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““INCREASED PROTHROMBIN, INCREASED PROTHROMBIN, APOLIPOPROTEIN A-IV, AND APOLIPOPROTEIN A-IV, AND
HAPTOGLOBIN IN THE HAPTOGLOBIN IN THE CEREBROESPINAL FLUID OF CEREBROESPINAL FLUID OF
PATIENTS WITH HUNTINGTONPATIENTS WITH HUNTINGTON´S DISEASE.”´S DISEASE.”
Yen-Chu Yen-Chu HuangHuang.
Maria Camila Montufar PantojaHeyly Milena OrtegaIII SEMESTER2011
UNIVERSIDAD PONTIFICIA BOLIVARIANA
GENERAL OBJETIVE
Demonstrate how Prothrombin, Apolipoprotein A-IV and Haptoglobin concentrations in CSF has an important role in the HD evolution and compare these concentrations in HD and control patients .
INTRODUCTION
HD is a hereditary, degenerative brain disorder.
It is caused by an unstable CAG trinucleotide.
Today there are others proteins involved in HD
development and symptoms and its important
to clarify through proteomics studies in CSF
wich are the concentrations (up or down) and if
this levels of biomarkers are correlated in HD.
PROTHROMBINPROTHROMBIN
Plasma protein synthesized in the liver.
It plays an important function in coagulation process by reaction with the thromboplastin enzyme , with Ca ++ cation and V activate factor as coenzymes.
In HD prothrombin proinflammatory agent : DISEASE STATUS BIOMARKER
Tromboplastina
Ca++
Xa
Va
APOLIPOPROTEIN AIV (APO AIV) Glycoprotein synthesized by the human intestine and
liver
The formation of chylomicrons acts as a signal for the
induction of Apo AIV synthesis
Alters the activity of the enzymes (LPL and LCAT) of
lipoprotein metabolism and cholesterol efflux from
extrahepatic tissues
Is involved in the long-term regulation of both food
intake and body weight
HAPTOGLOBIN
Protein produced by the liver
Haptoglobin binds free hemoglobin (Hb) released
from erythrocytes with high affinity and thereby
inhibits its oxidative activity
The haptoglobin-hemoglobin complex will then be
removed by the reticuloendothelial system
It prevents loss of iron through the kidneys and
protecting the kidneys from damage by hemoglobin
HUNITNGTON DISEASE - HD
Progressive neurodegenerative disorder with autosomaldominant inheritance:
- Movement Disorders.- Cognitive impairment.- Psychiatric disorders.
MATERIALES Y METODOS
LCR y suero extraido de 9 HD ( 5 hombres- 4 mujeres).
LCR y suero de 18 pacientes tipo control (7 hombres- 11 mujeres).
Cuatro grupos:- 1 Electroforesis de dos dimensiones.- 3 Cuantificacion de protrombina, Apo-A IV y
haptoglobina.
PREPARACION DE LAS MUESTRAS
ELECTROFORESIS DE DOS DIMENSIONES (2-DE )
Técnica de alta resolución cuyo objetivo es la separación de mezclas de proteínas
- Punto isoeléctrico
(isoelectroenfoque).
ánodo es ácida y la del cátodo es alcalina
- Masa molecular por electroforesis gel de poliacrilamida
DIGESTION EL GEL
Forma parte de la preparación de las muestras para la espectrometría de masas
- Decoloración.- Reducción y alquilación. - Corte de la proteína serina proteasa
tripsina
- Extracción del péptido generado.
ESPECTROMETRIA DE MASA
Técnica experimental que permite la medición de iones
derivados de moléculas
Se basa en el fenómeno conocido como desbastado
(sputtering, en inglés) de partículas centradas en un
blanco, que son bombardeadas por iones, átomos o
moléculas. Dependiendo del intervalo de energía de la
partícula primaria, ocurren colisiones.
El patrón de fragmentación resultante así como los
iones residuales constituyen el espectro de masas
WESTERN BLOT
Busco proteínas especificas
Uso ac Uso técnica SDS-
PAGE Separación de
proteínas por electroforesis en gel
ELISA
Ensayo por inmunoabsorción ligado a enzimas
Fosfatasa alcalina—peroxidasa de rabano
Detección ag-ac 3 tipos: Ensayo tipo sandwich Ensayo competitivo Ensayo indirecto
RESULTADOS
Representative Western blots of prothrombin (72 kD) and albumin (64 kD) in CSF and serum of HD patients and their corresponding controls.
Representative Western blots of Apo A-IV (46 kD) and albumin (64 kD) in CSF and serum, respectively, of HD patients and their corresponding controls (C).
RESULTADOS
Qalb, CSF haptoglobin, and serum haptoglobin concentration in HD patients.
RESULTADOS
DISCUSSION
AUTOR OPINION YES NO
Bjorkqvist M.
The mutant huntingtin may enchance the ability of microglia to produce proinflamatory mediators (IL-6, IL-8 and TNF-a) and contribute to neurodegeneration in HD.
Djoussé L.
In HD, weigth loss is a characteristic feature in advanced as well early stage.
Fang Q, Strand
In proteome analyses several the neuronal proteins were found to be reduced
Underwood BR
A serum metabolic profile indicative of a pro-catabolic phenotype in early HD
CONCLUTIONS
The proteomic studies in CFS in patients HD and control patients are very important to determinate if the increase levels of Haptoglobin, Apolipoprotein A-IV and Prothrombin are correlated with HD development.
It is important to recognize this proteins like specifics biomarkers in HD; thats why the concentrations of each one were quantified in CSF and serum; thus indirectly evaluate the BBB integrity.
CONCLUTIONS
To be able to correlate the levels of these proteins with other
mental diseases such as Alzheimer's, multiple sclerosis, among
others; allow us to determine whether the activity of proteins
mentioned in the study are a common phenomenon in a variety
of neurodegenerative-tive disease or are specifically for HD.
Becouse of the intact values of BBB and the increase levels of
proteins, it suggest that they are produced in the CNS itself and
that the relation within de concentrations of proteins in CSF has
nothing to do with the concentration of these in serum.
MAPA CONCEPTUAL
BIBLIOGRAFIA
MARTINEZ, L. Biología Molecular. ED UPB. Segunda edición. Medellín. 2011.
Yen-Chu Huang. Increased Prothrombin, Apolipoprotein A-IV, and Haptoglobin in the Cerebrospinal Fluid of Patients with Huntington’s Disease . Plos One. Vol 6, January 2011.
http://www.madrimasd.org/informacionidi/noticias/noticia.asp?id=44116