seminario molecular

““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