molecular,
Facultad de Ciencias Biológicas, Universidad
de Concepción.
Ethanol is the most widely used drug of abuse
in the world. Its effects go from desinhibition,
headaches, nausea, vomiting, even respiratory
depression and death. Recently, the glycine
receptor (GlyR) has been identified as one of
the targets in which this drug acts, enhancing
its inhibitory activity. This mechanism
involves the interaction of the cytoplasmic
domain of GlyR (GlyR-DC) with the βγ dimer
116
of the G protein (Gβγ). Through bioinformatic
studies, molecule M554 was selected, which
binds to Gβγ at the same site of interaction
for GlyR-DC inhibiting the effects of ethanol
in vitro and in vivo. In this project it was
studied whether this molecule inhibited the
interaction between these 2 proteins. For this
objetive, a fusion protein of GlyR-DC and
Glutathione S-transferase (GlyR-DC-GST)
was expressed and purified. Comparative
studies of GST pull-down showed that
GlyR-DC-GST retained its ability to interact
with Gβγ. At the same time GlyR-DC was
incubated with cell extracts, and the affinity
of GlyR-DC with Gβγ in the absence and in
the presence of 200 μM M554 was compared.
Densitometric analysis allowed to determine
that the interaction between both proteins
effectively decreased in the presence of this
molecule. Therefore, these results show that
this molecule decreases the binding capacity
of Gβγ with GlyR-DC, leaving clear that this
is the basal mechanism for the inhibition of
ethanol effects and supporting the projections
that M554 could have a pharmacological
potential to treat acute ethanol intoxication.
61. COMPLEX INHIBITION OF
OXPHOS AND Α-KETOGLUTARATE
DEHYDROGENASE COMPLEX BY
GENTISIC ACID-TPP+ INDUCES
CELL DEATH IN BREAST AND LUNG
CANCER CELL LINES.
López-Torres, C.; Fuentes-Retamal, S.;
Palominos, C.; Urra, F.; Catalán, M.; Ferreira,
J.
Clinical and Molecular Pharmacology
Program, Institute of Biomedical Sciences
(ICBM), Faculty of Medicine, University of
Chile.
Cancer cells have a more hyperpolarized
mitochondrial membrane potential than
normal cells, which allows selectively
guide towards mitochondria small cationic
molecules such as triphenylphosphonium
(TPP+), which participate as chemical
chaperones for pharmacophores moieties.
This property was used to synthesize from
the natural product, gentisic acid, derivatives
bound to TPP+ (GA-TPP+C10). This
mitocondriotropic compound is capable of
produce a time-dependent complex inhibition
of mitochondrial bioenergetics characterized
by 1) initial phase of mitochondrial
uptake with uncoupling effect of oxidative
phosphorylation, 2) inhibition of complex
I-dependent respiration and, 3) a late phase of
mitochondrial accumulation with inhibition
of
alfa-ketoglutarate
dehydrogenase
complex activity. This complex is part of the
tricarboxylic acid cycle composed of three
subunits that oxidizes and decarboxylates
alfa-ketoglutarate which is necessary for the
synthesis of aspartate, an essential amino acid
to proliferation and cell survival. The above
was verified using human breast and lung
cancer cell lines, by the addition of exogenous
permeable metabolites: alfa-ketoglutarate
(dm-alfaKG), aspartate (m-Asp) and pyruvate
(pyr). It was shown that dm-alfaKG and
m-Asp, but not pyr, produce a decrease in cell
death caused by GA-TPP+C10. Moreover, the
bioenergetic crisis induced triggers a drastic
mitochondrial membrane potential drop,
G1-phase cell cycle arrest with a significant
increase in ROS. In addition, this blockade of
mitochondrial functions triggers a metabolic
remodeling toward glycolysis and pro-survival
AMPK activation. Our results describe an
anti-cancer mechanism of GA-TPP+C10 that
induce a complex inhibition of mitochondrial
bioenergetics in a time-dependent manner
in breast and lung cancer cells that may have
relevance at therapeutic level.
62. ELECTROPHYSIOLOGICAL
RECORDINGS OF 3-(PYRIDIN-3YLMETHOXY)QUINUCLIDINE (Q-01),
A NOVEL SELECTIVE COMPOUND
FOR THE Α7Β2 NICOTINIC
ACETYLCHOLINE RECEPTOR.
López J.J.1; Mejía-Piedras J.2; HernándezAbrego A.2; García-Colunga J.2.
1, Department of Organic Chemistry,
Faculty of Chemical Sciences, University
of Concepcion, Concepcion, Chile. 2,
2Departamento de Neurobiología Celular
y Molecular, Instituto de Neurobiología,
Universidad Nacional Autónoma de México,
Querétaro, México.
A novel heteromeric alpha7beta2 nicotinic
acetylcholine
receptor
(nAChR)
with
functional properties different from those
of alpha7 and alpha4beta2 nAChRs, was
recently identified. Although its functions
are not known, it appears this nAChR may
be involved with Alzheimer’s disease. To date
there are no synthesis reports of ligands for
alpha7beta2 nAChR. Our work was based
on the design and synthesis of new ligands,
as well as on establishing their possible
effects with electrophysiological records in
interneurons from the stratum radiatum of
the rat hippocampal CA1 region. Thus, of
eleven synthesized ligands, only Q-01 and
EQ-01 inhibited the Choline-induced ionic
current: 51 and 100%, respectively; that is to
say, these acted as antagonists of alpha7 and
alpha7beta2. However, Q-01 presented an
inhibition similar to dihydro-beta-eritroidine
(selective antagonist of nAChR containing the
subunit beta2); suggesting that Q-01 might be
more selective for alpha7beta2 nAChR than
EQ-01. To understand the electrophysiological
results, molecular docking studies were
performed for the compound Q-01 at the
nAChRs α7 and alph7beta2. These studies
suggest that Q-01 would be more selective by
subtype alpha7beta2.
63. STRUCTURE-BASED VIRTUAL
SCREENING STUDIES TO IDENTIFY
NOVEL POTENTIAL AGONISTS FOR
SALMO SALAR GHSR1A-LR.
Macaya-Zapata L. 1; Starck-Méndez M. F. 1,3;
Toledo J. R. 2,3; Acosta J. 2; Sánchez O. 1,3.
1 Laboratorio de Biofármacos Recombinantes,
Departamento de Farmacología, Facultad
de Ciencias Biológicas, Universidad de
Concepción. 2 Laboratorio de Biotecnología y
Biofármacos, Departamento de Fisiopatología,
Facultad de Ciencias Biológicas, Universidad
de Concepción. 3 Centro de Biotecnología y
Biomedicina Spa.
Ghrelin is a growth hormone (GH)
secretagogue and functions primarily as a
GH-releasing hormone and as an orexigen. It
has also been documented to be involved in
the immune system, stress response, energy
metabolism and growth in fish. Its receptor,
Growth hormone secretagogue receptor
(GHS-R), is a class A G protein-coupled
receptor (GPCR) mostly expressed in the
hypothalamus. In Atlantic salmon (Salmo
salar) and other salmonids, a ghrelin receptor
isoform called GHSR1a-LR has been identified.
This receptor has an unique characteristic: the
second extracellular loop (ECL2) is notably
longer than in others GHS-R. In particular,
GHSR1a-LRs have the characteristic that
ghrelin or GH secretagogues treatment
either does not increase intracellular
Ca2+ or requires pharmacological doses
to activate the receptor. Given the high
conservation in folding and topology of class
A GPCR receptors, a comparative model of
GHSR1a-LR receptor from Salmo salar was
generated. This model was based on the
crystallographic structure of Neurotensin-1
receptor in active-like conformation (PDB
ID: 4XES). Subsequently, a conformational
exploration was carried out through
accelerated molecular dynamics simulations
(aMD). These simulations allowed us to
obtain diverse conformational variants
(inactive, intermediate and active), favoring
the selection of small molecules compatible
117
with the receptor active state. Finally, we
searched for potential agonists through
virtual screening, applying an ensemble
docking based strategy using multiple
snapshots of GHSR1a-LR receptor extracted
from aMD trajectories. A small molecule
library, containing 4997 positive or neutral
charged compounds obtained from ZINC12
database, was filtered under parameters of
drug-like properties and possible central
nervous system activity. From this campaign
four agonist-potential molecules, based
on predicted affinity in different receptor
structural samples, were identified.
that gelsemine did notmodify the fraction
of desensitized current or the decay time
constant of receptors. Our results that
gelsemine is able to negatively modulate the
synaptic activity of cortical neurons. Future
studies may contribute to shed light on the
mechanisms underlying the beneficial effects
of the Gelsemium alkaloids in the control of
pathological anxiety through the modulation
of inhibitory receptors.
65. CYTOTOXIC EFFECT OF
HYDROXYCHLOROQUINE,
ITRACONAZOLE AND CISPLATIN
ON SPHEROID CULTURE OF ORAL
SQUAMOUS CELL CARCINOMA.
Martínez D.; Yévenes S.
Universidad de Chile.
64. SYNAPTIC EFFECTS OF THE
ALKALOID GELSEMINE ON
CORTICAL NEURONS.
Marileo, A.M.1; Gavilan, J.1; Lara, C.O.1;
San Martín, V.P.1; Burgos, C.F.1; Sazo, A.1; Oral Squamous Cell Carcinoma (OSCC) is
the most common type of oral cancer and
Yévenes, G.E.1.
Cisplatin is the chemotherapeutic agent
Universidad de Concepción.
most commonly used, which induces cell
Several behavior studies have suggested that death by apoptosis. Furthermore, tumor cells
the natural alkaloid gelsemine has different (TC) acquire resistance against cytotoxic
biological effect, such as analgesia and effect of the drug. Therefore, it is necessary
anxiolytic. Nevertheless, until now there is to develop more effective treatments based
few information about neurophisyological on the metabolic changes that occur in TC.
mechanism, and pharmacological target Hydroxychloroquine and Itraconazole are
associated to this biological effect. Early two drugs traditionally used in traditional
as
immunomodulator
and
studies from our lab have shown that medicine,
gelsemine decreases the frequency of antifungal, respectively. Recently, it has
glycinergic and glutamatergic events in the been described that they may have antitumor
spinal cord neurons culture that suggest effect, because Hydroxychloroquine may
important effect in the synaptic function. inhibit autophagy, a mechanism of adaptation
Despite these advances, it is unknown if to metabolic stress, and Itraconazole would
gelsemine can modulate GABAARs and disturb energy metabolism. Tumor spheroid
GABAergic synapsis, which is relevant in the cultures are an in vitro model suitable
pathological anxiety phenomena. Here, we for antitumor activity evaluation because
examined the functional effects of gelsemine they can reproduce tumors in vivo main
on native sistem using electrophysiological characteristics, such as hypoxia-related
techniques. Studies performed on cultured drug resistance and the presence of Cancer
neurons was realized to explore the potential Stem Cell (CSC), which may be responsible
effect of gelsemine in the synaptic activity of of chemotherapy resistance and tumor
cortical neurons, which express the functional recurrence. In this project the expression of
component of a GABAergic synaptic. the markers of CSC CD44, CD56 and ALDHA1
Our electrophysiological result show that in spheroidal cultures of Cal-27 (COCE cells)
gelsemine 50 mM produced a significative was evaluated through flow cytometry. The
reduction of the frequency, but not in the results show greater expression of these
amplitud of the GABAergic and glutamatergic markers in spheroidal cultures, compared to
synaptic activity. I addittion, Effects of monolayer cultures. In addition, cytotoxicity
gelsemine on the agonist sensitivity and on for spheroidal cultures was determined by
the desensitization rates of GABAAR native, the MTT assay for Hydroxychloroquine,
are evaluated. Analysis of concentration – Itraconazole and Cisplatin. The IC50s were
response curves revealed that gelsemine 249, 472 and 577 micromolar at 48 hours and
significantly
decreased
the
apparent 272, 298 and 261 micromolar at 72 hours,
affinity for GABAAR without changing the respectively. A viability decrease induced by
maximal current amplitudes. Analyses of these drugs was observed in a concentrationthe GABA-activated currents stimulated by dependent manner. At present is being
saturating agonist concentrations indicated evaluated if the drug combinations have
118
a synergy effect reducing the individually 67. SIMVASTATIN AND 15-EPILIPOXIN A4 INDUCE CARDIAC
required concentration.
REPAIR THROUGH NOTCH 1
ACTIVATION IN CHRONIC CHAGAS
66. NEONATAL EXPOSURE TO
CARDIOMYOPATHY.
TESTOSTERONE PROPIONATE
Guzmán-Rivera; D.1; González-Herrera; F.1;
INDUCES AN INCREASED
Lapier; M.1; Carrillo; I.1; Quinteros; H.1;
EXPRESSION OF RGS9-2 AND
Fuentes; S1.; Pesce; B1.; Castillo; C2.; Liempi;
PKCΒ2 IN NACC AND VTA OF ADULT
A2.; Kemmerling U2.; Maya; J.D.1.
FEMALE RATS.
Martínez-Pinto, J.; Müller, E.; Sotomayor- 1Molecular and Clinical Pharmacology
Program, Biomedical Sciences Institute
Zárate, R.
Laboratorio
de
Neuroquímica
y (ICBM), Faculty of Medicine, University of
Neurofarmacología, Centro de Neurobiología Chile, Santiago, Chile 2 Program of Anatomy
y Fisiopatología Integrativa (CENFI), and Developmental Biology, Biomedical
Instituto de Fisiología, Facultad de Ciencias, Sciences Institute (ICBM), Faculty of
Universidad de Valparaíso, Valparaíso, Chile. Medicine, University of Chile, Santiago, Chile.
Research in programming is focused on the
study of stimuli that alters sensitive periods in
development, such as prenatal and neonatal
stages, that can produce long-term deleterious
effects in various organs or tissues such as
the brain, affecting brain circuits and related
behaviors. Previously, we have demonstrated
that neonatal programming with sex
hormones affects the mesocorticolimbic
circuitry, increasing the synthesis and release
of dopamine (DA) in striatum and Nucleus
accumbens (NAcc); also, we have observed a
reduction of locomotor activity in response to
methylphenidate in female rats treated with
testosterone propionate (TP). However, is not
clear if the alterations observed in our model
are related to modifications in the signaling
pathway or DA release/uptake. Interestingly,
RGS9-2, which is expressed in dopaminergic
neurons, can inhibit the signal transduction
of dopamine receptor 2 (D2) and have been
related to drug addiction and movement
disorders. Also, PKCβ2 can increase the
amphetamine-stimulated dopamine efflux
regulating the Dopamine Transporter (DAT)
activity. The objective of this work was to
evaluate if the neonatal reprogramming
with Estradiol Valerate (EV) or TP affects
the expression of Rgs9-2 and Pkcβ2 in
NAcc and Ventral Tegmental Area (VTA)
in adult rats using qPCR. The expression of
Rgs9-2 and Pkcβ2 was increased in NAcc
and VTA of female rats treated with TP; no
significant changes were observed in males
under any condition. These results suggest
that the neonatal exposure to TP modifies
the expression of Rgs9-2 and Pkcβ2 in female
rats, and this modification can account for the
modifications in response to methylphenidate
observed in our model. Further analysis using
WB or IHC are needed to depict the functional
alteration in this model.
Chagas Disease, caused by Trypanosoma
cruzi, is endemic in Latin America and
worldwide because of migration. Without
appropriate treatment this disease progress
to a chronic phase that could affects the
heart. Chronic Chagas Cardiomyopathy
(CCC), the most severe clinical manifestation,
involves
a
progressive
inflammatory
myocarditis affecting ventricular wall
causing cardiovascular complications due to
diminished cardiac function and heart failure.
Despite intense research no one drug can
stop or reverse the progressive heart damage.
Simvastatin, a drug that decreases blood
cholesterol, has anti-inflammatory effects
and inhibits platelet aggregation. Previously,
we described that simvastatin reduces
myocardial inflammation caused by T. cruzi
through 15-epi-lipoxin A4 (15-epi-LXA4)
production, a pro-resolutory inflammation
molecule. Several reports suggest that
simvastatin activates Notch pathway after a
stroke enhancing blood flow by promoting
angiogenesis. CCC progress with myocardial
inflammation, endothelial damage with
micro focal ischemia and fibrosis. We propose
that simvastatin reverts cardiac damage in
the chronic T. cruzi infection by 15-epi-LXA4
production and Notch 1 pathway activation.
BALB/c mice were chronically infected
with T. cruzi Dm28c strain and treated with
simvastatin 1 mg/Kg/day and 15-epi-lipoxin
A4 25 μg/Kg/day for 20 days. At day 80
post-infection animals were euthanized to
analyze the heart, Notch pathway, fibrosis,
and angiogenesis process. In chagasic mice,
the cardiac function was restored with
simvastatin and 15-epi-lipoxin A4 treatment.
The Notch signaling pathway was active
in cardiac tissue, a finding that correlated
with drug treatment, the fibrosis process
was decreased, and angiogenesis was also
evidenced in this model. Thus, we concluded
119
that simvastatin and 15-epi-LXA4 improve
cardiac architecture and function through
Notch 1 activation by increasing blood flow
and decreasing cardiac remodeling. Thus,
it could be incorporated rapidly in CCC
treatment.
68. INHIBITORY ACTIVITY OF
ACETYLCHOLINESTERASE AND
BUTYRYLCHOLINESTERASE
FROM AMARYLLIS BELLADONNA
ALKALOIDS.
Mella M. 1,4; Iturriaga-Vásquez P. 2,4;
Quiroz A. 3,4; Moraga F. 3,4; Mutis A. 3,4;
Hormazábal E. 3,4.
1, Estudiante de Bioquímica, Universidad
de La Frontera. 2, Laboratorio de
Farmacoquímica y Síntesis Orgánica,
Universidad de La Frontera. 3, Laboratorio
Química Ecológica, Departamento Ciencias
Químicas y Recursos Naturales, Universidad
de La Frontera. 4, Centro de Excelencia en
Investigación Biotecnológica Aplicada al
Medio Ambiente (CIBAMA).
Alzheimer’s disease, a neurodegenerative
disorder characterized by an irreversible and
progressive loss of memory. Traditionally
the pharmacological treatment associated
with the disease in its medium to moderate
stages, or similar diseases related to a deficit
of the neurotransmitter acetylcholine are
mainly guided through inhibitors of the
enzyme acetylcholinesterase. Only four
compounds have been approved as treatment
against alzheimer diseases: donepezil,
rivastigmine, galantamine and memantine.
In most cases these drugs are well tolerated,
however, various side effects such as:
nausea, vomiting, among others may occur.
That is why it is necessary to search for new
molecules with pharmacological potential
for their treatment. A potential source of
acetylcholinesterase inhibitors is Amaryllis
belladonna, belonging to the Amaryllidaceae
family, widely distributed worldwide.
Several studies support the presence of
alkaloids in this species with varied biological
activities. Considering the variation in the
production of metabolites reported in this
species, depending on the geographical
distribution, it is interesting to analyze the
alkaloids present in the representative of
this family introduced in our country and its
pharmacological potential. The objective of
this research was to evaluate the inhibitory
activity of alkaloids isolated from Amaryllis
belladonna on enzymes acetylcholinesterase
and butyrylcholinesterase.The alkaloids
present in the plant’s bulbs were obtained by
120
maceration with methanol and subsequently
fractionated, for the isolation. The putative
alkaloid composition of the fraction was
analyzed by GC-MS, highlighting the
presence of type licorin and crinamine. The
inhibitory activity of the alkaloid extract
and the isolated compounds was evaluated
by the Ellman method, finding IC50 values
(ug / mL) for hexane, chloroform and
butanol extract of 17.12, 8.89 and 19.09 for
acetylcholinesterase and 77.27, 55.44 and
200 for butyrylcholinesterase respectively.
69. USE OF MEDICINES BY
SOUTHERN BRAZIL FARMERS
AND ITS RELATIONSHIP WITH
EXPOSURE TO PESTICIDES FROM
DIFFERENT CROPS.
Calinca Skonieski Karina Raquel Fagundes
Matheus Henrique Machado Bento Anderson
Joel Martino Andrade Daniel Barbosa de
Chaves Samara de Cesaro Cavaler Andressa
Talita Nunes Maiara Grasiela Rossi Fabiana
Elias Dalila Moter Benvegnú. Universidade
Federal da Fronteira Sul - Campus Realeza.
Agriculture in its development model has
not been addressing issues such as the
environment.
Consequently,
countless
substances ended up being released into
the environment, plenty of them with
the ability to alter the behavior of several
physiological systems, inducing numerous
pathologies. Concomitantly, several studies
have demonstrated a growing use of
medicines. Therefore, the goal of this study
is to investigate the relationship between
the use of medicines by farmers and their
exposure to pesticides. Upon approval by the
Research Ethics Committee from the Federal
University of the Southern Border, farmers
were randomly selected in two cities: Mafra,
Santa Catarina and Planalto, Paraná, both
in Southern Brazil. The subjects were then
asked to fill up a form for data collection. A
total number of 251 farmers participated in
the research, being 123 from Mafra and 128
from Planalto. The average age of the subjects
in this study was 48,4 ± 14,4 and 114 were
female while 137 were male. Out of these,
23,1% (58) are making use of neuropsychiatric
drugs, 32,7% (82) of cardiovascular drugs,
17,5% (44) of metabolic disorder drugs,
0,8% (2) of respiratory disorder drugs, 1,6%
(4) of gastrointestinal drugs and 2% (5)
of musculoskeletal purposes drugs. When
correlation tests were performed between
the type of crops and the drugs used by the
respective farmers, the results showed a
greater use of medicines for metabolic (p =
0.014) and musculoskeletal disorders (p =
0.025) from wheat crops farmers in Mafra.
Thus, the data suggests that farmers in wheat
crops are more likely to make use of drugs for
metabolism and musculoskeletal disorders,
as it might be related to the specific pesticides
used in this crop.
71. ANTITUMOR PROTOTYPE BASED
ON POLYMERIC NANOPARTICLES
WITH APPLICATION IN GENE
THERAPY.
Ñacato A.1; Cerro R.P.1; Rivas V.2; Rivas C.1;
Ramos T. 1; Gómez-Gaete C.2; Toledo J.R.1.
1, Biotechnology and Biopharmaceuticals
70. SUCRALOSE INTAKE IMPAIR
Laboratory, Pathophysiology Department,
THE HIPPOCAMPAL POSTSYNAPTIC
School of Biological Sciences, Universidad de
INHIBITORY CURRENTS.
Concepción. 2, Laboratory of Pharmaceutical
Muñoz-Perez de Arce A. 1,2,3; Bravo J.A. 2; Technology, Department of Pharmacy, School
Fuenzalida M. 1.
of Pharmacy, Universidad de Concepción.
1,
Laboratorio
Plasticidad
Neuronal,
Centro de Neurobiología y Fisiopatología Gene therapy is a therapeutic strategy mainly
Integrativa (CENFI), Facultad de Ciencias, focused on correcting altered or mutated
Universidad de Valparaíso; 2, Laboratorio genetic sequences, which can induce the
Neurogastrobioquimica,
Instituto
de development of hereditary or acquired
Química, Pontificia Universidad Católica de pathologies, such as cancer. Nanoparticles
Valparaíso; 3, Programa de Magíster Ciencias based on biocompatible polymers have been
Biológicas mención Neurociencia, Facultad used as carriers for therapeutic molecules,
due to their ability to encapsulate labile
de Ciencias, Universidad de Valparaíso.
molecules such as linear or plasmidial DNA
Non-caloric sweeteners (NCS) are widely by electrostatic interactions. Thus, it could
used in foods with the aim of reducing sugar prevent their degradation by nucleases present
consumption and caloric intake. Sucralose in the environment. Safety and effectiveness
is the most used NCS worldwide and in of the polymers make feasible in vivo tests
Chile, with the current “Labeling Law”, its and future commercial products as described
consumption has been increasing. Sucralose and approved by regulatory agencies, such
intake causes alterations in the composition as the FDA. The aim of this work was to
of the intestinal microbiota, which is design and evaluate a formulation based on
closely related to mental health through polymeric nanoparticles as gene therapy
the crosstalk communication between gut applied to prostate cancer. Nanoparticles
and brain. Considering that microbiota can were elaborated using a double emulsion
affect behavior and modulate GABA levels, method, with solvent evaporation, and
brain plasticity and cognitive function, we PLGA as the main matrix agent, associated
wonder whether NCS affect the integration with a cationic polymer. A model plasmid,
and synaptic function in hippocampal CA1 which transcribes a tumor progression
pyramidal neurons in adult Sprague Dawley blocker was encapsulated. Physicochemical
rats treated with 0.5% sucralose in the characteristics of nanoparticles were analyzed
drinking water for a period exceeding 17 by Zetasizer Nano, and their effect was
days. Using patch-clamp recordings in whole evaluated in vitro, in a human tumor cell line.
configuration we observe that membrane Nanoparticles were obtained in nanometric
potential pyramidal neurons NCS treated rats size range, with a polydispersion index (PdI)
have a more depolarized value than control less than 0.2, and the surface charge was
group and no effect on the trigger threshold positive. Morphologically, nanoparticles were
of action potentials. Also, we observe that spherical according to Transmission Electron
frequency of the spontaneous inhibitory Microscope images. It was also observed an
synaptic currents in PYNs is lower than control increase in the genetic transformation rate
slices. The paired pulse protocol did not show for human tumor cells, and an alteration in
differences between animals treated with the characteristic tumor progression markers
NCS and control, suggesting that NCS intake expression. These results are promising
modify the presynaptic and postsynaptic for the development of new therapeutic
excitability, no apparent effect on the release candidates based on nano delivery system for
of GABA. These results show for the first time complementary treatment in different types
that permanent consumption of sucralose of cancer.
may have affect GABAergic synaptic efficacy
in the central nervous system.
121
72. RESOLVIN D1 PREVENTS
CARDIAC HYPERTROPHY AND
FIBROSIS IN ANGIOTENSIN IIINFUSED C57BL/6 MICE.
Olivares-Silva F. 1,2; De Gregorio N. 2;
Sánchez-Ferrer C. 3,4; Peiró-Vallejo C. 3,4;
Díaz-Araya G.
1,2
1, Laboratorio de Farmacología Molecular,
Departamento de Química Farmacológica y
Toxicológica, Facultad de Ciencias Químicas
y Farmacéuticas, Universidad de Chile. 2,
Centro Avanzado de Enfermedades Crónicas
(ACCDiS), Facultad de Ciencias Químicas
y Farmacéuticas y Facultad de Medicina,
Universidad de Chile. 3, Departamento
de Farmacología, Facultad de Medicina,
Universidad Autónoma de Madrid. 4,
Instituto de Investigación Sanitaria Hospital
Universitario La Paz (IdiPAZ), Madrid,
España.
Background: Resolvin D1 (RvD1) is an
endogenous specialized lipid mediator
enzymatically derived from docosahexaenoic
acid and synthesized locally in acute
inflammatory processes, where it exerts proresolving effects demonstrated in diverse
pathological models. Angiotensin II (Ang
II), in cardiac tissue, contributes to the
development of cardiac hypertrophy and
fibrosis. To date, there are not studies on the
potential protection that RvD1 may provide
at the structural and functional level in an
Ang-II infusion model. Purpose: To evaluate
RvD1 effects in Ang II-induced cardiac
hypertrophy and fibrosis. Methods: Alzet®
osmotic mini-pumps filled with Ang II (1.5
mg/kg/day) were implanted in C57BL/6
mice for 14 days, previous basal left ventricle
(LV) functionality assessment. RvD1 (3 ug/
day) was injected intraperitoneally. At the
end of the infusion period, the animals were
sacrificed, and functional and histological
parameters were studied. Results: 14-day
Ang II infusion increased heart weight/tibia
length ratio, LV thickness, ejection fraction,
shortening fraction and collagen deposition
at the interstitial and perivascular area.
Treatment with RvD1 significantly prevented
LV dysfunction, hypertrophy and collagen
deposition in both areas. Conclusions: RvD1
prevents Ang II-induced cardiac hypertrophy
and fibrosis demonstrating cardioprotective
properties. Further studies will be performed
to elucidate the possible mechanisms
of action of RvD1. Ethics approval: The
Institutional Animal Care and Use Committee
of the University of Chile (CICUA) approved
the protocol (CBE2018-12).
122
73. ANTIBIOTIC SUSCEPTIBILITY
PROFILE OF HELICOBACTER PYLORI
IN THE ARAUCANÍA REGION.
Oporto M. 1.; Troncoso C.1; Cerda A.1;
Hofmann E.;2,3, Sierralta A.2,4; Ríos E.2,3;
Coppelli L.5; Barrientos L.1 Pavez M.1.
1 Centro de Excelencia en Medicina
Traslacional CEMT- UFRO, Temuco. 2
Departamento medicina Interna, UFRO,
Temuco. 3 Endoscopia CAT, Temuco. 4
Endoscopia HHHA, Temuco. 5 Endoscopía
H. Villarrica.
Introduction: Antibiotic resistance is one
of the main causes of therapeutic failure
in eradication treatments of Helicobacter
pylori (Hp), which currently and according to
Maastricht consensus the standard consists of
a triple scheme of a proton-pump inhibitors
(PPI) +2 antibiotics. In recent years, high
resistance rates to the main antibiotics used
in these treatments have been reported in
several countries. Even varying between
geographical areas of the same country, which
prevents generalizing efficient therapies
in certain populations without previous
susceptibility studies Aim: To evaluate the
susceptibility profile of Hp, in the Araucania
region, against antibiotics used in eradication
therapy. Methods: A descriptive study on
37 Hp, isolates from gastric biopsy samples
on dyspeptic patients was performed in
main health centers of the Araucania. The
susceptibility profile against amoxicillin,
clarithromycin, levofloxacin, metronidazole
and tetracycline was performed by agar
dilution. Minimum inhibitory concentration
values were evaluated according European
Committee on Antimicrobial Susceptibility
Testing, using Hp ATCC 43504 as a quality
control strain. Results: All isolates reported
resistance at least one antibiotic and 81.08%
showed resistance to two or more antibiotics.
13.8% of the Hp isolates were resistant to
amoxicillin, 45.94% to clarithromycin, 41.66%
to levofloxacine, 81.08% to metronidazole
and 16.66% to tetracycline. Conclusion:
The resistance rates to metronidazole,
clarothromycin and amoxicillin were higher
to reported in Chile and there are not previous
reports to LVZ. These results show the need
of future studies of therapeutic efficacy in the
Araucanía as well a new review of current
eradication strategies.
75. INHIBITORY ACTIVITY ON
GLYCOGEN PHOSPHORYLASE A OF
PHENOLIC EXTRACTS FROM LEAVES
AND FRUITS OF 8 UGNI MOLINAE
TURCZ GENOTYPES.
Ordóñez, J.L.1; González, J.1; Pérez, R.;
Bugueño, I.1; Guzman, P. 1; Seguel, I.2;
Delporte, C.1.
1, Laboratorio de Productos Naturales,
Departamento de Química Farmacológica y
Toxicológica, Facultad de Ciencias Químicas
y Farmacéuticas, Universidad de Chile; 2,
Instituto de investigaciones agropecuarias
The movement of glucose and other sugars and (INIA) Carillanca.
polyols with essential functions in the energy
metabolism of living beings through biological One of the strategies used for the discovery of
membranes is carried out by transporter natural products with hypoglycemic activity
proteins belonging to the family of sugar is the analysis of species used by traditional
transporters (SP family, TC code 2.A.1.1), medicine, such as Ugni molinae, Myrtaceae,
where we find the human GLUT transporters, popularly known as murtilla. In this context,
the hexoses transporters in yeasts, among the analysis of the inhibition on the activity of
others. There are highly specific transporters Glycogen phosphorylase A (Gpa), an enzyme
for a physiological substrate, and there are expressed in brain, muscle and liver, which has
others much more promiscuous. There is major role on post-prandial hyperglycemic
an increasing interest in pharmacology to peaks in diabetic patients, could account for
design GLUTs inhibitors, whose development potential candidates for the development
has risen since the determination of the of new treatments. Therefore, the aim of
crystallographic structure of human GLUT1. this work was to demonstrate and compare,
For glucose homeostasis diseases that through an in vitro spectrophotometric
occur with hyperglycemia and cancer, the methodology, the Gpa inhibitory activity of
therapeutic use of these molecules has been phenolic-rich extracts obtained from leaves
proposed. In the first case, its potential use and fruits of 8 murtilla genotypes from the
is in the downregulation of the incorporation INIA-Carillanca germplasm bank, which
of glucose to the blood, while in cancer the were cultivated at the same edaphoclimatic
aim is to avoid the dispensation of glucose conditions. Compared to caffeine (IC50 =
to cells with active proliferation. Several 5,3 ug / mL), the leaves ethanolic extracts
inhibitors of transporters are designed from were more potent (EETs; IC50 between 1,03
substrate/solute modifications. To advance – 3,52 ug / mL; p ≤0,05), while the fruit
in the understanding of the molecular acetonic extracts were less potent (EACs;
bases that explain the varied selectivity of IC50 between 27,9 – 86,1 ug / mL; p ≤ 0,05)
the SP family, we carried out the present than the reference substance, as well as less
work, which consists of the identification potent than the leaves extracts. Based on our
and characterization of sequence profiles results, the leaves and fruits of U. molinae
related to the specificity of substrates for could be a potential source of bioactive
the SP family. To achieve this goal, we first phenolic compounds for the treatment of
characterized the functional diversity of the hyperglycemia, through the development of
family by identifying functional subclasses functional foods or phytopharmaceuticals.
using supervised methods that build the On the other hand, based on the results
functional classification based on empirically from this work, INIA-Carillanca will be able
obtained transport activities reported for to classify the genotypes for their potential
the transporters from databases. From hypoglycemic effects, which will allow the
the analysis of the specificity determinant future to promote the cultivation of murtilla
positions (SDPs), we build structures to find for its agronomic and commercial value, as
inhibitors in a pharmacophore-like mode to well as for its medicinal properties.
accelerate the identification of new inhibitors
and predict the selectivity of other GLUT1
orthologues.
74. ANALYSIS OF THE FUNCTIONAL
SPECIFICITY IN THE SUGAR
PORTER FAMILY TO IDENTIFY NEW
INHIBITORS OF GLUT1.
Oppliger M.1; Ormazabal, V.A.1,2; Zuñiga,
F.A.2; Salas-Burgos, A.1.
1, Nanocell Laboratory, Pharmacology
Department, Biological Science Faculty,
Concepción
University.
2,
Exosome
Laboratory, Clinical Biochemistry and
Immunology Department, Pharmacy Faculty,
Concepción University.
123
77. ANXIOGENIC EFFECT OF
AMPHETAMINE ON ZEBRAFISH
USING A NOVEL TANK DIVING TEST
AND MONOAMINE TRANSPORTER
GENES EXPRESSION.
Paillali, P.1; Viscarra, F.1 ; Reyes-Parada,
M.2; Iturriaga-Vásquez, P.1.
1, Laboratorio de Farmacología Molecular
y Síntesis Orgánica, Depto. Cs. Químicas
y Recursos Naturales, Fac. de Ingeniería y
Ciencias, Universida de La frontera. 2, Centro
de Investigación Biomédica y Aplicada
(CIBAP), Escuela de Medicina, Facultad de
Ciencias Médicas, Universidad de Santiago
Cancer is one of the most frequently diagnosed de Chile, Chile.
diseases worldwide, being the second most
frequent cause of death. Previous studies have Monoamine
Transporters
regulate
established that omega-3 fatty acids, mainly neurotransmission via the reuptake of
DHA (docosahexaenoic acid), have protective dopamine, serotonin and norepinephrine in
effects against various types of cancer, among the brain and regulate the neurotransmitters
these, gastric cancer, which is one of the homeostasis. This class of protein are target
most common cancers in the world, with for a wide number of compounds including
one of the highest mortality rates in Chile. antidepressants, drugs for neuropsychiatric
On the other hand, Carica papaya protein and neurodegenerative disorders, and
fraction P1G10 also has proven anti-cancer substance of abuse, such as amphetamine.
properties due to its proteinase activity. Our This drug of abuse has been described that
data indicate that gastric adenocarcinoma produce anxiogenic effect on rodents and it
cells (AGS) are more sensitive to DHA than is well known that interacts with monoamine
non-tumor gastric epithelium cells (GES-1), transporters
inducing
monoaminergic
determining, through MTT, an IC50 of 40.47 release. In our group we have used zebrafish
μM in AGS. Through Hoechst/Annexin V/ as models for behaviour using different
IP was found that DHA promotes apoptosis drugs that acts over nicotinic receptors and
in AGS cells, but not in GES-1. It was also monoamine transporters. The novel tank
determined that DHA decrease procaspase-3 diving test has been used as a model for to
protein levels in AGS cells only. In vivo assays test anxiolytic behaviour on zebrafish, the
in BALB/c NOD/Scid mice conclude that time spending on the bottom of the tank has
DHA treatment for 6 weeks significantly been describe as anxiogenic-like behaviour in
decreases the volumes of tumors generated this model. This work shows the anxiogenicby AGS cells xenografts. To assess the effects like effects produced by amphetamine on the
of DHA+P1G10 in vitro, we determined cell novel tank diving test. Additionally, we design
proliferation through MTT, treating cell lines the primers and detect the genes expression
derived from the main types of cancer in our of Crebs, DAT, NET and SERTa, SERTb by
country: gastric, lung, gallbladder and breast. PCR. Also we measure the expression changes
To observe cell apoptosis/necrosis visually, using qPCR for this monoamine transporter
we will stain treated cells with Hoechst/ using a chronic dose of amphetamine. Our
Annexin V/PI solution, and to gain further results indicate that, amphetamine induce
insight into the mechanism of DHA+P1G10 anxiogenics-like effects of diving behaviour
-induced apoptosis, we will examine protein and increase genes expression of MAT´s at
levels of procaspase-3 and caspase-3/7 different level.
activity using Western blot and luminescence
assay, respectively. Thus, this research seeks
to determine and validate the joint use of
DHA and the protein fraction P1G10, on cell
lines of the main types of cancer in Chile.
76. EVALUATION OF ANTICANCER
POTENTIAL OF DHA + P1G10 IN CELL
LINES DERIVED FROM CANCERS
WITH HIGH INCIDENCE IN CHILE.
Ortega L.A.1,2; Reyna-Jeldes M.A.1; Lobos L.
3; Schnaiderman A.2; Coddou C.1.
1, Laboratorio de Señalización Purinérgica,
Departamento de Ciencias Biomédicas,
Facultad de Medicina, Universidad Católica
del Norte, Coquimbo, Chile; 2, Schnaiderman
Abraham & Cía; 3, Centro de Medicina
Regenerativa, Universidad del Desarrollo,
Santiago, Chile.
124
78. MITOCHONDRIOTROPHIC
POLIHYDROXY-BENZOATES
DERIVATIVES AGENTS MODIFY
THE EXPRESSION OF METASTATIC
BIOMARKERS IN HUMAN
COLORECTAL METASTATIC CELLS
IN VITRO.
Palominos, C.1, Ruz, D.1, Fuentes-Retamal,
S.1, Jara, J.A.2, Castro-Castillo, V.3, Vivar,
R.1, Ferreira, J.1, Catalán, M.1.
1 Programa de Farmacología Molecular
y Clínica, ICBM, Facultad de Medicina,
Universidad
de
Chile,
Santiago,
Chile. 2 Programa de Farmacología y
Farmacogenética, Instituto de Investigación
de
Ciencias
Odontológicas
(ICOD),
Facultad de Odontología, Universidad de
Chile, Santiago, Chile. 3 Departamento de
Fisicoquímica y Química, Facultad de Ciencias
Químicas y Farmacéuticas, Universidad de
Chile, Santiago, Chile.
with a consequent decreased of the maximal
respiration. In addition, concomitant use
of GA-TPP+C10 and doxycycline is able
to generate a selective synergic cytotoxic
effect on the activation of apoptotic
processes in BC cells, which suggest that this
combined strategy based on the blockage
of mitochondrial bioenergetics inhibitioninduced adaptive response may have
therapeutic relevance in breast cancer.
79. AMYLOID BETA OLIGOMERS
INDUCE MITOCHONDRIAL
DYSFUNCTION BY ITS
DIRECT INTERACTION WITH
MITOCHONDRIAL MEMBRANES ON
HIPPOCAMPAL SLICES.
J. PANES-FERNÁNDEZ1, J. GAVILAN 1,
P.A. GODOY1, O. RAMIREZ-MOLINA1, T.
SILVA-GRECCHI, N. MUNOZ-MOLINA, C.
MUNOZ-MONTECINO1, J. FUENTEALBA1.
Department of Physiology, Faculty of
The metabolic plasticity of cancer cells is Biological Sciences, University of Concepción,
the main limiting factor in the research of Concepción, Chile.
effective pharmacologic treatments, for
disease
(AD)
is
a
development of drug resistance, being one of Alzheimer’s
the major obstacles in the clinical treatment, neurodegenerative disorder characterized
as a promising target for new anti-cancer by impaired learning and memory loss.
drugs therapies. Mitochondria have been Amyloid beta peptide (Aβ) plays a key role
the main factor in the metabolic plasticity. in the pathogenesis of AD, especially soluble
This organelle also participates promoting oligomers (SO-Aβ) because can reproduce the
metastasis, tumor-initiating cells and survival major aspects of the disease. In vitro studies
and propagation of cancer stem cells, which have associated mitochondrial dysfunction
transforms it into an attractive therapeutic with an early role in the AD; however, the
target. Previously reports have demonstrated molecular events are not understood with
that GA-TPP+C10 triggered a mitochondrial precision. In this work, we have studied the
dysfunction, characterized by an inhibition intracellular effects of SO-Aβ treatments, on
of electron transport chain (ETC) and mitochondrial morphology and mitochondrial
AKGDH complex inhibition which triggers potential (ΔΨm). We found that the degree
cell death. In order to increase this effect, of colocalization between Aβ and TOM20
we have analyzed mitochondrial resistance was increasing at 24 h of SO-Aβ treatments,
mechanism generated by the action of with a Manders coefficient (0.640 ± 0.1).
this compound, highlighting an increased Furthermore, we evaluated the ΔΨm using
expression of PGC1α and ETC components- the JC-1 probe, we observed that at chronic
related genes encoded by mitochondrial DNA. treatments (24h), SO-Aβ shown a decrease on
In order to inhibit the resistance generated by ΔΨm near to 50% of the control conditions.
this inhibitory mechanism of action, we have Additionally, at the same times (SO-Aβ, 24h)
incorporated a second agent, doxycycline, strong changes were observed in the size of the
which demonstrated inhibits the synthesis mitochondrial network in primary cultures,
mitochondrial proteins by blockage of only displacing the equilibrium towards a more
mt-ribosome activity. This effect inhibits the granular pattern in mitochondria that present
adaptive survival response generated to the a positive colocalization with Aβ. Secondly,
action of GA-TPP+C10 evidenced by inhibition the intracellular distribution of SO-Aβ
of ETC-related protein. Interestingly, the (2.5uM) in a mouse hippocampal slices model
combined therapy increments significatively was evaluated by immunohistochemistry
the mRNA levels, both ETC-components and electron transmission microscopy
and mitochondrial biogenesis signaling (TEM), where we observed the presence
factors (PGC1a-TFAM-NRF1-NRF2), which Aβ targeted with gold nanoparticles in
suggests a greater mitochondrial damage, an intramitochondrial zone. On the other
evidenced by a decreased mitochondrial mass hand, it was observed that ΔΨm showed
125
a progressive decrease in time manner on
under SO-Aβ treatments (JC-1590/520 C:
1.01 ± 0.01; SO-Aβ 3h: 0.78 ± 0.04). This
study suggest a new pathogenic mechanism
in AD, where cytotoxic effects of SO-Aβ are
related with their direct interaction with the
mitochondria, and reveals a novel therapeutic
strategies for neuroprotection.
81. ANTIMICROBIAL
SUSCEPTIBILITY TESTS OF
HELICOBACTER PYLORI ISOLATES
FROM PATIENTS IN THE BIOBÍO
REGION: COMPARISON OF AGAR
DILUTION AND DISK DIFFUSION.
Parra-Sepúlveda C. 1; Sánchez-Alonzo K.1;
Arellano L. 1; Olivares J. 1; Manríquez C. 2;
González C.1; Garcia A.1.
1, Laboratorio de Patogenicidad Bacteriana,
Departamento de Microbiología, Facultad
de Ciencias Biológicas, Universidad de
Concepción. 2. Departamento de Obstetricia
y Puericultura, Facultad de Medicina,
Universidad de Concepción.
80. CYTOTOXIC EFFECT OF
COMBINATIONS OF ITRACONAZOLE,
HYDROXYCHLOROQUINE AND
CISPLATIN IN HEAD AND NECK
CARCINOMA IN LOW GLUCOSE
CULTURES.
Pardo A.; Vidal D.; Jara J.
susceptibility
testing
Laboratorio de Farmacología, Facultad de Antimicrobial
for Helicobacter pylori is increasingly
Odontología, Universidad de Chile.
important due to resistance to the most
Head and neck cancer (HNC) is the sixth most commonly used antimicrobial agents. The
common malignancy in the world, corresponds Gold Standard proposed by the CLSI is the
to 6% of cancer cases and is responsible for agar dilution method, but it is difficult to
1-2% of deaths worldwide. The most prevalent perform routinely. The objective of this work
HNC subtypes are laryngeal cancer and oral was to determine the concordance of disc
squamous cell carcinoma. These pathologies diffusion in comparison to the agar dilution
are very aggressive and have poor prognosis method for: clarithromycin, metronidazole,
and recurrences, which can be caused by a levofloxacin, amoxicillin and tetracycline,
possible resistance to chemotherapy. Cisplatin using 44 strains of H. pylori from patients
is the chemotherapeutic most used to treat in the BioBío region. Univariate analysis was
these pathologies, however it has been high performed, and the Kappa test was applied for
rates of resistance. This resistance may be concordance using the Stata V.14 program.
caused partially by “Cancer stem cells”, which The resistance rates were for clarithromycin
are resistant to stress stimuli such as starvation 29.5% and 25.0%; metronidazole 45.4%
and low oxygen levels. It has been described and 56.8%; Levofloxacin 31.8% and 25.0;
in some studies that there are drugs, such Amoxicillin 2.2% and 0% respectively by
as Itraconazole and hydroxychloroquine, an disk diffusion and agar dilution. Tetracycline
antifungal drug that acts at the mitochondrial showed no resistance with any of the 2
membrane of the tumor cell by inhibiting methods used. Clarithromycin presented a
the VDAC1 receptor, and an antimalarial/ considerable degree of concordance with a k
immunosuppressive that has been described = 0.6571 (p <0.0001). Metronidazole did not
with antineoplastic potential by inhibiting show concordance for the techniques under
autophagy, respectively. In this way it is study (p=0.1586). Levofloxacin presented an
proposed that the combination of these drugs almost perfect concordance with a k=0.8333
sensitize the effect of Cisplatin. Cell viability (p<0.0001). On the other hand, the Kappa
tests were performed with the compounds at test was not calculated for amoxicillin
24, 48 and 72 hours under normoxia conditions and tetracycline, since 97.3% and 100%
with low glucose medium (1,0 g/L) in two concordance were obtained respectively.
cell lines, laryngeal squamous cell carcinoma The disc diffusion method presented a high
(HEp-2) and squamous tongue carcinoma degree of agreement with the Gold Standard
(CAL-27), using as a control oral dysplastic for clarithromycin, levofloxacin, amoxicillin
cells (DOK). This will be carried out in order and tetracycline. This is an easy method to
to obtain the IC50 of each compound and assess susceptibility to H. pylori especially if
determine their cytotoxic effect. A cytotoxic it is performed routinely. For metronidazole
effect has been observed for all compounds there was a high degree of disagreement
assessed on tumor cells, highlighting the with agar dilution, which has already been
efficacy of Hydroxychloroquine over Cisplatin reported. Finally, other studies with a greater
and Itraconazole. The combination of number of isolations are necessary to assess
hydroxychloroquine or itraconazole with whether the method of disk diffusion, which
cisplatin, improve the cytotoxic effects on is simpler and cheaper, can be continued
routinely in our region.
tumor cells.
126
82. POLYMERIC BIOCOMPATIBLE
NANOCARRIERS FOR DRUG
DELIVERY APPLICATIONS SHOWS
PRESERVED BIOLOGICAL ACTIVITY
OF LOADED PROTEINS, IN VITRO
AND IN VIVO.
Pedroso-Santana, S.1; Fleitas-Salazar, N.1;
Gancino-Guevara, M.1,2; Lamazares, E.1;
Gómez-Gaete, C.3; Toledo, J.R.1.
1. Biotechnology and Biopharmaceuticals
Laboratory, Pathophysiology Department,
School of Biological Sciences, Universidad
de Concepción, Chile. 2. School of
Biological
Sciences
and
Engineering,
YachayTech
University,
Ecuador.
3.
Pharmacy Department, School of Pharmacy,
Universidad de Concepción, Chile.
In the search for new and more effective
therapies,
polymeric
nanoparticulate
systems which protect the drugs and increase
bioavailability have been developed. The use
of biocompatible and biodegradable polymers
could guarantee the harmless character of
the formulation while allows the controlled
release of the active principle. Using ionotropic
gelation method, we synthesized chitosanTPP nanoparticles loading recombinant and
model proteins, in a reproducible way. This
nanoparticulate system showed a peak of
protein released around the fourth day, in
vitro, and promoted the internalization of
loaded BSA-FITC conjugates by Hep-2 cells
after 24 hours of incubation. Cytotoxicity
assay evidenced the benign character of the
formulation, while experiments of biological
activity in vitro and in vivo, showed a specific
biological response due to the system loading.
Visualization of the nanoparticles was
possible thanks to transmission electronic
microscopy. This procedure proved to be an
effective method to formulate proteins, and,
potentially, other molecules, in a safe way,
while the release of the active principle can be
delayed over time. This type of systems can
be used in drug delivery applications in which
pharmacological interaction with the cell is
required.
83. LEUKEMIA INHIBITORY
FACTOR, A NEW MODULATOR
OF THE OVARIAN CHOLINERGIC
SYSTEM IN SUBFERTILE RAT.
Peña S., Vargas C. Rubio M. and Paredes A.H.
Laboratory of Neurobiochemistry, Center
for Neurobiochemical Studies of Endocrine
Diseases.
Faculty
of
Chemical
and
Pharmaceutical Sciences, University of Chile,
Chile.
Leukemia Inhibitory Factor (LIF) is a
proinflammatory cytokine that participates
the regulation of ovarian functions. LIF
participation in the subfertility period has
not been described and the mechanism of
action is unknown. In vitro studies have
shown that LIF increase the acetylcholine
(ACh) synthesis, choline acetyltransferase
(ChAT) expression and its activity in the
upper cervical ganglion. In our laboratory,
an intrinsic ovarian cholinergic system has
been determined recently, which participates
in the regulation of ovarian function. The aim
was to evaluate the LIF/LIF Receptor (LIFR)
levels and its effect on the ovarian cholinergic
system in subfertile rats. We measured LIF
and LIFR mRNA and protein levels by qRTPCR and western-blot at 3 (fertile) and 9
months old (subfertile) Sprague-Dawley
rats. To evaluate the LIF effect on the
ovarian cholinergic system, rat ovaries were
incubated in vitro for 3 and 8 h with LIF
(100ng/ml) and buffer Krebs (vehicle). ACh
production and the mRNA content of the
genes encoding the ChAT and AChE enzymes
it was determined by fluorometry and qRTPCR respectively. The results show increase
in LIF protein levels and increase of LIFR
mRNA in ovaries in fertile period in subfertil
period. Incubation with LIF increases ACh
in incubation medium, without observing
changes in ovarian ACh levels. The ChAT and
AChE mRNA content enzymes significantly
decrease at 3h of incubation (40% and 50%,
respectively). In contrast, in ovaries incubated
for 8 h, LIF does not affect ovarian ACh levels
nor in the incubation medium. These results
suggest that LIF regulates ovarian cholinergic
function during reproductive aging, pending
as LIF and the cholinergic system regulate
follicular development at this period.
127
84. IDENTIFICATION OF RESIDUES
INVOLVED IN THE DOPAMINE
TRANSPORTER-GBETAGAMMA
PHYSICAL/FUNCTIONAL
INTERACTION.
Pino J.A. 1; Nuñez-Vivanco G. 2; Hidalgo G.
3; Quiroz M. 4; Reyes-Parada M. 5; Torres
G.E. 4.
1, Biomedical Sciences Laboratory, Faculty
of Medicine, University of Atacama. 2,
Center for Bioinformatics, Simulations
and Modelling, Universidad de Talca. 3,
Department of Pharmacology & Therapeutics,
School of Medicine, University of Florida.
4, Department of Molecular, Cellular &
Biomedical Sciences, City College of the City
University of New York. 5, School of Medicine,
Faculty of Medical Sciences, Universidad de
Santiago de Chile.
The dopamine transporter (DAT) plays
a crucial role in the regulation of brain
dopamine (DA) homeostasis. Through reuptake of DA, DAT serves two important
functions: the termination of synaptic
transmission at dopaminergic terminals, and
the replenishment of vesicular DA pools. In
addition to uptake, DAT can also function to
release DA. This process, which is referred to
as DAT-mediated efflux, is the mechanism
used by potent and highly addictive
psychostimulants, such as amphetamine and
its analogues, to increase extracellular DA
levels in motivational and reward areas of the
brain. It has long being recognized that DA
neurons release DA through exocytotic and
non-exocytotic processes. However, the exact
mechanism by which physiological signals
or psychostimulants, such as amphetamine,
induce DA efflux through DAT still remains
a complex and not completely understood
area of research. Recently, we discovered
that the G protein betagamma subunits
bind to the intracellular carboxy-terminus
of DAT and regulate transporter activity.
More importantly, we have observed that
activation of Gbetagamma promotes DATmediated DA efflux. However, the amino acid
residues involved in Gbetagamma interaction
site(s) in DAT and their role in transporter
regulation remain largely unknown. Here,
we used a combination of bioinformatics,
mutagenesis, immunoprecipitations, and
functional assays to identify the Gbetagamma
binding site on DAT and its role in transporter
regulation. Preliminary functional studies
are consistent with previous biochemical
evidence indicating that the sequence FREKL
located in the carboxy-terminus of DAT plays
a role in Gbetagamma interaction with DAT
128
and promotion of DA efflux. Thus, this study
provides a starting point for a further detailed
characterization of the DAT-Gbetagamma
interaction and a better understanding of its
contribution to DAT-mediated efflux.
85. AMYLOID BETA OLIGOMERS
INTERRUPT NUCLEAR CA2+
TRANSIENTS AND GENE
EXPRESSION INDUCED BY
GABAZINE IN HIPPOCAMPAL
NEURONS.
Lobos P. 1; Vega I. 1; Bruna B. 1; Henriquez N.
1; Hidalgo C. 1 , 2; Paula-Lima A. 1,3.
1, Laboratorio de Señales Mediadas por
Calcio, Instituto de Neurociencia Biómedica
(BNI), Facultad de Medicina, Universidad de
Chile.; 2, Laboratorio de Señales Mediadas
por Calcio, Instituto de Ciencias Biomedicas
(ICBM), Departamento de Neurociencia
Facultad de Medicina, Universidad de Chile. ;
3, Laboratorio de Biología Celular y Molecular
, Instituto de Ciencias Odontologicas (ICOD),
Facultad de Odontología, Universidad de
Chile.
Ca2+ signals are essential mechanisms
that regulate neuronal plasticity. Nuclear
Ca2+ transients generated by neuronal
activity induce changes in gene expression
and in dendritic spine remodeling, which
are mediated by the rapid activation and
expression of transcription factors. Among
them, Npas4 is known for inducing distinct
activity-dependent gene programs that
regulate the expression of neurotrophic
factors and antioxidant enzymes. Thus,
Npas4 may provide a molecular link between
neuronal activity and the activation of
memory and neuroprotection signaling
pathways. Amyloid-beta oligomers (A-beta
Oligomers) are synaptotoxins that induce
aberrant Ca2+ signals and promote Reactive
Oxygen Species (ROS) generation,leading to
synaptic plasticity disruption. In this work,
we studied the effects of AβOs in nuclear
Ca2+ signals production and gene expression
induced by Gabazine, a GABA(A) receptor
blocker that functions as an inductor of
synaptic activity. To this aim, we transfected
primary hippocampal neuronal cultures with
a genetically encoded Ca2+ indicator with
nuclear destination (GCaMP3-NLS). We preincubated these cultures with AβOs for 6 h and
applied Gabazine at the microscope stage, to
record nuclear Ca2+ signals by live-imaging.
We also performed immunocytochemistry
to evaluate CREB phosphorylation and RTqPCR to evaluate the mRNA expression of
Npas4, BDNF and of the antioxidant enzymes
Glutamate-Cysteine-Ligase
(GCL)
and
NADPH-Quinone-Oxidoreductase
(Nqo1)
in these conditions. Our results indicate
that neurons treated with A-beta Oligomers
showed reduced nuclear Ca2+signals and
diminished Npas4, GCL and Nqo1 mRNA
expression levels in response to GBZ. In
summary, the present results indicate that
A-beta Oligomers altered the activation of
signaling pathways induced by gabazine,
leading to a disruption of neuroprotective
gene expression pathways essential to
memory and learning processes, which are
affected in neurodegenerative diseases.
86. EVALUATION OF SINAPTIC
COMPONENTS DURING
NEURULATION OF XENOPUS LAEVIS
EMBRYOS.
Ingrid Pinto Borguero1, Nicolás Zúñiga S.1,
Claudio Retamal U.1, Patricio Castro M.1.
1 Laboratory of Physiology and Pharmacology
for Neural Development, Department of
Physiology, Faculty of Biological Sciences,
Universidad de Concepción, Concepción,
Chile.
In chordates, neurulation and neural tube
formation is the first step in the central
nervous system (CNS) development. Failures,
by genetic or environmental alterations,
in this process may induce neural tube
defects (NTDs). It has been described in
the literature, that the use of anti-epileptic
drugs (AEDs) during pregnancy, increase
the probability of NTDs by mechanisms not
completely identified. Recently, glutamate
signaling through NMDAR has been proposed
to participate in neurulation process. Here
we hypothesized that glutamate would be
released by a vesicular-related mechanisms
which contribute to cellular responses
necessary for normal neural tube formation.
For evaluate this, we use Xenopus laevis
embryos, collected in different neurulation
stages (9 - 20 hours post-fertilization
(hpf)) for obtain RNA transcripts. Then we
performed PCR and qPCR for assess relative
expression studies, focalized in evaluate the
presence of vesicular release-related and
synaptic receptor proteins. We observe the
presence of vesicle related proteins, such as
SNAP25, VAMP2, Syntaxin and VGLUT1,
as well as glutamate receptor MGLuR2 and
AMPAR during neurulation. Then, to evaluate
the functionality of AMPAR in neurulation,
we perform pharmacological studies, using
the antagonist CNQX. We observe that CNQX
don’t provoke any evident alteration in neural
tube formation. Finally, we performed an
induction of epileptogenic behavior using
Pentylenetetrazol to evaluate CNS health
after neurula-treatments. We observe a
decrease of almost ~50% in the seizure
latency onset necessary for epileptogenic
behavior vs not treated controls. Our results
suggest that, glutamate could be released
using vesicles proteins and the expression
of AMPAR don’t participate in the normal
neural tube development but could regulate
additional later process important for the
CNS establishment on Xenopus laevis.
87. TRIPANOCIDAL ACTIVITY OF
CASTANEDIA SANTAMARTENSIS
(ASTERACEAE) AGAINST
TRYPANOSOMA CRUZI.
Quintero H.1; Lapier M2; Carbonó, E3; Torres
O4; Liempi A5; Maya J2; Delporte C1.
1,Laboratorio de Productos Naturales,
Departamento de Química Farmacológica y
Toxicológica, Facultad de Ciencias Químicas
y Farmacéuticas, Universidad de Chile.
2,Laboratorio de Bioquímica, Metabolismo y
Resistencia a Fármacos, Instituto de Ciencias
Biomédicas ICBM, Facultad de Medicina,
Universidad de Chile. 3,Herbario UTMC,
Universidad del Magdalena, Colombia.
4,IDEFARMA, Programa de Regencia en
Farmacia, Facultad de Ciencias de la Salud,
Universidad de Córdoba. 5,Laboratorio
de mecanismos de infección parasitaria,
Instituto de Ciencias Biomédicas ICBM,
Facultad de Medicina, Universidad de Chile.
Chagas disease (CD) an endemic disease from
Latin America, is caused by Trypanosoma
cruzi infection. More than 7 million people
are infected. Currently, there are two drugs
derived from nitro compounds for the
treatment of CD with important side effects,
that cause the treatment to be abandoned,
furthermore, the effectiveness in the chronic
phase is still controversial. Therefore, is
necessary the search for new drugs that are
more effective and better tolerated. Castanedia
santamartensis R. M. King & H. Rob, is
known for their properties to treat skin sores.
The objective of this study was to evaluate the
trypanocidal activity of an ethanolic extract
(ETE) of C. santamartensis and its fractions.
Air-dried and powdered leaves, were
extracted at room temperature with ethanol
and concentrated and dried by evaporation
at reduced pressure. The fractionation was
obtained by chromatographic separation
methods, using solvents of different polarity.
The in vitro trypanocidal activity of the ETE
and the fractions was determined against
T. cruzi trypomastigotes (Dm28 strain)
129
using MTT and flow cytometry techniques.
Nifurtimox was used as a reference drug.
The IC50 (concentration that produce
a 50% parasitic death) was calculated
using the least squares method. The ETE
presented trypanocidal activity (IC50 of
197.3 micrograms/mL). The CS200; CS300
and CS400 fractions, presented trypanocidal
activity with IC50 values of 91.2; 63.9 and 15,4
microgramos/mL respectively. The IC50 of
the reference drug was 5.7 micrograms/mL.
The results indicate that C. santamartensis
contains secondary metabolites with activity
against T. cruzi.
88. EFFECTS OF P2X2R
OVEREXPRESSION IN CELL LINES
AND ITS IMPACT IN SIGNALING
PATHWAYS ASSOCIATED TO AMPK/
CAMKII.
Ramírez-Molina, O; Godoy, PA; SilvaGrecchi, T; Mennickent, D, Gavilán J; PanesFernández, J, Muñoz-Molina, N; Cuevas, ME;
Fuentealba, J.
Departamento de Fisiología, Facultad
de Ciencias Biológicas, Universidad de
Concepción, Concepción, Chile.
One of the main toxic agents in Alzheimer’s
Disease (AD) are the soluble oligomers of
the Aß peptide (OS-Aß). Chronic treatments
with OS-Aß have been shown to increase the
expression of the P2X2 receptor (P2X2R)
in PC12 cells and rat hippocampal cells,
participating in increasing intracellular
calcium and allowing a leak of ATP to the
extracellular environment. AMPK protein
kinase has several roles on protein, energy and
mitochondrial metabolism and is regulated
by changes in the levels of intracellular
Ca2+ and AMP/ATP ratio. AMPK is
capable of phosphorylate PGC-1a, which is
a transcription co-activator that, when is
phosphorylated, is activated and translocates
to the nucleus, promoting mitochondrial
biogenesis. Using PC12 cells to overexpress
P2X2R, the effect of its activation was
assessed by ATP treatments, on AMPK activity
and the subcellular distribution of PGC1a. From functional experiments (calcium
microfluorimetry and electrophysiology),
immunocytochemistry and Western blot,
it was concluded that overexpression and
activation of P2X2R by ATP, prevents an
increase in AMPK activity and generates
changes in the subcellular distribution of
PGC-1a, which suggests that P2X2R would
be related to the toxicity generated by the
Aß peptide and the intracellular calcium
overload.
130
89. NEW LIPOPHILIC CATIONS
DERIVED FROM CAFFEIC ACID
INDUCE CYTOTOXIC EFFECT IN
HUMAN COLORECTAL CANCER
CELLS.
Ramírez D1,2, Rojas D1, Cortez G1,2, Escobar
B1,3, Jara JA3, Catalán M1.
1 Laboratory of Biochemistry, Metabolism
and Drug Resistance, ICBM, Facultad de
Medicina, Universidad de Chile, Santiago,
Chile. 2 Department of Biology, Faculty of
Basic Sciences, Metropolitan University
of Education Sciences, Santiago, Chile.
3 Pharmacology Laboratory, Research
Institute of Dental Sciences (ICOD), School
of Dentistry, University of Chile, Santiago,
Chile.
One of the deadliest pathologies worldwide
is cancer. Colorectal cancer is the third
most common type of cancer. A few drugs
are provided for treatment this disease, like
5-fluorouracil, oxaliplatin and irinotecan,
as standard therapy. However, this
therapy several times failed due to high
drug resistance and side effects, leading
cancer progression. There are several risk
factors both exogenous and endogenous
that increase the incidence of this disease
in the organism, hence the importance of
characterize the cancer cells from cytological,
metabolic and molecular aspects. These
features give them the differences between
normal epithelium cells, becoming with high
proliferative rates in an uncontrolled manner.
In this sense, the mitochondria appear as
a new target for new molecules against
cancer, since they have high mitochondrialtransmembrane potential than normal cells,
capable to accumulate cationic compounds.
This work is focused in the evaluation of a
new set of molecules derivatives from caffeic
acid attached to a different size length of
triphenylphosphonium-aliphatic chain and
their effect on human colorectal cancer cells.
We evaluated cytotoxic effect by MTT assay,
the decrease of mitochondrial potential by
flow cytometry and the decrease of cellular
of ATP levels by luminescence. The results
showed that the compounds were cytotoxic in
colorectal cell lines (HCT-15 and COLO 205),
decreasing
mitochondrial-transmembrane
potential and cellular ATP levels. In
conclusion, these new compounds may
induce cytotoxic effect by a mitochondrial
mechanism, inducing bioenergetics stress,
suggesting the importance of studying new
pharmacological agents taking advantage of
the cellular singularities like mitochondrial
metabolism.
90. BIOLOGICAL EFFECT OF NOVEL
TRIAZOLOPYRIDINES AGAINST
MACROPHAGES MURINE.
Ramos-Aguayo A.1; Lapier M.1-2; Olea-Azar
C.1; Maya J.2.
1Laboratorio de Antioxidantes y radicales
libres, Departamento de Química Inorgánica
y Analítica, Facultad de Ciencias Químicas
y Farmacéuticas, Universidad de Chile.
2Laboratorio de Bioquímica, Metabolismo
y Resistencia a Fármacos, Instituto de
Ciencias Biomédicas ICBM, Facultad
de Medicina, Universidad de Chile.
3Laboratorio de Productos Naturales,
Departamento de Química Farmacológica y
Toxicológica Facultad de Ciencias Químicas y
Farmacéuticas, Universidad de Chile.
understanding the mechanisms that trigger
abnormal proliferation rates and subsequent
tumor migration in gastric epithelia. Between
these possibilities, purinergic signaling
emerges as promising pathway that regulate
cell growth, proliferation and migration
according to the expression rates of its many
receptor classes and subclasses. Among these,
P2Y2 receptor (P2Y2R) is widely known by its
contribution to cell invasion and metastasis in
prostate, colorectal and colon cancer; which
is in contrast to the antiproliferative effects
reported for P2X4 receptor (P2X4R) on cancer
models in vitro. Despite all this background,
purinergic signaling involvement in gastric
cancer remains unknown. For this reason,
our investigation was focused to characterize
the expression profile of P2Y2R and P2X4R,
in terms of protein levels by western blot and
gene expression by qPCR, in cell lines derived
from primary gastric adenocarcinoma
(AGS), moderately and mildly differentiated
metastatic gastric adenocarcinoma (MKN74 and MKN-45, respectively) and healthy
gastric epithelia (GES-1). Moreover, to
assess P2Y2R and P2X4R contribution
to gastric cancer growth and invasion, we
evaluated the effect of different agonists and
antagonists on cell proliferation by Resazurin
assay, and stablished metastatic potential
by transepithelial electrical resistance
(TEER) measurements in the gastric cell
lines described above after overexpressing
or silencing P2Y2R and P2X4R. Our results
provide preliminary insights on gastric cancer
pathophysiology that can be used as future
pharmacological approaches for treatment.
Recently, in our group we have synthesized and
characterized triazolo pyridine derivatives,
based on a rational study (structure-activity),
where we found that compound 1 has an
antiproliferative effect against the replicative
form of the Trypanosome cruzi parasite.
However, to evaluate the cytotoxic activity
of the new [1.2.3] triazolo [1.5a] pyridine
in a murine cell model, which is the first
defense system against T. cruzi, we find
the macrophages. Viability results by MTT
indicate that compound 1 and 2, have cytotoxic
activity in this system. On the other hand, by
flow cytometry and propidium iodide, we
have found that compound 2 can stop the cell
cycle, consequently, stop cell proliferation
and induce apoptosis death processes. This
effect is observed in the literature with classic
sterols synthesis inhibitors. This indicates
that they are possibly affecting an enzyme of
92. INTRACELLULAR AMYLOIDthe p450 complex in mammalian cells.
BETA OLIGOMERS DECREASE
EXCITABILITY AND AMPA
91. P2Y2R AND P2X4R EXPRESSION
MEDIATED CURRENT IN NUCLEUS
PROFILE AND ITS ROLE IN
PROLIFERATION AND METASTATIC
ACCUMBENS NEURONS.
N.O. Riffo, E.J. Fernandez and L.G. Aguayo.
POTENTIAL IN GASTRIC CANCER
Laboratory of Neurophysiology, Department
CELL LINES.
Reyna-Jeldes M.A.; Cerda-Barraza D.C.; of Physiology, University of Concepcion.
Coddou C.
Laboratorio de Señalización Purinérgica, Alzheimer disease (AD) is a progressive
Departamento de Ciencias Biomédicas, neurological disorder that causes dementia in
Facultad de Medicina, Universidad Católica an increasingly aging worldwide population.
Despite the current dogma of AD, where
del Norte, Coquimbo, Chile.
extracellular aggregates of amyloid beta
Gastric cancer is considered a major health peptide (Aß) initiates neurotoxicity, growing
concern due to its unspecific symptomatology evidence shows synaptic dysfunction and
on early stages and complex pathophysiology loss of limbic functions in early stages
that hinders any attempts for targeted before amyloid plaque deposition, where the
therapeutic approaches. This disease has presence of intracellular Aß has been reported.
high incidence and mortality rates worldwide, The nucleus accumbens (NAc), a central
being the third cancer-related cause of death integrative brain area of the limbic system,
in Chile, which focuses scientific work in is particularly affected in AD in humans and
131
transgenic mice models. However, the effect
that intracellular Aß may have on neuronal
function has still not been examined.
Therefore, in this study we analyzed the
effects of intracellular Aß oligomers (iAßo)
on acutely dissociated NAc neurons. To
evaluate if iAßo could modulate components
of the neurotransmission, we used a modified
whole-cell patch clamp technique to dialyze
Aßo intracellularly through the recording
electrode. The effects of iAßo were study
on the maximum evoked current (Imax)
where under control conditions, the AMPA
current was 149 ± 18 pA and decreased to 73
± 15 pA after the application of iAßo 1 μM.
Interestingly, GABA and GLY currents were
not affected. Furthermore, iAßo was able
to decrease accumbal neurons excitability,
diminishing the number of action potential
spikes and its amplitude. Overall, these
findings showed that iAßo inhibited the
amplitude of AMPA receptors in accumbal
neurons and also decreased neuronal
excitability. These effects support the notion
that iAβo is able to impair neurotransmission
in limbic areas.
93. FUNCTIONAL MODULATION
AND MOLECULAR INTERACTION
OF THE ALKALOID KOUMINE WITH
GLYCINE RECEPTORS.
Riquelme, C.R., Sazo, A.E., Lara, C.O.,
Marileo, A.M., San Martín, V., Petermann, A.,
Flaig, D., Soto, P., Pineda, B., Moraga-Cid, G.,
Yévenes, G.E.
Departamento de Fisiología, Universidad de
Concepción.
Koumine is one of the main alkaloids of the
Gelsemium genus plants. Behavioral studies
have reported that the administration of
koumine exerted analgesic and anxiolytic
effects. The mechanisms underlying these
beneficial effects are not well defined.
However, behavioral studies have shown that
the analgesic and anxiolytic effects of koumine
are inhibited by strychnine, a selective
antagonist of inhibitory glycine receptors
(GlyRs), which are chloride-permeable
pentameric ligand-gated ion channels
expressed in the central nervous system. To
date, whether koumine is able to modulate
the function of GlyRs is unknown. Here, by
using biochemical, electrophysiological and
bioinformatics approaches, we studied the
potential modulation of GlyRs by koumine.
Our electrophysiological studies showed
that koumine negatively modulates the GlyR
function. For example, the acute application
of 25 micromolar of koumine inhibited
132
the glycine-activated current through
recombinant alpha1-GlyRs and alpha3-GlyRs
by ≈35%. Molecular docking studies based
on the alpha3-GlyR crystal structure suggest
that koumine interacts with the orthosteric
pocket of the receptor, favoring a closed state
of the ion channel. Ongoing biochemical
studies will determine whether koumine
directly interacts whit the extracellular
domain of alpha3-GlyRs. Overall, these
results demonstrate the actions of koumine
on the GlyR function. These results, together
with ongoing studies, may contribute to
understand the mechanisms underlying the
koumine-induced analgesia and anxiolysis.
94. PHARMACOLOGICAL
INHIBITION “IN VIVO” OF OVARIAN
ACETILCOLESTERASE REVERTS
POLIQUISTIC OVARY PHENOTYPE IN
RAT.
Riquelme R., Ruz F., Lara HE.
Laboratorio
de
Neurobioquímica,
Departamento
de
Bioquímica
y
Biología Molecular, Centro de Estudios
Neurobioquímicos
para
Enfermedades
Endocrinas, Facultad de Ciencias Químicas y
Farmacéuticas, Universidad de Chile.
Ovarian function is subject to endocrine and
nerve regulation. An increase in sympathetic
tone by cold stress (CS) induces a phenotype
like polycystic ovarian condition (PCO). On
the other hand, a local cholinergic system has
been described in rat ovary, in which we find
acetylcholine (ACh), the muscarinic receptor
M1, and the enzyme acetylcholinesterase
(AChE). Chronic treatment with the AChE
inhibitor Huperzine A (Hup-A) has been
reported to increase the fertility on the rat.
In this context, the purpose of this study is
to determine whether the long-term changes
induced by CS on ovarian function can be
reversed by increasing ACh chronically by
administering Hup- A. In this study, SpragueDawley rats were subjected to CS subsequently
hemiovarioectomized and implanted with a
miniosmotic pump with Huperzine A (10 μM)
or subjected to the procedure but without the
implantation of miniosmotic pump (Sham).
28 days after the procedure the ovary and
the serum were collected to measure steroid
hormones Testosterone (T), Progesterone
(P4) and Estradiol (E2) by enzyme
immunoassay and the follicular development
by morphometry. A second group of rats were
used to measure the fertility after mate with
males of proven fertility. The results show
that CS generates a polycystic phenotype with
cysts, hyperandrogenism and low fertility.
The administration of Hup-A reverses the
alterations in follicular development and
hyperandrogenism produced by CS but not
increase the fertility. The pharmacological
potential of these findings gives to the
cholinergic local system relevance in the
treatment of PCO.
of the release kinetics showed that the
encapsulation of ACPV-56 within MPs delays
its release at least 10 times when compared to
the free drug. As a conclusion, the developed
microparticles represent a promising
alternative for treatment of RA.
96. DEVELOPMENT OF CHEMICALLY
CROSS-LINKED HYDROGELS
WITH POTENTIAL BIOMEDICAL
APPLICATIONS.
Rivas B. 1; Pedroso S. 1; Fleitas N. 1; Sandoval
C. 2; Gómez C. 3; Toledo J.R. 1.
1, Laboratorio de Biotecnología y Biofármacos,
Departamento de Fisiopatología, Facultad
de Ciencias Biológicas, Universidad de
Concepción; 2, Departamento de Química
Analítica e Inorgánica, Facultad de Ciencias
Químicas, Universidad de Concepción;
3, Departamento de Farmacia, Facultad
de Química y Farmacia, Universidad de
Rheumatoid arthritis (RA) is a chronic Concepción.
disease whose worldwide incidence is
increasing. Currently, there are non- Hydrogels based on natural origin polymers
pharmacological
and
pharmacological have shown promising results as scaffolds
approaches for the therapeutic management for cell encapsulation and drug delivery in
of RA. From the latter, the drug ACPV-56 tissue engineering. In this work, biopolymeric
has demonstrated anti-arthritical activity hydrogels, from natural and biocompatible
due to its ability to inhibit the proliferation polymers, were produced by two methods,
of activated lymphocytes, which results chemical cross-linking with glutaraldehyde
in a marked anti-inflammatory effect. and freeze-thawing. The hydrogels were
Clinical studies have proven that upon oral characterized using scanning electron
administration of therapeutic doses, ACPV- microscopy (SEM) and Fourier-transform
56 causes gastrointestinal adverse effects infrared spectroscopy (FTIR). The SEM
that negatively influence the compliance images showed that the structure and
to chronic treatments. A strategy to avoid morphology of the hydrogels produced by
the adverse gastrointestinal effects of chemical cross-linking differed from those
ACPV-56 and increase the dosing time produced by freeze-thawing, while FTIR
intervals is by incorporating the drug into analysis also revealed different chemical
a controlled release system administered composition between them. Their potential
by intramuscular injection. As none of the biomedical application was also assessed.
available formulations containing ACPV-56 First, their biocompatibility with HEP-2 cell
is intended for parenteral administration, line was tested using an MTT assay. The
the objective of this research is to develop a results showed that the chemically crossdrug delivery system based on biodegradable linked hydrogels did not affect the cell viability
microparticles (MPs) encapsulating ACPV- compared to the freeze-thawing-produced
56. The MPs, elaborated by spray drying of a hydrogels. We further tested the potential of
mixture of anionic polysaccharides, cationic chemically cross-linked hydrogels to retain
and phospholipids, were characterized and release bioactive compounds in the cells
in terms of its in vitro release kinetics, by loading the hydrogels with BSA protein
employing conditions that emulate the conjugated with FITC. Using a fluorescence
physiological environment. The formulation microscope, we observed that the HEP-2 cells
parameters were optimized in order to obtain were stained green, indicating a successful
MPs suitable for injection. The obtained release of the conjugate. These data suggest
micro particles were spherical, with a that our chemically cross-linked hydrogels
medium diameter close 20 μm, relatively have the potential to be used for drug delivery
mono-disperse and with a minor tendency to in tissue engineering applications.
aggregation. The incorporation of ACPV-56
did not affect the physicochemical properties
of the developed MPs. Preliminary findings
95. DEVELOPMENT OF ACPV-56
LOADED MICROPARTICLES, FOR
THE TREATMENT OF RHEUMATOID
ARTHRITIS. PRELIMINARY STUDY.
Riquelme C.1 ; Gómez-Gaete C.1 ; Torres P.1 ;
Chávez-Santoscoy RA.2 ; Arellano-Villaseñor
N.2.
1 Departamento de Farmacia, Facultad
de farmacia, Universidad de Concepción,
Concepción. 2 Facultad de Ciencias Químicas
e Ingeniería, Universidad Autónoma de Baja
California-Campus Tijuana, México.
133
97. ANTIDEPRESSIVE LIKE
EFFECT INDUCED BY THE ACUTE
ADMINISTRATION OF IBOGAINE
AND NORIBOGAINE IN RATS AND
ITS POSSIBLE MECHANISM OF
ACTION.
Rodríguez P.,1,2 Urbanavicius J.,2 Prieto
J.P.,2 Fabius S.,2 Reyes A.L.,3 Sames D.,4
Scorza C.2, Carrera I.1.
1 Laboratorio de Síntesis Orgánica,
Departamento de Química Orgánica, Facultad
de Química -Universidad de la República,
Montevideo – Uruguay; 2 Departamento de
Neurofarmacología Experimental, Instituto
de Investigaciones Biológicas Clemente
Estable, Montevideo – Uruguay; 3 Centro
Uruguayo de Imageneología Molecular,
Montevideo – Uruguay; 4 Department of
Chemistry, Columbia University, New York –
United States.
data. Interestingly, noribogaine 40 mg/kg
elicited an antidepressant-like effect per se
with a higher potency than fluoxetine. Our
data support the possibility that this potent
antidepressive-like action could collaborate,
at least in part, to explain the ibogaine´s
previous anti-addictive effects.
98. MODULATION OF ANTIOXIDANT
ACTIVITY IN DERIVATIVES OF
AMINOETHYL PHENANTRENE
AND HALO- APORPHINES WITH
ANTINEOPLASTIC ACTIVITY.
Rodríguez O. 1, Garrido-Ayala L. 2, SalgadoCamacho C. 1, Santos J.C. 3, Vallejo E. 1,
Georges N. 1, Asencio M. 1.
1. Laboratorio de Investigación e Innovación
Química, Facultad de Ciencias Naturales,
Matemáticas y Medio Ambiente, Universidad
Tecnológica Metropolitana. 2 Laboratorio
de Tecnología de Alimentos. Universidad de
Previous human subjective data and animal Santiago de Chile 3. Laboratorio de Síntesis
studies demonstrated that the psychedelic y Química Teórica, Universidad Andrés Bello.
alkaloid ibogaine, and its metabolite
noribogaine, have potent antiaddictive In the search for new molecules with
effects. The biological mechanism through antineoplastic and antioxidant properties
both compounds elicit this beneficial derived from the natural product boldine (1),
effect remains still unclear. Among several it was proposed to increase the molecular
molecular targets, ibogaine and noribogaine lipophilicity of the precursor molecule with
inhibit the serotonin transporter (SERT). the expectation of improving the above
This action and a longterm increase of brain- mentioned bioactivities. Using (1) as starting
derived neurotrophic factor RNAm levels matherial was prepared: secoboldine (N, Nin the rat prefrontal cortex that we found in (methyl) (ethyl) phenanthrene; seco-boldine
our previous study after the acute ibogaine (2)) and a series of halo derivatives (halo =
i.p. administration, lead us to hypothesize Cl or Br) (3-6), which were characterized by
that the anti-addictive property of ibogaine NMR-1H and -13C and the measurement of
and noribogaine could be related to a potent their melting point. Boldine (1) has the lowest
putative antidepressant-like effect. Consistent dissociation energy in the phenolic group of
with this possibility we characterized C-9 with respect to C2-OH which is consistent
the behavioral effects (dose and time- with the highest acidity recorded for first
dependence) induced by the acute ibogaine phenolic group. The insertion of one halogen
(20 and 40 mg/kg) and noribogaine (20 atom (3,4) maintains the energy value to break
and 40 mg/kg) administration in rats using the O-H bond, but in compounds that bearing
the forced swimming test (FST). Fluoxetine two halogen atom (5,6) the dissociation
(40 mg/kg/i.p.) a standard antidepressant energy markedly increases while antioxidant
drug, was used as a control. We found that capacity decrease in these compounds. This
ibogaine and noribogaine induced a dose- experimental evidence is correlated with the
and timedependent antidepressive-like effect. “local softness” (LS) exhibited by boldine
To know if the antidepressive-like effect and halogenated compounds. In conclusion,
induced by ibogaine was due to an effect the antioxidant activity in these compounds
per se or by the presence of its metabolite can be modulated by the insertion of halogen
(noribogaine) we intravenously injected atoms in the appropriate positions.
animals with ibogaine. Ibogaine 1 and 5 mg/
kg after an i.v. injection on animal behavior 99. GALLIC AND GENTISIC ACID
immediately evaluated in the FST. Under DERIVATES INDUCE AUTOPHAGY IN
these conditions, ibogaine did not generate MURINE AND HUMAN COLORECTAL
an antidepressant effect. Ibogaine seems to CANCER CELL LINES.
depend on noribogaine content to induce the Rojas D.1, Ramirez D.1-2, Cortes G.1-2,
beneficial effect. All the behavioral responses Escobar B.1-3, Catalán M.1.
were consistent with the pharmacokinetic 1 Laboratory of Biochemistry, Metabolism
134
and Drug Resistance, ICBM, Facultad de
Medicina, Universidad de Chile, Santiago,
Chile. 2 Department of Biology, Faculty of
Basic Sciences, Metropolitan University
of Education Sciences, Santiago, Chile.
3 Pharmacology Laboratory, Research
Institute of Dental Sciences (ICOD), School
of Dentistry, University of Chile, Santiago,
Chile.
Colorectal cancer is the third most common
neoplasm in the world. The standard
treatment consists mainly in surgery and
the use of three first-line chemotherapies,
5-fluorouracil, oxaliplatin and irinotecan.
The high drug resistance, several side effects
and high costs of the treatments, give an
opportunity to search for new molecules
with new pharmacological targets. In the
recent years, cancer cell mitochondria have
become in an interesting pharmacological
target due to their high mitochondrialtransmembrane potential that allows
accumulated cationic probes conjugated
with cytotoxic pharmacophore. In our
laboratory, the gallic and gentisic acid
derivatives as conjugated with lipophilic
cationic triphenylphosphonium through
an aliphatic chain of ten carbons have been
tested in breast and colorectal cancer cells.
These compounds triggered a series of events
that leads cell apoptosis. The objective of
this work is described how the decrease of
ATP levels induces the activation of AMPK,
which promotes death by autophagy in
colorectal cancer lines. Through western
blot and luminescence assay, we observed
that in human and murine colorectal cell
lines, the derivatives induce the reduction in
cellular ATP levels followed by the activation
of AMPK and LC3B, leading to cell death by
autophagy. In conclusion, our compounds
may be inducing apoptosis by triggering
mitochondrial unbalance, energy stress and
autophagy.
is a bioactive plant with antiparasitic activity
related with its content of sesquiterpene
lactones (SL). Most antiparasitic studies of
chicory have been focused on nematodes, but
poorly explored against parasitic protozoa.
The aim of this study was to evaluate the
antiprotozoal effects of SL-rich chicory
extracts against Trypanosoma cruzi, the
etiological agent of Chagas disease. SL of
chicory leaves and roots were extracted
from 5 chicory cultivars (Spadona, Goldine,
Larigot, Measeto and Benulite) using
methanol/water and purified by solid-phase
extraction. The extracts were dissolved
in DMSO. SL profiles of the extracts were
characterised by UHPLC-MS metabolomics.
The cytotoxicity of extracts was tested on
T. cruzi trypomastigotes (Y strain) and
mammalian Vero cells. Trypomastigotes were
incubated for 24 h with serial dilutions of
extracts (100-6.3 μg/mL), and benznidazole
was used as positive control. Vero cells were
exposed to extracts for 24 h at 100 and 50
μg/mL to evaluate cytotoxicity. Cell viability
was evaluated by resazurin reduction test.
Chemical profiling showed that chicory
extracts have distinct content of SL among
cultivars and between plant parts. All the
extracts had dose-dependent effect against
isolated trypomastigotes. However, Spadona
leaf extract was the only with no toxicity
against Vero cells at 100 μg/mL, suggesting
a selective trypanocidal activity. Taken
together, these results revealed that chicory
Spadona leaf warrants deeper exploration
regarding the relationship between its
chemical profile and antiprotozoal activity.
Consequently, these results encourage further
investigation of chicory as a source of SL with
antiparasitic therapeutic potential.
100. ANTIPROTOZOAL ACTIVITY OF
CHICORY (CICHORIUM INTYBUS)
AGAINST TRYPANOSOMA CRUZI.
Romero-Uzqueda Y. 1; Peña-Espinoza, M. 1;
Valente, A. 2; Williams, A. 2; Thamsborg, S.
2; López-Muñoz, R. 1.
1, Instituto de Farmacología y Morfofisiología,
Facultad
de
Ciencias
Veterinarias,
Universidad Austral de Chile.
101. AUTISTIC AUTOANTIBODIES
ABSORBED FROM BREAST
MILK GENERATES COGNITIVE
IMPAIRMENT IN BREEDING FEMALE
BUT NOT MALE RATS.
Rossi G.1,2; Cobarrubias A.1; Arancibia M.1;
Araya G.1; Uribe F.1; Gonzalez-Gronow M.1;
Sandoval R.1.
1.
Environmental
Neurotoxicology
Laboratory, Department of Biomedical
Sciences, Faculty of Medicine, Universidad
Católica del Norte; 2, PhD. Program in
Applied Ecology and Biology, Universidad
Católica del Norte.
Chagas disease is an endemic parasitosis in
Latin America. However, its drug treatment
frequently induces adverse effects. Thus, it
is urgent to develop new therapies. Chicory
Autism spectrum disorders (ASD) involve
a range of complex neurodevelopmental
disorders,
characterized
by
social
impairments, communication difficulties,
135
and restricted, repetitive and stereotyped
patterns of behavior. ASD exerts a significant
physiological, emotional and financial
burden on the families of the individual
and society as a whole. Recently, beside the
knowledge about genetic factors involved
in this pathology, there is new evidence
related to immunological causes of ASD.
Therefore, it is of outmost importance to
elucidate the molecular and physiological
mechanisms of ASD pathology. Data from us
and others have shown that normal young rat
hippocampal slices incubated with purified
IgA autoantibodies from ASD patients and
breeding rats from pregnant mothers injected
with the same antibodies, impairs LTP as well
as disrupts learning and memory. Taking
this into account, we hypothesized that ASD
autoantibodies are absorbed from breast
milk and generates autoimmune-related
cognitive impairment characteristic of ASD
pathology. To achieve this aim, we used
a rat model where mothers were injected
with ASD autoantibodies during breast milk
period and the breeding was tested after
that period using learning and memory
test together with electrophysiological and
immunohistochemical studies. We found
that both LTP and learning and memory were
significantly impaired in female but not male
breeding rats and this alteration are correlated
with the presence of ASD autoantibodies
in hippocampus and Cortex. These results
demonstrate that ASD autoantibodies are
incorporated from breeding milk, cross both
intestinal and blood-brain barrier and impairs
learning and memory in a sex-preference
fashion. They also give us new knowledge
about possible causes of autism and opening
a new line in pharmacological therapies.
102. MDMA (3,4-METHYLENEDI
OXYMETHAMPHETAMINE) AND
HELPING BEHAVIOR: PRELIMINARY
CHARACTERIZATION IN SPRAGUEDAWLEY RATS.
Vilches-Lagos,
M.J.1,4,
AlbornozBustamante, J. 1,4, Castro-Castillo, V.2;
Hernández, A.3; Sáez-Briones, P. 1,4.
1 Laboratory of Neuropharmacology and
Behavior, Faculty of Medical Sciences,
Universidad de Santiago de Chile. 2
Department of Organic Chemistry and
Physical Chemistry, Faculty of Chemical and
Pharmaceutical Sciences, Universidad de
Chile. 3 Laboratory of Neurobiology, Faculty
of Chemistry and Biology, Universidad de
Santiago de Chile. 4 School of Medicine,
Faculty of Medical Sciences, Universidad de
Santiago de Chile.
136
MDMA (3,4-methylenedioxymethampheta
mine, “ecstasy”) is a psychotropic drug that
induces an “open mind” state in healthy
humans characterized by heightened selfacceptance, openness to communication
and a fear threshold decrease known as
the entactogenic syndrome. Disregarding
its therapeutic potential, direct evaluation
of MDMA-like effects in animal models
remained limited to the pro-social paradigm,
a model that recreates different stereotyped
rodent behaviors. In contrast to these classical
pharmacological criteria, helping behavior is a
complex type of pro-social paradigm that has
been recently described in rodents. It stands
out because of its pertinence to develop a
more sophisticated pharmacological model to
study human-like behaviors, as it may occur
in rats as a result of the interaction between
psychomotor capabilities and the amount of
stress experienced by the animal, even in the
absence of reward. Despite of its relevance,
the behavioral characterization of the effects
of MDMA in this model remains unexplored.
In the present work, a preliminary
characterization of the effects of MDMA on
helping behavior in male rat pairs (helper rat
+ victim rat; with/without previous individual
housing) after 12 days-administration/
training cycles at two different dose levels (5
mg/kg; 10 mg/kg i.p.) has been attempted
using a slightly modified water-trap model
developed ad hoc. The results obtained
indicated that MDMA might not enhance
helping behavior compared to controls when
the acting roles of each pair member has not
been interchanged. In contrast, current data
seems to be in agreement with the notion
that helping assistance may rather depend
on if the rat pair met each other previously or
not and/or the experience of being trapped
in the water trap, at least at the dose ranges
evaluated.
103. EFFECT OF A LACTOBACILLUS
ADMINISTRATION ON ANXIETYLIKE BEHAVIORS IN ADULT RATS.
Salazar-Contreras, C.1, Escobar-Luna, J.1,
Barrera-Bugueño, C.1, Julio-Pieper, M.1,
Gotteland M.2, Bravo, JA.1.
1Grupo
de
NeuroGastroBioquímica,
Laboratorio de Química Biológica. Instituto
de Química, Facultad de Ciencias, Pontificia
Universidad
Católica
de
Valparaíso,
Valparaíso. Chile. 2Departamento de
Nutrición, Facultad de Medicina, Universidad
de Chile, Santiago. Chile.
There is increasing evidence that gut
microbes affect central nervous system
(CNS) function. For instance, there are some
Lactobacillus strains with proven anxiolytic
and antidepressant like effects in rodents.
However, there is also evidence that other
Lactobacillus have anxiogenic effects in rats.
In this regard, our previous findings show
that 2 week administration of the potential
probiotic bacteria Lactobacillus casei L-54-233 to healthy pre-pubescent Sprague-Dawley
male rats, increased anxiety like behaviors,
while lowering hippocampal expression of
5-HT1A receptor. Therefore, we asked if
this anxiogenic-like effect was due to the
rat’s young age (post-natal day [PND] 35).
To test this, we administered 104 CFU/ml
of L. casei L-54-2-33 in the drinking water
of male Sprague-Dawley rats from PND65
till PND76, and compared their anxiety-like
behaviors with age and sex matched control
rats fed with vehicle (sucralose and MRS
broth) in the drinking water for the same
amount of time. Anxiety-like behaviors were
then evaluated using the open field (OF)
test and elevated plus maze (EPM). Rats fed
with the bacteria spent significantly less time
in the central zone of the OF in comparison
to controls, while there were no differences
between bacteria fed and controls rats in the
EPM. These results match with our previous
findings in younger male rats, suggesting that
the anxiogenic effects of L. casei L 54 2 33 are
strain specific, and that these effect do not
depend on the age of the animal. Together
our findings suggest that bacteria known to
promote changes in the CNS, might exert its
strain-specific effects regardless of the age of
the host, which is a novel feature in probiotic
(or in this case psychobiotic) interventions
104. ANXIOLYTIC EFFECTS OF A
CHILEAN EXTRACT OF HUMULUS
LUPULUS.
Godoy, J (1); Sáez, S.(1); Ríos M(2); Silva,
M.E(2).; Rivera, F(3); Simirgiotis, M.J.(2)
Sánchez-Montoya, E.L.(2).
(1)Escuela de Química y Farmacia, Facultad
de Ciencias, Universidad Austral de Chile (2)
Instituto de Farmacia, Facultad de Ciencias,
Universidad Austral de Chile., Universidad
Austral de Chile. (3)Instituto de Anatomía,
Histología y Patología, Facultad de Medicina,
Universidad Austral de Chile.
Introduction: Humulus lupulus is broadly
cultivated in the world both for beer
manufacture, but also for its medicinal
properties, for the treatment of excitability
and restlessness. There are important regional
differences in metabolome composition of
the plants that influence its response. The
present study investigated whether a Chilean
Humulus lupulus extract, previously selected
by demonstrate antioxidant properties
(enzymatic and in vitro assays), can elicit
effects on the central nervous system, using
various experimental models in rats.
Methods: a) Treatments: Two different doses
(low or high) of Humulus lupulus extract
from a regional variety, or Medi-Dropsucralose used as vehicle (control group),
were orally administered for 42 days to
adults male Sprague-Dawley rats. Extracts
were administrated one hour before the
behavioral tests performed in this study.
b) Open field was used for the evaluation of
locomotor activity. Anxiety was evaluated
by elevated plus-maze test (EPM). Results:
No significant differences were observed on
the locomotor behavior of rats, following
the oral administration of two doses of
extract or control, measured by total distance
travelled and average speed, on the open field
apparatus. But rats treated with low dose of
extract significantly increased (16,75 +5,1
sec) the time spent on the central zone of the
open field, compared to control (4,7+ 1,7 sec);
this parameter is correlated with statistically
difference observed with open arm spent time
on the EPM between control and medium
dose of extract. Conclusion: These results
show that the low dose of this Chilean lupulus
extract, could exert and anxiolytic effect.
105. PRODUCTION OF
RECOMBINANT HUMAN
INTERLEUKIN-4 EXPRESSED IN
ESCHERICHIA COLI AS INCLUSION
BODIES.
Urrutia J.1,3; Herrera P.1,3; Mansilla R.1,3 ;
Toledo J.2,3 ; Sánchez O.1,3.
1,
Laboratorio
de
Biofármacos
Recombinantes, Departamento fr Facultad
de Ciencias Biológicas, Universidad de
Concepcion1; Laboratorio de Fisiopatología,
Facultad de Ciencias Biológicas, Universidad
de Concepcion2; Centro de Biotecnología y
Biomedicina SPA.3
Interleukin-4 (IL-4) is a potent lymphoid cell
growth factor that stimulates the growth and
survivability of certain B cells and T cells. It
exhibits anti-inflammatory responses and
participates in immune processes by providing
protection from intracellular pathogens. IL-4
also plays an important role in T helper cells
differentiation. Additionally, it can suppress
pro-inflammatory cytokines. In this work,
a His-tagged recombinant human IL-4 was
overexpressed in Escherichia coli under
the control of a T7 promoter. The resulting
137
inclusion bodies were separated from cellular
debris by centrifugation and solubilized by
8M urea. The denatured IL-4 was refolded
in a single chromatographic step by gradual
removal of denaturant agent. This protocol
yielded 4.5 mg of IL-4 from 40g of biomass.
The refolded protein was highly pure and
subsequent biological activity assay that was
measured in the human erythroleukemia
cell line TF-1 suggested that IL-4 had similar
activity profile to the commercial produced
protein. The results of this study suggest that
on-column refolding represent a convenient
and low-cost process for the refolding of
IL-4 and may be a promising candidate for
development as commercial reactive for
cancer research.
106. A-KINASE ANCHORING
PROTEIN AKAP79 INTERACTS WITH
THE INTRACELLULAR DOMAIN OF
THE GLYCINE RECEPTORS ALPHA
SUBUNITS.
Sazo, A.E.1,2, Lara, C.O.1, Marileo, A.M.1, San
Martín, V.1, Peterman, A. 1, Flaig, D. 2, Soto,
P2., Pineda, B2., Riquelme, C.R.2., MoragaCid, G.2, Yévenes, G.E.1.
1Laboratory
of
Neuropharmacology,
2Laboratory
of
Structural
Neuropharmacology.
Department
of
Physiology, University of Concepción,
Concepción.
Glycinergic inhibition is critical for
breathing control, muscle tone regulation
and nociception. Studies showed that PKA
phosphorylation in the residue S346 located
in the intracellular domain (ICD) of the
glycine receptor (GlyRs) containing the alpha
3 subunit. This posttranslational modification
produces inhibition of glycinergic function
in the spinal cord, which was related to the
generation of inflammatory chronic pain.
Noteworthy, the molecular mechanisms
associated with the inhibition of a3GlyR by
phosphorylation are not yet elucidated. In
this context, the interaction of the A-kinase
anchoring protein (AKAP79) to partners
involved in nociceptive pathways has been
recently reported. Specifically, it has been
observed that disruption of the interaction
between AKAP79 and TRPV1 decreases
sensitization
in
nociceptive
neurons.
Furthermore, the direct interaction between
AKAP79 and the beta3 and beta2 subunits
of the GABAAR promote the PKA-mediated
phosphorylation of serine residues located
in the ICD of those subunits. Nonetheless,
whether AKAP79 is able to bind GlyRs and
modulates its function is still unknown.
138
Here, by using immunocytochemical and
GST pull-down assays, we reported a direct
association between the a1, a2 and the a3
subunits of the GlyRs with AKAP79. Confocal
imaging showed that AKAP79 specifically
co-localized with all the GlyRs subunits. In
addition, in pull-down studies we observed
that a GST-ICDa3GlyR fusion protein are
able to bind AKAP79.Thus, our experimental
data contributes to the characterization of a
new intracellular partner of the GlyRs. This
open new avenues in the searching of new
therapeutic targets for the inflammatory
chronic pain treatments.
107. PERINATAL ASPHYXIA INDUCES
LONG-TERM DEMYELINATION,
OLIGODENDROCYTES DAMAGE
AND NEUROINFLAMMATION IN RAT
BRAIN: PREVENTION BY NEONATAL
MESENCHYMAL STEM CELLS
TREATMENT.
Andrea Tapia-Bustos1, Carolyne LespayRebolledo1, Ronald Perez-Lobos1, Valentina
Vio1, Emmanuel Casanova1, Rosario Matte1,
Emilia Licci1, Diego Bustamante1, José Luis
Valdés1,2, Fernando Ezquer3, Mario HerreraMarschitz1, Paola Morales1,2.
1 Programme of Molecular & Clinical
Pharmacology, ICBM, 2Department of
Neuroscience, Medical Faculty, University
of Chile. 3Center for Regenerative Medicine,
Faculty of Medicine-Clínica Alemana,
Universidad del Desarrollo, Santiago, Chile.
The effect of perinatal asphyxia (PA) was
evaluated on myelination, oligodendrocytes,
neuroinflammation and cell death in rat
telencephalon and hippocampus from
postnatal (P)1 up to 14 days, a period
characterized by a spur of neuronal
networking, finding a sustained injury
that may have profound adverse effects on
neuronal development. The study evaluated
whether that injury could be prevented by
mesenchymal stem cells (MSCs) treatment.
PA was induced by immersing foetuscontaining uterine horns into a water bath
at 37°C for 21 min. Asphyxia-exposed (AS)
and sibling caesarean-delivered (CS) foetuses
were resuscitated and nurtured by surrogate
dams. Animals were euthanized at P1, 7 or
14, dissecting samples from telencephalon
and hippocampus to be assayed for (i)
myelin (MBP and transcriptional factors
involved
in
repairing
demyelination,
Olig-1and
2;
immunofluorescence,
RT-PCR);
(ii)
oligodendrocyte
density
(immunofluorescence);
(iii)
neuroinflammation
(RT-PCR,
ELISA,
immunofluorescence), and (iv) cell death
(TUNEL). Two hours after delivery, AS
and CS neonates were injected with either
5 μl of vehicle or 5x104 MSCs into the
left lateral ventricle. It was found that PA
produced: (i) a decrease of MBP density and
oligodendrocyte/mm3 at P7 in telencephalon,
but not in hippocampus; (ii) an increase of
Olig-1, in telencephalon at P7; (iii) an increase
of IL-6 mRNA levels in telencephalon at
P7, and of IL-1β mRNA in hippocampus at
P14; (v) an increase of cell death, including
oligodendrocyte at P7 in telencephalon;
(vi) MSCs treatment prevented the effect
of PA on demyelination, oligodendrocyte
density, neuroinflammation and cell death.
It is proposed that PA induces regionally and
developmental-dependent changes in brain
regions, and MSCs treatment can prevent
the changes induced by PA on myelination,
oligodendrocyte density, neuroinflammation
and cells death.
108. MHC-CLASS I POLYPEPTIDERELATED SEQUENCE A (MICA) AS
AN IMMUNOTHERAPEUTIC TARGET
IN CANCER.
Toledo-Stuardo K. 1; Ribeiro C.1; Rodríguez
J. 1; Jerez B.1; Tello S.1; Farías C. 1; González
P. 2; Molina M.C.1.
1,
Laboratorio
de
Anticuerpos
Recombinantes e Inmunoterapia. Centro
de
Inmunobiotecnología,
Programa
Disciplinario de Inmunología, ICBM,
Facultad de Medicina, Universidad de Chile.
2, Programa de Genética Humana, Instituto
de Ciencias Biomédicas, Universidad de
Chile.
MICA is a ligand to NKG2D, an activation
receptor that triggers natural killer (NK)
cells effector functions for early tumor
elimination; however, the normal function
of MICA/NKG2D axis is compromised in
cancer, including gastric adenocarcinoma
(GA). Several mechanisms have been
proposed to explain this response, including
the presence of released MICA, either as
soluble proteins or in exovesicles, which
may favor down-modulation of NKG2D in
cytolytic cells, resulting in desensitization
of NK cells as the tumors progress. MICA is
a highly polymorphic molecule that codifies
allelic variants, which have been described
to affect NKG2D binding avidity and cell
cytotoxicity, while some MICA-STR variants
located in the transmembrane domain
promote NKG2D internalization. MICA-STR
A5.1 variant acquires a GPI-anchor which is
recruited in exosomes. The aim in this work
was to evaluate the MICA expression in gastric
adenocarcinoma and their relationship with
the allelic variants and effect on the regulation
of NKG2D receptor. We study the MICA
expression and release in samples of tumor
tissue by flow cytometry and ELISA assay.
We isolated DNA genomic to determinate the
MICA allele by sequence based-typing PCR
using specific primers. Also, we evaluate the
expression of NKG2D in tumor- infiltrating
NK cells by flow cytometry. Our results
indicated that the expression of NKG2D
on NK cells was inversely proportional to
the levels of MICA on tumor cells, and that
not all patients showed detectable levels of
soluble MICA (sMICA) in their serum and,
while the diminished expression of NKG2D
on cytolytic cells did not correlate with the
concentration of sMICA in the serum of GA
patients, this could be due to the presence
of MICA in exovesicles as the MICA-STR
A5.1 variant. In conclusion, we propose
that MICA is an immunotherapeutic target
in gastric adenocarcinoma and the MICA
allelic variants should be considered in the
therapeutic strategies.
109. INTERNALIZATION
MECHANISM OF FOLATE-MODIFIED
PAMAM DENDRIMERS IS MEDIATED
BY MORE THAN ONE ENDOCYTOSIS
PATHWAY.
Torres J.; Vásquez P.; Vidal F.; Guzmán L.;
Alderete, J.
Laboratorio de Neurobiología Molecular,
Facultad de Ciencias Biológicas, Universidad
de Concepción.
Nowadays, central nervous system (CNS)
diseases affect 1.5 billion people worldwide
and there is a continuous development of new
therapies. However, in many cases efficiency
of therapies is low because of biological
barriers and deficient biodistribution of
drugs. New advances in the nanomedicine
have allowed the creation of nanotransporter
systems. Among them, polyamidoamine
(PAMAM) dendrimers have demonstrated
a great potential in drug delivery to
CNS. PAMAM dendrimers are polymeric
structures composed by an ethylenediamine
core that branches creating layers, called
generations, which end in primary amines
protonated at physiological pH and can be
modified with other terminal groups, such
as folate. Considering the current difficulty
of delivering drugs to the CNS, we examined
the internalization mechanism of folateconjugated PAMAM dendrimers mediated
by folate receptor α (FRα), a membrane
139
protein overexpressed in choroid plexus
that once it binds to folate is internalized
by the caveolae endocytosis pathway, and
is postulated as a target tissue for drug
delivery to CNS. In this study, we selected
the HeLa cell line for internalization
experiments, based on confocal and westernblot results. One unmodified (G4) and two
folate-modified (PFO25 and PFO50) fourth
generation PAMAM dendrimers were used.
Confocal images showed that PFO50 was
not able to entry HeLa cells, unlike PFO25
and G4, which were visualized after one
hour incubation. Quantification of Mander’s
coefficients indicated only a slight increase
of colocalization of PFO25 with FRα than
unmodified G4, which suggests that the
internalization pathway of folate-modified
dendrimers is possibly mediated by more
than one endocytosis mechanism.
110. EFFECT AQUEOU EXTRACT
OBTAINED FROM LEAVES OF U.
MOLINAE AND THEIR RESPECTIVE
PRODUCTS OF GASTOINTESTINAL
DIGESTION ON THE VIABILITY OF
COLON CANCER CELLS.
Torres E. R. 1; Avello M. 1; Pastene E. 2.
1,
Laboratorio
de
Farmacognosia,
Departamento de Farmacia, Facultad de
Farmacia, Universidad de Concepción. 2,
Laboratorio de Síntesis y Biotransformación
de productos naturales, Departamento de
Ciencias Básicas, Facultad de Ciencias,
Universidad del Bío-Bío.
Colorectal cancer is the third most common
diagnosis in men (10%) and second in
women (9.4%). The use of chemotherapy to
fight this disease leads side effects, this the
main reason for investigations of possible
antiproliferativity of different natural sources.
In that regard, active compounds U. molinae
and their products the gastrointestinal
metabolized could act as prophylactic and
complementary because effcts anticancer
has been reported.The aims is To asses the
effect aqueou extract obtained from leaves
of U. molinae and their respective products
of in vitro gastrointestinal digestion on the
viability of colon cancer cells. Ugni molinae
leaves were used to prepare an aqueous
extract, with this a gastrointestinal disgestion
was performed, obtaining also a final residue.
These samples were evaluated in different
viability tests, such as trypan blue exclusion,
metabolic activity (MTT) and cytotoxicity
(LDH), on colorectal cancer cells (CaCo2) and healthy cells (HEK) for a period
of 24 hours. When treating the cells, it is
140
observed that the count of viable CaCo-2 cells
decreases as the concentration increases.
In the case of HEK cells no changes in the
count are observed. MTT assay only with the
gastrointestinal digestion samples observated
an effect of inhibition of the metabolic activity
in the case of caco-2 cells, in hek cells there is
no significant effect. Cytotoxicity assays using
LDH do not show significant changes in the
activity of the enzyme lactate dehydrogenase
in any case. Finally, it is concluded that the
samples have positive effect on viable cell
count and MTT assay, because damage
colorectal cancer cells (caco-2) but not healthy
cells (HEK), while very mild cytotoxicity was
observed at through the LDH assay
111. ASSESSMENT OF DIFFERENT
PHARMACOLOGICAL ACTIVITIES
OF PEUMUS BOLDUS EXTRACTS
USING CHEMICAL SUBTRACTION
STRATEGY.
Torres J. P. 1; Correa D.1; Alarcón J. 3;
Gómez-Alonso S.2; Silva C.1; Pastene. E.1,3.
1 Laboratory Pharmacognosy, Department of
Pharmacy, Faculty of Pharmacy, University
of Concepción, Concepción, Chile 2 Regional
Institute for Applied Scientific Research,
Faculty of Chemical Sciences, University of
Castilla-La Mancha, Castilla-La Mancha,
Spain. 3 Laboratory of Synthesis and
Biotransformations of Natural Products,
Department of Basic Sciences, University of
Bio-Bio, Chillán, Chile.
Peumus boldus Mol., (Monimiaceae)
is a Chilean medicinal three used for
gastrointestinal
and
liver
diseases.
Phytochemical profiling of this plant is
based on its aporphine alkaloids, phenolic
compounds and essential oil. However, in
herbal infusions some authors thought that
flavonoids are responsible for its antioxidant
and chemopreventive effects rather than
alkaloids and essential oil. The objective
of this study was to evaluate different
knock-out extracts prepared by chemical
subtraction oriented to selectively remove
alkaloids and essential oils from crude
extracts. These extracts were obtained by
means of conventional centrifugal partition
chromatography (CPC) and pH-zone-refining
CPC. DPPH bleaching test, cytotoxicity in
AGS cells, DNA damage in monocytes (Comet
assay) and inhibition of Acetylcholinesterase
were determined for all extracts. Solutions
of the different lyophilized extracts were
prepared at different concentrations (1-1000
ug/mL). The results of DPPH assay indicated
an IC50 of 63.05, 109 and 43.73 ug/ml for
total extracts, alkaloids and polyphenols
respectively, the fraction containing the
polyphenols having greater antioxidant
capacity. In turn, cytotoxicity tests showed
that polyphenols at concentrations lower
than 1000 ug/mL protected AGS cells. On
the contrary, alkaloid fraction reduced cell
viability from 400 ug/mL whereas fraction
containing essential oil displayed higher
toxicity from 125 ug/mL. Only the fraction
with alkaloids displayed an expected
acetylcholinesterase inhibition.
to induce neural tube defects. In addition,
in silico studies using molecular docking
techniques, we determine the possible site of
cbx and enx association in the protein, located
in the extracellular domain (E2). Taken
together these results, we suggest that Cx 46
and Cx 32 will participates as hemichannel in
neural tube closure and their blockade results
in NTDs.
113. INHIBITION OF ENDOPLASMIC
RETICULUM EXIT RESCUES A
NIEMANN PICK TYPE C DISEASE
MODEL.
112. PARTICIPATION AND ROLE
Urbina J.; Astete G.; Milla L.A.
OF CONNEXINS IN THE RELEASE
Centro de Investigación Biomédica y
OF GLUTAMATE AND ATP IN
Aplicada, Escuela de Medicina, Facultad de
THE NEURULATION PROCESS IN
Ciencias Médicas, Universidad de Santiago
XENOPUS LAEVIS.
Tovar L.M 1; Benavibes C.I 1; Gonzalez A.A 1; de Chile.
Castro P.A 1.
Laboratorio de fisiología y farmacología Currently, more than 70 lysosomal diseases
been
identified,
accumulating
para desarrollo neural, Departamento de have
Fisiología, Facultad de Ciencias Biológicas, substrates in lysosomes and late endosomes.
Within this group we find the NiemannUniversidad de Concepción.
Pick type C (NPC) disease, that generates
Neurulation is an important process in aberrant accumulations of cholesterol and
the formation and development of CNS. other lipids within cells, resulting in early
This event correspond to the first step neuronal death. NP-C1, the most common
of neural embryonic development (stg protein showing disease-causing mutations,
12,5–20 in Xenopus laevis) and implicates codes for a transmembrane protein NPC1,
different cellular process like, migration and present in lysosomes and late endosomes
proliferation. Alterations in the signaling of membranes. NP-C2, is caused by a mutation
this period could result in neural tube defects in the NPC2 gene that encodes the NPC2
(NTDs). Several studies has demonstrated protein, which is soluble and present in the
the participation of connexins (Cxs) as same organelles. The disease produced by
hemichannels in cellular communication loss of function of NP proteins generates
through the release of ATP and glutamate, hepatomegaly and splenomegaly. Based on
regulating cellular migration and stabilizing preliminary laboratory data, we hypothesize
synaptic transmission. In this investigation, that proteins related to the organization
we identified the presence of several Cxs of the endoplasmic reticulum (ER) are
during neurulation. To evaluate its relative necessary to maintain the disease phenotype.
expression, we identified their RNA In order to identify other proteins involved,
transcripts in different stages of Xenopus we studied Tango1, a transmembrane ER
laevis neural embryonic development such protein that organizes vesicle cargo. Its loss
as: stg 10; stg 12,5; stg 14 and stg 20. Our of function produces disorganization and
results revealed the presence of transcripts stress of the ER. It was analyzed in a model
of Cxs 43, 45, 46, 32 and 26 in different of Drosophila melanogaster where NP-C1
stages of Xenopus laevis development. The is replicated with a knock-down of dnpc1a
more important proteins correspond to Cx gene, NPC1 ortholog, through RNAi. Using
46 (GJA3) which has 6 fold expression vs Cx this system, we determined that tango1
45 (GJA7) and Cx 43 (GJA1). In turn, Cx 32 loss of function reverts NP-C1 phenotype,
(GJB1) have a significant presence of 3 fold improving Drosophila larval development
vs Cx 26 (GJB2), the second more abundant, progression. Also, the effect of the inhibition
during neurulation. Later, we decided to of ER secretion was analyzed using Flievaluate the functionality of these Cxs as 06, a compound that inhibits exportation.
hemichannels through pharmacological We tested a pharmacological model that
blockage assays, using inhibitors such as phenocopies NP-C1, completely reverting
carbenoxolone (cbx) and enoxolone (enx). We NPC phenotype. The study corroborates that
found values of IC50 of ~30 μM and ~20 μM the organization of the secretory pathway is
for cbx and enx respectively in their capacity determinant to maintain the phenotype of
141
this disease and that by itself an alteration 115. CLINICAL CHARACTERISTICS
in it results in a phenotype equivalent to the OF NEUROLOGICAL PATIENTS
deficiency of NPC1.
INFECTED WITH HTLV-1 AND
DETERMINATION OF THE LOCATION
114. ADDITIVE EFFECT OF
OF TAX VIRAL PROTEIN.
MODAFINIL AND CAFFEINE ON THE Valenzuela M.A.1; Puente J.1; Cartier L.2;
LOCOMOTIVE ACTIVITY OF ADULT
Ramírez E.3. 1 Departamento de Bioquímica
RATS.
Y Biología Molecular, Facultad de Ciencias
Urbina, A.1,2 ; Sotomayor-Zárate, R.2.
Químicas y Farmacéuticas, Universidad
1Programa de Magíster en Ciencias Biológicas de Chile. 2 Departamento de Neurología,
mención Neurociencias, Facultad de Ciencias, Facultad de Medicina, Universidad de Chile.
Universidad de Valparaíso, Valparaíso, Chile. 3 Departamento de Virología, Facultad de
2Centro de Neurobiología y Fisiopatología Medicina, Universidad de Chile. Instituto de
Integrativa (CENFI), Instituto de Fisiología, Salud Pública (ISP).
Facultad de Ciencias, Universidad de
Valparaíso, Valparaíso, Chile.
Tropical
Spastic
Paraparesis
neuropathogenesis (abbreviated HAM / TSP,
Currently, many people are subject to a high “HTLV-1-associated myelopathy / tropical
work and academic load that leads them spastic paraparesis”), endemic in Chile,
to consume psychotropic substances to be shows by anatomo-pathological studies
able to carry out these highly demanding spinal cord injuries due to axonal loss and
activities. In Chile, it has been observed demyelination of cortical spinal beams,
that some of these substances commonly visualized by NMR. 70% of patients start their
used are modafinil and caffeine to promote disease with paretospastic gait. Since 2009
wakefulness and concentration. Concomitant the screening of HTLV-1 in blood banks.
use of modafinil and caffeine could trigger The prevalence in donors studied in the ISP
anxious
symptoms
and
psychomotor in PBMCs (“Peripheral Blood Mononuclear
agitation in humans. Modafinil has different Cells”) containing T-CD4+ lymphocytes,
action mechanisms, being the blocking of the main viral reservoir, showed real-time
dopamine transporter (DAT) the most known prevalence of 1.2 healthy / 1000 individuals.
and relevant. On the other hand, caffeine Neuropathogenesis is attributed to the viral
is an adenosine receptor antagonist and protein Tax because the virus does not infect
inhibitor of phosphodiesterase. Therefore, neurons and 40% of patients with paraparesis
the concomitant use of modafinil and caffeine are seronegative for the virus, but express a tax
could promote a higher effect on neural gene. In cerebrospinal fluid (CSF) of patients
activity compare to the use of caffeine or we detect Tax (by ELISA) and in isolated cells
modafinil alone. The objective of this work (by immunofluorescence) and in plasma (by
was to measure the additive effects of the “Western Blot”). Tax secreted from patient
administration of modafinil and caffeine on PBMCs agrees with the extracellular role
the horizontal and vertical locomotor activity. that we propose, because we know that it
To assess the traveled distance and number interacts with semaphorin-4D that triggers
of bipedestation, we used rats treated with the disassembly of microtubules and actin
caffeine (20 mg/kg, i.p.), modafinil (80 mg/ fibers through Plexin-B1.
kg, i.p.) and caffeine plus modafinil. Our
preliminary results show that rats treated 116. POLYMERIZATION ACTIVITY
with caffeine plus modafinil produce an AND CYTOTOXICITY OF MOLECULES
increase on locomotor activity (horizontal WITH AFFINITY FOR LAU/PLA
and vertical) compared to the administration BINDING SITE OF TUBULIN AS
of caffeine or modafinil alone. The effect NOVEL STABILIZING AGENTS.
induced by caffeine plus modafinil was Vásquez Pilar 1, Zúñiga Matías 2, Guzmán
additive. To correlate these behavioral effects Leonardo 1, Jiménez, Verónica 3.
with an increase in dopaminergic activity in (1) Departamento de Fisiología, Facultad
the mesolimbic and nigrostriatal pathways, de Ciencias Biológicas, Universidad de
we will measure the dopamine release in Concepción, Concepción, CL (2) Center for
striatum and nucleus accumbens using in Bioinformatics, Simulations and Modelling,
vivo brain microdialysis and fast scan cyclic Facultad de Ingeniería, Universidad de Talca,
voltammetry
Talca, CL (3) Departamento de Ciencias
Químicas, Facultad de Ciencias Exactas,
Universidad Andrés Bello, Concepcion, CL.
142
The importance of microtubules in cellular
division set these proteins as pharmacological
targets for antimitotic agents, known as
tubulin binding agents (TBA), which can
promote stabilization or destabilization of
tubulin polymerization. The occurrence
of adverse drug reaction associated to
several of these agents drives the need for
the development of new TBAs with a safer
pharmacological profile. In this regard,
a combination of computational virtual
screening, molecular dynamics and binding
free energy estimations was performed by
our group, based on the stabilizing LAU/PLA
binding site of tubulin. A set of 7 candidates
were proposed as potential stabilizing agents
with affinity for the site. In this work, we
confirm the polymerization capacity for these
7 candidates in vitro at concentrations of 50
and 100 μM. Also, we observed an additive
effect of the compounds when co-treating with
Taxol, confirming a non-competitive binding
with taxane-site binders. Finally, viability
assays in a cancer cell line were developed
showing a cytotoxic effect of molecules at
100 μM. These results set a starting point
of further studies for the characterization of
the novel agents that will open possibilities
for the rational screening of new tubulin
stabilizing agents.
117. RELEASE AND UPTAKE
KINETICS OF DOPAMINE ARE
PRESERVED IN STRIATUM OF
ADULT FEMALE RATS EXPOSED
DURING FIRST HOURS OF
POSTNATAL LIFE TO ESTRADIOL
VALERATE.
Velásquez, V.B.1; Escobar, A.P.2; España
R.A.3; Sotomayor-Zárate, R.1.
1Centro de Neurobiología y Fisiopatología
Integrativa (CENFI), Instituto de Fisiología,
Facultad de Ciencias, Universidad de
Valparaíso, Valparaíso, Chile. 2Centro
Interdisciplinario de Neurociencias de
Valparaíso (CINV), Facultad de Ciencias,
Universidad de Valparaíso, Valparaíso, Chile.
3Department of Neurobiology and Anatomy,
Drexel University College of Medicine,
Philadelphia, USA.
Sex hormones play an important role
in regulating reproductive and nonreproductive tissues, such as the brain. In the
nervous system, sex hormones are important
in its development and neural plasticity,
however changes in the sex hormones milieu
during fetal or neonatal stages affect brain
function and generate persistent changes
until adulthood. During last 7 years our lab
has been interested in study how neonatal
exposure to sex hormones such as estradiol
valerate (EV) affect the functionality of
midbrain dopaminergic neurons of adult
male and female rats. The aim of this work
was to evaluate the release and uptake
kinetics of striatal dopamine (DA) induced
by methylphenidate (MPH: 5.0 mg/kg i.p.)
of adult female rats exposed during the first
hours of postnatal life to estradiol valerate
(EV: 0.1 mg/50 uL of sesame oil s.c.) or
vehicle (50 uL of sesame oil s.c.). Our results
did not show significant differences in
the voltammetry parameters such as peak
amplitude, area and tau (time constant of
decay). Despite these results, we cannot rule
out changes in the voltammetry parameters
in nucleus accumbens, a key nucleus of the
reward circuit, where we have previously
observed a reduction in DAT expression of
animals programmed with sex hormones.
118. CYTOTOXIC EFFECTS CAUSED
BY DELOCALIZED LIPOPHILIC
CATIONS DERIVED FROM
POLYHYDROXY-BENZOIC ACIDS IN
COMBINATION WITH DOXYCYCLINE
ON LUNG CANCER CELLS.
Vidal D.A. 1; Pardo A. 1; Jara J.A. 1; Ferreira
J. 2.
1, Laboratorio de Farmacología, Instituto
de Ciencias Odontológicas, Facultad de
Odontología, Universidad de Chile. 2,
Laboratorio de Bioenergética y Cáncer,
Instituto de Ciencias Biomédicas, Facultad de
Medicina, Universidad de Chile.
Lung cancer has the highest mortality
between all neoplasms, being the second
leading cause of death in Chile. These cancers
are classified as small cell carcinoma or nonsmall cell carcinoma, being Smoking the
main risk factor. Conventional treatments
are radiotherapy, chemotherapy and surgery;
however, 5-year survival rates remain
extremely poor, due to the development of
resistance and eventual relapse. The Cancer
Stem Cells hypothesis suggests that they are
responsible for tumor initiation and growth
and are resistant to conventional treatments.
Therefore, it is necessary to develop new
therapies that allow us to effectively eliminate
resistant tumor cells (TC). Mitochondria
may be considered as a therapeutic target in
the treatment of cancer, because it exhibits a
greater transmembrane potential in TC, being
susceptible to being the target of positively
charged molecules. The delocalized lipophilic
cations of triphenylphosphonium (TPP+) are
molecules synthesized from gallic acid, mono
143
and polybenzoates decyl esters. Therefore,
we evaluated 4 decyl polyhydroxybenzoate
compounds linked to TPP+ as potential
cytotoxic agents in two lung cancer cell
lines (NCI-H727 and NCI-H1299) and in
pulmonary fibroblasts (PH) as control.
Doxycycline is an antibiotic of the tetracycline
group; recently its has been studied this
antineoplastic use producing the inhibition of
mitochondrial biogenesis in TC. Cell viability
assay was performed with the compounds
at 24, 48 and 72 hours in normoxia and
hypoxia with 5% oxygen to determine IC50,
to subsequently perform a combination test
of compounds with doxycycline. The results
showed that the TCT analyzed are sensitive to
the cytotoxic action of all compounds and this
effect is increased as time goes by. In addition,
there are no significant differences in IC50
between hypoxia and normoxia cultures.
119. TARGETING MITOCHONDRIAL
METABOLISM IN
NEURODEGENERATIVE DISEASES
THROUGH NCLX BLOCKADE.
Viejo L. 1,2; L. Arribas R. 1; Palomino A. 2;
Egea J. 2; Martinez-Ruiz A. 2; de los Ríos C.
1, 2.
1 Instituto Teófilo Hernando, Universidad
Autónoma de Madrid, C/ Arzobispo Morcillo,
4, 28029, Madrid, Spain. 2 Instituto
de Investigación Sanitaria del Hospital
Universitario de La Princesa, C/ Diego de
León, 62, 28006, Madrid, Spain.
The loss of mitochondrial function is
part of the almost all neurodegenerative
diseases. Therefore, there’s a reduction in
ATP synthesis ending up in a dysfunction of
neuronal dynamics. At least three proteins
from the Krebs’ circle are calcium sensitive. In
our laboratory, we’re focus in the design and
synthesis of CGP37157 derivatives, reference
blocker drug of the mitochondrial sodium/
calcium exchanger (NCLX), which also has
neuroprotective properties. Our aim is the
discovering of new pharmacological tools able
to handle calcium flux between mitochondria
and cytosol, lowering the calcium overload
descried in the cytosol. Putting together
these two ideas, we wondered if the partial
blockade of mitochondrial calcium efflux
could improve not only mitochondrial
metabolism but also calcium handling. The
CGP57137 derivative selected was ITH12575,
a benzothiazepine with a different aromatic
substitution. First, the calcium movements
were studied by the fluorescent dye Fluo4,
in the human neuroblastoma SH-SY5Y
cell line and in embryonic cortical neuros
144
of rat primary culture. In order to confirm
ITH12575 selectivity for NCLX, the exchanger
was silenced by a siRNA and neuroprotective
assays were evaluated. Finally, mitochondrial
stress assays were performed using the
seahorse method, in presence/absence
of both a toxic stimulus (high potassium
concentration) and the compound. Data from
ATP synthesis, mitochondrial respiration and
respiratory maximal capacity were obtained.
The results show that, by the regulation of
mitochondrial calcium, the mitochondrial
metabolism is partially recovered thanks to
ITH12575.
120. FOXO1 MEDIATES HIGH
GLUCOSE-CARDIAC FIBROBLASTS
DIFFERENTIATION
Vivar R 1, Anfossi R 1; Cárdenas S 2; Contreras
A 2.
1) Faculty of Medicine, University of Chile;
2)Department of Biology, Faculty of Basics
Sciences,
Metropolitan
University
of
Educational Sciences.
Normally cardiac fibroblasts (CF) maintain
the homeostasis of the extracellular matrix
(ECM) in the heart, whereas in pathological
conditions such as diabetes, become more
active promoting cardiac fibrosis. High
glucose (HG) induces CF differentiation,
where TGF-beta1 has a crucial role. TGF-beta1
requires FoxO1 to induce CF differentiation,
whereas FoxO1 is deregulated in diabetes,
resulting in its hyperactivation, oxidative
stress and cell differentiation. Therefore, in
this work we wanted to determine the role
of FoxO1 in CF differentiation promoted
by high glucose. CF obtained from adult
Sprague-Dawley rats were incubated in HG,
as a in vitro model of hyperglycemia. CTGF
and alpha-SMA expression was determined
by RT-PCR, whereas CTGF and alpha-SMA
protein were evaluated by westernblot
(WB). The activation of FoxO1 was analyzed
evaluating its phosphorylated forms, its
nuclear localization and the expression of
FoxO1 specific genes targets (p21cip and
p15ink) by RT-PCR. The oxidative stress was
evaluated analyzing the expression of the
FoxO3a, catalase and SOD2 proteins by WB,
and ROS production by colorimetry. The role
of FoxO1 was demonstrated using AS1842856
(FoxO1 inhibitor) and FoxO1 silencing using
siRNA. HG increased the protein and mRNA
of CTGF and alpha-SMA (CF differentiation
marker), whereas HG decreased of AKT
activation, decreased phospho-s256-FoxO1
level, increased FoxO1 nuclear localization
and increased FoxO1 genes target expression
(FoxO1 activation marker). Likewise, HG
decreased FoxO3a, catalase and SOD2
protein, and increased ROS production. In
addition CF differentiation induced by HG
was completely abolished by FoxO1 inhibition
using AS1845628 and FoxO1 silencing.
Collectively these data suggest that FoxO1 is
necessary to CF differentiation induced by
high glucose and suggest that FoxO1 would be
a pharmacological target for new treatments
against diabetic cardiomyopathy.
121. PHARMACOLOGICAL
COMPARISON BETWEEN
PREFRONTAL CORTEX AND
NUCLEUS ACCUMBENS
NEUROTRANSMITTER LEVELS
AFTER BASOLATERAL AMYGDALA
STIMULATION.
Zegers J.A.1; Yarur H.E.1; Bastías C.P.1;
Gysling K.1.
Departemt of Cell and Molecular Biology,
Faculty of Biological Sciences, Pontificia
Universidad Católica de Chile.
Glutamatergic neurons of the basolateral
amygdala (BLA) innervate both, the nucleus
accumbens (Nac) and prefrontal cortex (PFC)
(McDonald, 1991). The relation between BLA
and Nac has been implicated on the control
of motivated behavior (Stuber et. al., 2011).
Furthemore, the triade between BLA, Nac
and PFC has also been shown to be critical in
the regulation of goal-directed behavior by an
inhibitory control of PFC on Nac dopamine
release during amygdala activation (Jackson
et. al. 2018). BLA has been implicated in
fear, anxiety and stress (Simon et. al., 2014;
Janak and Tye, 2015). The stress response
is centered in the corticotrophin releasing
factor (CRF) system (Bale and Vale, 2004).
There are two major receptors for CRF in
the brain, type-1 and type-2 CRF receptors
(CRF-R1 and CRF-R2). Several studies have
shown a significant role of CRF-R1 in the
stress response; however, the role of PFC
and Nac CRF-R2 in the stress response is
poorly understood. We studied the role of
CRF-R2 in PFC and Nac neurotransmitter
levels after BLA stimulation by double in vivo
microdialysis in PFC or Nac of anesthetized
adult rats. Local infusion of antisauvagine
30 (CRF-R2 antagonist) in the PFC or
Nac significantly increased PFC and Nac
dopamine and glutamate extracellular levels
induced by BLA stimulation. Our results
suggest that there is an inhibitory tone
mediated by CRF-R2 controlling dopamine
and glutamate extracellular levels in PFC and
Nac that dependon BLA stimulation.
122. ACTIVATION OF NMDA
RECEPTORS DURING CHRONIC PAIN
RECRUITS PROTEIN SRC KINASES
TO OPEN PANNEXIN1 CHANNEL IN
NEUROPATHIC RAT MODEL.
Zepeda K. D 1; Hernández A. 1; Pelissier T. 1;
Constandil L. 1.
1 Laboratorio de Neurobiología, Facultad de
Química y Biología, Universidad de Santiago
de Chile. 2 Prog. Farmacología Molecular
y Clínica, ICBM, Facultad de Medicina,
Universidad de Chile.
In experimental pain models the upregulation
of NMDA receptor (NMDAR) appears to be
crucial in the enhanced responsiveness of
nociceptive neurons of dorsal horn of the
spinal cord for initiation and maintenance of
pain. In the chronic pain model performed
by our laboratory, it was shown that
intrathecal injection i.t. of 10Panx (Inhibitor
panx1 channel peptide) prevents the effect
of hyperalgesia caused by i.t of NMDA in
neuropathic rats suggesting an interaction
between Panx1 channel and NMDAR. Our
work is based on elucidating whether the
regulation of the Panx1 channel by posttranslational modifications is carried out
by Src kinases in neuropathic rats model.
In dye uptake experiments (used to assess
Panx1 channel opening) of the spinal cord
slices of neuropathic rats have demonstrated
that pharmacological inhibition of PP2 (Src
tyrosine kinase protein inhibitor), and 10Panx
decreased dye uptake in neurons stimulated
by NMDA. Likewise, rats treated with
10panx-NMDA or PP2-NMDA decreases the
expression of phosphorylated Panx1 (pPanx1)
and phosphorylated Src527 (pSrc527) by
western Blotting. Algesymmetric test (Randall
Selitto) results have shown that intrathecal
administration of 10Panx (300 μM, 10μl i.t )
-NMDA (0.6 mM, 10μl i.t) and PP2 (3.3 mM,
10μl i.t )-NMDA (0.6 mM, 10μl i.t) inhibit
pain in neuropathic rats compared to control.
We conclude that Panx1 or Src inhibition
prevents nociceptive signaling induced by
upregulation of NMDAR in neuropathic rats,
suggesting that the pronociceptive effects
of pharmacological activation of NMDAR
induce the opening of the Panx1 channel
probably mediated by Src kinase.
145
123. PARTICIPATION OF VGLUT AND
GLUTAMATE SIGNALING DURING
NEURULATION IN XENOPUS LAEVIS.
Nicolás Zúñiga S.1, Camilo Venegas L.1,
Patricio Castro M.1.
1Laboratory of Physiology and Pharmacology
for Neural Development, Department of
Physiology, Faculty of Biological Sciences,
Universidad de Concepción, Concepción,
Chile.
Introduction: The development of the
nervous system begins with the closure of
the neural tube, one of the morphological
changes present in the neurulation process.
Several signaling pathways participate in
the formation of the neural tube, such as
FGF, Wnt, Chordin and glutamate, recently
described. Failures in this process lead
to the formation of neural tube defects
(NTDs), the second more prevalent birth
defect worldwide. Use of antiepileptic drugs
during pregnancy had shown an increase
in the incidence of NTDs by mechanism
not fully understood yet. Here, we propose
that glutamate, the principal excitatory
neurotransmitter of the nervous system and
specifically its vesicular transporter VGLUT1,
participate in the normal closure of neural
tube. Methods: X. laevis embryos were treated
with Rose Bengal, a VGLUT1 antagonist, at
early neurula stage (14 hpf, neural plate)
until end of neurulation (21 hpf). Then,
we perform a morphological evaluation of
neural tube closure and immunofluorescence
experiments. Additionally, after neurula
treatments, behavior studies using Xenopus
tadpoles (stg 45-49), were performed to
evaluate epileptic sensibility by measure
seizure latency onset using Pentylenetetrazol
(PTZ). Results: We observe and incomplete
neural tube closure in embryos treated with
Rose Bengal in a dose-response manner, with
an EC50 of 3.5±0.5μM. Furthermore, we
observe a decrease of ~43% (p<0.01, ANOVA)
in the seizure latency onset necessary for
epileptogenic behavior induced by PTZ and a
5-fold increase (0.4±0.3 m vs 5±0.2m) in the
distance traveled at 2 minutes after treatment
(p<0.05, ANOVA). Conclusions: VGLUT1mediated glutamate signaling participate
in normal neural tube development. Partial
blocking of this pathway at neurula modifies
the epileptogenic-induce response in tadpoles,
possibly by alter the normal establishment of
the nervous system.
146
124. INDOMETHACIN IMPAIRS
POLYAMINE METABOLISM IN LUNG
CANCER CELLS: A KRAS MUTATIONASSOCIATED FEATURE?
López-Contreras F1, Muñoz-Uribe M1, PerezLainez J1, Ascencio-Leal L1, Rivera-Dictter
A1, Martin-Martin A1, Burgos Aguilera R1,
Alarcon Uribe P1, López-Muñoz R1.
1 Instituto de Farmacología y Morfofisiología,
Facultad
de
Ciencias
Veterinarias,
Universidad Austral de Chile.
Non-small cell lung cancer (NSCLC) is the
most lethal and prevalent type of lung cancer.
NSCLC patients carrying mutations in the
Kirsten rat sarcoma viral oncogene homolog
gene (KRAS) still lack targeted therapies.
Also, the levels of polyamines (putrescine,
spermidine, and spermine) are increased
in cancer, playing a pivotal role in tumor
proliferation. Indomethacin increases the
levels of the polyamine-catabolic enzyme
spermidine/spermine-N1-acetyltransferase
(SSAT). Consequently, the aim of this study
was to compare the effect of indomethacin
in the polyamine metabolism of two NSCLC
cell lines, with different KRAS mutation
status. A549 and H1299 NSCLC cells (KRASmutated and wild-type, respectively) were
exposed to indomethacin. Evaluations
included SSAT expression and protein levels,
and metabolic analysis of cells by CG-MS
metabolomics. Moreover, the difference in
polyamine synthesis enzymes among cell lines
and the synergistic effect of indomethacin
combined with inhibitors of these enzymes
were investigated. Indomethacin increased
the expression and levels of SSAT in both
cell lines. In A549 cells, indomethacin
significantly impairs polyamine metabolism.
However, in H1299 cells, the impact of
treatment on the polyamine pathway was
non-significant. Evaluation of the levels of the
polyamine synthesis enzymes showed that
ornithine decarboxylase (ODC) is increased in
A549 cells, whereas S-adenosylmethioninedecarboxylase (AMD1) and polyamine
oxidase (PAOX), are increased in H1299
cells. Finally, indomethacin demonstrated
a synergistic effect with the PAOX inhibitor
MDL72527 in A549 cells, whereas in H1299
had a synergistic effect with the AMD1
inhibitors SAM486. Collectively, these results
indicate that indomethacin alters polyamine
metabolism in NSCLC cells and enhances
the effect of polyamine synthesis inhibitors
such as MDL72527 or SAM486. However,
this effect varies depending on the basal
metabolic fingerprint of each type of NSCLC
cell. FONDECYT-1160807.
147
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